WO2014205074A2 - Devices, compositions and methods for imaging with raman scattering - Google Patents

Devices, compositions and methods for imaging with raman scattering Download PDF

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Publication number
WO2014205074A2
WO2014205074A2 PCT/US2014/042936 US2014042936W WO2014205074A2 WO 2014205074 A2 WO2014205074 A2 WO 2014205074A2 US 2014042936 W US2014042936 W US 2014042936W WO 2014205074 A2 WO2014205074 A2 WO 2014205074A2
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Prior art keywords
bond
edited
compound
imaging
vibrational
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PCT/US2014/042936
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French (fr)
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WO2014205074A3 (en
Inventor
Wei MIN
Lu WEI
Zhixing CHEN
Fanghao HU
Yihui Shen
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The Trustees Of Columbia University In The City Of New York
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Publication of WO2014205074A2 publication Critical patent/WO2014205074A2/en
Priority to US14/974,992 priority Critical patent/US11408892B2/en
Publication of WO2014205074A3 publication Critical patent/WO2014205074A3/en
Priority to US15/927,463 priority patent/US20180372632A1/en
Priority to US16/717,634 priority patent/US11408894B2/en
Priority to US17/027,515 priority patent/US20210068663A1/en
Priority to US17/882,938 priority patent/US20230128716A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/4795—Scattering, i.e. diffuse reflection spatially resolved investigating of object in scattering medium
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N2021/653—Coherent methods [CARS]
    • G01N2021/655—Stimulated Raman

Definitions

  • the present disclosure relates generally to imaging technology, and in particular to vibrational microscopy and spectroscopy with Raman scattering technology.
  • fluorescent microscopy such as confocal microscopy, t o-phoion microscopy, single molecule microscopy and superresolution microscopy have enabled detection, of structures that can be much deeper and finer than before.
  • quantum mechanics e.g. particle in the box
  • the chromopSiore within a fluorophore have to be a large conjugation, system in order for the efficient absorption i the visible spectrum.
  • fluorescent tags intrinsically cannot be properly used for tagging small molecules such as glucose, nucleosides, amino acids, choline, fatty acids and small molecule drags, for their relatively large size perturbs with the small molecule dynamics.
  • An opposite strategy for visualizing these important building block small molecules in biological systems can be label-free imaging.
  • Representative imaging procedures of the kind can include vibration microscopies based on infrared absorption and Raman scattering detecting the characteristic vibrational mode of specific chemical bond from the molecules themselves (See, e.g., References 5-9).
  • Other label-free procedures can be second harmonic generation ("SHG"), imaging special. «on-cetUrosym.m.eirk structures, third harmonic generation. (“THG”), sensing interfaces and optical heterogeneities and optical coherence tomography (“OC '), measuring the backscattered light from tissues through low-coherence ioterfero etry.
  • label-free imaging can suffer from two fundamental problems; first, there can be insufficient specificity because small molecules usually do not have unique spectroscopic signature in the vast pool of other biomolecuSes; second, there can be unsatisfying sensitivity due to usually low concentration of the small molecules in the biological systems.
  • alkyne e.g. carbon-carbon triple bond
  • alkyne can offer three advantages over others.
  • alkyne is only a chemical bond
  • second alkyne can enable background-free detection
  • alkyne can be inert to react with any intrinsic bio-molecules in the biological systems, in fact, a!kyne can be widely used in the powerful hioorthogonai chemistry utilizing ajkyne-azide specific click-chemistry reaction for various purposes (See, e.g.. References 10-12).
  • aztde tagged detection reagent e.g. affinity probes or fluorescent tag
  • the proteome of a cell can be highly dynamic in nature and tightly regulated by both protein synthesis and degradation to actively maintain homeostasis. Many intricate biological processes, such as ceil growth, differentiation, diseases and response to environment stimuli, can require protein synthesis and translatk ai control (See, e.g., Reference 24). In particular, long-lasting forms of synaptic plasticity, such as those underlying long-term memory, can need new protein synthesis in a space- and time- dependent, manner (See, e.g., References 26-30). Therefore, direct visualization and quantification of newly synthesized proteins at a global level can be indispensable to unraveling the spatial-temporal characteristics of the proteomes in live cells.
  • Newly synthesized proteins ca then be visualized through subsequent conjugation of the reactive amino acids to .fluorescent tags via click chemistry. (See, e.g.. Reference 29). Unfortunately, these fluorescence-based methods generally use non-physiological fixation and subsequent dye staining and washing.
  • radioisotope labeling can be another powerful tool to trace and quantify proteome dynamics.
  • Classical radioisotope-labeled amino acids e.g., 35S-meth.ion.ine
  • 35S-meth.ion.ine can provide vigorous analysis of global protein synthesis.
  • One exemplary aspect of the present disclosure relates to a method for obtaining biological information in a living cell or a living organism with bond-edited compounds using stimulated Raman scattering.
  • the method comprises the steps of introducing one or more bond-edited compounds into a li e cell or a living organism, and detecting a vibrational tag in the cell or organism with stimulated Raman scattering,
  • Another exemplary aspect of the present disclosure relates to methods for making a bond-edited compound.
  • Another exemplary aspect of the present disclosure relates io a method for detecting a disease condition in a subject, comprising: administering to said subject a composition comprising a bond-edited compound targeting a disease tissue or pathogen, and detecting said bond-edited compound by stimulated Raman scattering.
  • Another exemplary aspect of the present disclosure relates to a method for monitoring a treatment for a disease condition.
  • the method comprises administering to the subject
  • composition comprising a bond-edited compound and detecting the bond-edited compound by stimulated Raman scattering at a first time point, performing the treatment after the first time point, further administering to said subject the composition comprising a bond-edited compound, and detecting the bond-edited compound by stimulated Raman scattering at a second time point, and comparing images obtained at the two time points,
  • Another exemplary aspect of the present disclosure relates to a method for screening an agent.
  • the method comprises administering the agent and at least one bond-edited compound to a live cell or organism, detecting the bond-edited compound in the live cell or organism using stimulated Raman scattering, and selecting a candidate agent based on one or more predetermined criteria, such as the uptake, accumulation, trafficking or degradation of the bond-edited compound by the said live cell or organism.
  • Another exemplary aspect of the present disclosure relates to a device for imaging bond-edited compounds by stimulated Raman scattering.
  • the device comprises a first
  • single-wavelength laser source that produces a pulse laser beam of a first wavelength
  • second single- wavelength laser source that produces a pulse laser beam of a second wavelength
  • a modulator that modulates the pulse laser beam of one of the first or second laser source
  • a pbotodetector that is capable of or configured to detect stimulated Raman scattering from a biosample, and a computer.
  • Another exemplary aspect of the present disclosure relates to a non-transitory computer-accessible medium having stored thereon computer-executable instructions for determining data associated with at least one tissue, wherein, when a computer hardware
  • the computer arrangement executes the instructions, the computer arrangement is configured to perform procedures comprising; receiving first information related to at least one bond between at least two atoms attached to a metabolite; and determining the data based on the at least one bond,
  • Another exemplary aspect of the present disclosure relates to a method for determining data associated with at least one tissue, comprising: receiving first information related io at least one bond between at least two atoms attached to a metabolite; and using a computer hardware arrangement, determining the data based on the at least one bond.
  • Another exemplary aspect of the present disclosure relates to a system for determining data associated with at least one tissue, comprising: a computer processing arrangement configured to receive first information related to at least one bond between at least two atoms attached to a metabolite; and determine the data based on the at least one bond.
  • Another exemplary aspect of the present disclosure relates to a pre-mixed essential amino acid combination, comprising: at least one non-denierated essential amino acid: and at least 5 deuterated essential amino acids.
  • FIG. 1 shows imaging complex protein metabolism by stimulated Raman scattering (SRS) microscopy in live cells, tissues and animals.
  • SRS stimulated Raman scattering
  • FIG. la illustrates a cartoon for SRS imaging following metabolic labeling of deuterated amino ac ids (D-AAs) in live organisms (e.g. mice), which are first administered with D-A As for certain period of time and then imaged by SRS to probe protein metabolism.
  • D-AAs deuterated amino ac ids
  • lb illustrates spontaneous Raman spectra from HeLa cells incubated with medium containing either regular amino acids (gray, dashed) or D-AAs illustrate three distinct ways to probe complex protein metabolism: imaging newly synthesized proteins by targeting 2133 cnvi from carbon-deuterium bonds (C-D), imaging degradation of pre-existing proteins by targeting the pure methyl group (Clh) distribution, and two-color pulse-chase protein imaging by labeling with two sub-groups of D-AAs (i.e., group I and group II).
  • FIG. 2 depicts sensiti vity optimization and time-lapse imaging of the de novo proteome synthesis dynamics.
  • FIG. 2a illustrates spontaneous Raman spectra of C-D peaks in HeLa cells incubated in optimized deuteration medium display a 50% increase when compared to the previously reported partial deuteration medium, and about 8 times higher than using leucine-dj f t only.
  • FIG. 2b illustrates SRS images of newly synthesized proteins in live HeLa cells confirm a 50% average signal increase.
  • FIG. 2c illustrates SRS images of newly synthesized proteins in live neurons in optimized deuteration medium for 20 h. The zoom-in image highlights the fine dendritic structures (likely dendritic spines, arrow-headed).
  • FIG. 2d illustrates SRS image of newly
  • FIG. 2e illustrates time-lapse SRS images of protein synthesis dynamics in a same set of live HeLa cells with
  • FIG. 3 shows time-dependent SRS images of protein degradation.
  • FIG. 3a illustrates adopting linear combination algorithm between 2940 cm " and 2845 cm ' channels, the obtained SRS image exclusi vely from 03 ⁇ 4 vibration dispiay gradual degradation of pre-existing proteins in live HeLa cells cultured in optimized deuteration medium for 0 h, 24 h, 48 h and 96 h
  • FIG, 3b illustrates SRS images exclusively from (3 ⁇ 4 vibratio dispiay the total lipid distribution at the corresponding time point.
  • FIG. 3c illustrates a single exponential decay fitting from averaged image intensities from pre-existing protein in FIG. 3a, yielding a protein degradation time constant of 45 ⁇ 4 h. Error bars., standard deviation. Scale bar, 10 ⁇ .
  • FIG. 4 depicts pulse-chase SRS imaging of temporally defined proteins.
  • FIG. 4a illustrates structures and spontaneous Raman spectra of group I D-AAs (i.e. the branched chain amino acids).
  • FIG. 4b illustrates structures and spontaneous Raman spectra of three examples of group II non-branched D ⁇ AAs.
  • FIG. 4c illustrates spontaneous Raman spectra of HeLa cells cultured with group I D-AAs ⁇ element 405), showing multiple peaks with the first around 2067 cm '1 , and with group 11 D-AAs ( element 410), showing a common peak around 2133 cm '1 .
  • FIG, 4d illustrates two-color pulse-chase imaging by sequential labeling of group and group I D-AAs in time with simultaneous expression of mutant himtingtin (mHtt94Q-m£os2) proteins.
  • Cartoon displays experimental timeline of plasn.iid transaction and D-AA medium exchanges.
  • the fluorescence image (overlaid with bright field) indicates the formation of a large aggregate
  • the retrieved signals from linear combination of the original images at 2067 and 2.133 channels display a large aggregation of mBtt proteins solely labeled by group II D-AAs during the first 22 h (pulse 415) and mHtt only labeled by group 1 D-AAs during the following 20 h (chase 420).
  • the merged image, as well as the intensity profile, from, the pulsed (element 415) and chased (element 420) images confirms with a yellow core and a green shell. Scale bar, 10 um.
  • FIG . 5 shows SR S imaging of newly synthesized proteins in live mouse brain tissues.
  • FIG. 5a illustrates SRS images at dentate gyrus of a. live organotypic brain slice (400 pin thick, from a ⁇ 0 mouse) after culturing in D-AA medium for 30 hr. 2133 cm “1 (C-D) image presents the distribution of newly synthesized proteins.
  • the C3 ⁇ 4 and CI L images show the old protein pools and total lipids, respectively.
  • FIG. 5a illustrates SRS images at dentate gyrus of a. live organotypic brain slice (400 pin thick, from a ⁇ 0 mouse) after culturing in D-AA medium for 30 hr. 2133 cm “1 (C-D) image presents the distribution of newly synthesized proteins.
  • the C3 ⁇ 4 and CI L images show the old protein pools and total lipids, respectively.
  • 5b illustrates a 4-by-3 ram large field view overla image of new proteins (C-D, element 505), old proteins ( €3 ⁇ 4, element 510) and total lipids CC3 ⁇ 4, element 515) for a brain slice (400 ⁇ thick, from a PI 2 mouse) cultured in D-AA medium for 30 h.
  • Scale bar 100 ⁇ ,
  • FIG. 6 shows SRS imaging of newly synthesized proteins in viva.
  • FIG. 6a illustrates SRS images of a 24-hpf (hpf: hours post fertilization) zebrafish. Wild-t pe zebrafish embryos were injected at I -cell stage with I nL D-AA solution and allowed to develop normally for another 24 h before imaging. Bright field image shows the gross morphology of embryonic zebrafish at 24 hpf (dashed boxes ). 2133 cm '1 (C-D) image presents the distribution of newly sy nthesized proteins (Supplemental FIG. 2a) in the somites of an embryonic zebrafish tail.
  • C-D 2133 cm '1
  • FIG. 6b and 6c SRS images of live mouse liver are shown in FIG. 6b and. intestine tissues
  • FIG. 6c harvested from mice after administered with D-AA containing drinking water for 12 days, 2133 cm *1 (C-D) channel shows newly synthesized proteins (Supplemental FIG. 2b ⁇ 2c that resemble the distribution of total protein as shown in the 1 55 cm 4 image (Amide ⁇ ). Scale bar, 10 ⁇ .
  • FIG, 7 depicts SR images at 2067 cm ! and 2133 cm "1 channels of proteins labeled with grou 1 D-AA only shown in FIG, 7a and group 0. D-AA only shown in FIG. 7b.
  • FIG. 8 shows raw C-D on-resonance (2133 cm *5 ) and off-resonance (2000 cm *1 ) SRS images of newly synthesized proteins in vivo in FIG. 6a, which illustrates SRS C-D on-resonance and off-resonance images of a 24 hpf embryonic zebrafish. The difference image between C-D
  • FIG. 6 shows pure C-D labeled protein distribution i the somites of an embryonic zebrafish tail, as in FIG. 6,
  • FIG, 8 b and 8c illustrate SRS C-D on-resonance and off-resonance images of live mouse liver FIG. 6b and intestine FIG. 6c tissues harvested from the mice after administering with D-AA containing drinking water for 12 days.
  • the difference image between C-D on-resonance and off-resonance shows pure C-D labeled protein distribution in the liver and intestine tissues, shown in FIG. 6b and FIG. 6c, respectively.
  • the residual signal presented m the off-resonance images mainly comes from
  • FIG. shows SRS imaging for newly synthesized proteins in vivo with
  • FIGS. 9a and 9b illustrate SRS images of live mouse liver FIG, 9a and intestine FIG. 9b tissues harvested from mice after
  • FIG. 9c and 9d illustrate corresponding raw C-D on-resonance (2133 cm *1 ) and off-resonance (2000 cm") images are shown as references for liver FIG. 9c and intestine FIG. 9d tissues. Scale bar, 10 ⁇ .
  • FIG, 10 depicts bond-selecti ve SRS imaging of alkynes as nonlinear vibrational tags.
  • FIG. 1 a illustrates Spontaneous Raman spectra of HeLa ceils and 10 mM EdU solution. Inset: the calculated SRS excitation profile (FWHM 6 cm-') is well fitted within the 2125 cm" alkyne peak (FWHM 14 era ', magenta).
  • FIG. 10b illustrates linear dependence of stimulated ' Raman loss signals (2125 cm '* ) with EdU concentrations under a 100 ps acquisition time.
  • F.1G. 10c illustrates the metabolic incorporation scheme for a broad spectrum of Jkyne-tagged small precursors. a.u. arbitrary units.
  • FIG, 1 1 illustrates the working mechanism of a stimulated Raman scattering with A Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second).
  • the Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second) are both temporally and spatially synchronized before focused onto cells that have been metabolically labeled with alkyne-tagge small raolecoies of interest.
  • the energy difference between the Pump photon and the Stokes photon matches the vibrational .frequency (-3 ⁇ 4&) of alkyne bonds, alkyne bonds are efficiently driven from their vibrational ground state to their vibrational excited state, passing through a virtual state.
  • a photon in the Pump beam is annihilated (Raman loss) and a photon in the Stokes beam is created (Raman gain).
  • the detected pump laser intensity changes through a lock-in amplifier targeted at the same frequency as the modulation of Stokes beam serve as the contrast for alkyne distributions,
  • FIG. 12 shows live SRS imaging of de novo synthesis of DNA, RNA, proteomes,
  • FIG. 12a illustrates Raman spectra of cells incubated with EdU, EU, Hpg, propargylehoiine and i 7-Qctadecynoic acid ( 17-ODYA).
  • FIG, 12b illustrates live HeLa cells incubated with 100 uM EdU alone
  • FIG. 12c illustrates time-lapse images of a dividing ceil incubated with 100 ⁇ EdU.
  • FIG. 12d illustrates live HeLa cells incubated with 2 mM EU alone
  • FIG. 12e iilosiraies pulse-chase imaging of RNA turnover in HeLa cells incubated with 2 mM EU for 12 h followed by EU-frce medium.
  • FIG. 12f illustrates live HeLa cells incubated with 2 raM Hpg alone (alkyne-on) and with 2 mM methionine (Control).
  • FIG. 12g illustrates live neurons incubated with i mM propargylehoiine (alkyne-on).
  • FIG. I h illustrates live macrophages incubated with 400 ⁇ 17-ODYA (alkyne-on).
  • FIG. 12i illustrates C. egctns fed with
  • FIG. 12j illustrates dual-color images of simultaneous EdU ' (2125 cm “1 ) and propargylehoiine (2142 cm") incorporation.
  • alkyne-off and amide images display the same set of cells as the alkyne-on images; lipid images capture the same cells as controi images.
  • Scale bars .10 um. Representative images of 10-15 trials, a.o, - arbitrary units.
  • FIG. 13 shows SRS imaging of distal mitotic region of e!egam gerrnlrae incorporated with EdU.
  • the composite image shows both the protein derived 1655 cm “1 (amide) signal, from all the germ cells, and the direct visualization of alkynes (2125 cm “1 (EdU)) highlighting the proliferating germ cave ls.
  • White circles show examples of EdU positive gertn cells in the mitotic region of C iegcms gerailine. Scale bar, 5 pm,
  • FIG. .1 shows SRS imaging of fixed HeLa cells after incorporating with.2 rnM Hpg.
  • the alkyne-on image displays the Hpg distribution for the newly synthesized proteins.
  • the off-resonant (alkyne-ofT image shows vanishing signal
  • the amide image shows total protein distribution. This result confirms that the detected signal is not from freely diffusive precursor Hpg itself (which is eliminated during the fixation process).
  • FIG. 15 shows click-chemistry based fluorescence staining of fixed HeLa ceils. Fluorescence images of HeLa cells incorporated with FIG, 15a, EdU (for D ' A); FIG. 15b Ell (for R A): FIG, 15c Hpg (for protein). Scale bars, 10 ⁇ .
  • FIG, 16 shows SRS imaging of propargylcholine incorporation in NIH3T3 cells and control experiments.
  • FIG. 16a illustrates fixed NIH3T3 cells after culturing with 0.5 x»M propargylcholine for 48 hours.
  • the alkyne-on image shows alkyne-tagged choline distribution.
  • FIG. 1 b illustrates treatment of fixed IH3T3 cells with phospholipase C, which removes Choline head groups of phospholipids onl in the presence of calcium.
  • the alkyne-on image shows the strong decrease of incorporated propargylcholine signal, supporting its main incorporation into membrane phospholipids.
  • FIG. 16a illustrates fixed NIH3T3 cells after culturing with 0.5 x»M propargylcholine for 48 hours.
  • the alkyne-on image shows alkyne-tagged choline distribution.
  • FIG. 1 b illustrates treatment of fixed IH3T3 cells with phospholip
  • FIGS, 16a-16c illustrate images in the same set of cells as in alkyne-on images, the alkyne-ofT images show a clear background.
  • the amide images display total protein distribution. Scale bars, 10 ⁇ ,
  • FIG. 17 shows m vim delivery of an alkyne-bearin drug (TH in DM SO) into mouse ear.
  • FIG. 1 a illustrates Raman spectra of a drug cream, Lamisil, containing 1% TH and mouse ear skin tissue.
  • FIGS. I 8b-18e illustrate SRS imaging of tissue layers from stratum corneum (z :::: 4 pm) to viable epidermis (z - 24 pm), sebaceous gland (z ⁇ 48 ⁇ ) and subcutaneous fat (r - 88 ⁇ ).
  • DMSO solution containing 1 % TH wa applied onto the ears of an anesthetized live mouse for 30 mm and the dissected ears are imaged afterwards.
  • alkyne-on images display TH penetration; alkyne-off images show off-resonant background (The bright spots in FIG. 18d are due to two-photon absorption of red blood cells).
  • the composite images show protein (1 55 cm *5 ) and lipid (2845 cm *1 ) distributions. Scale bars, 20 pm, a.u. arbitrary units.
  • FIG. 18 shows in vivo delivery of an. alkyne-bearing drag (TH m Lamisli cream, a FDA approved drug cream) into mouse ear.
  • I S(a-b) illustrates SRS imaging of the viable epidermis layer (z - 20 pm) and the sebaceous gland layer (z - 40 ⁇ ).
  • F IG. 18a and FIG. 18b illustrates the alkyne-on images display the TH penetration into mouse ear tissues through lipid phase.
  • the composite images show both protein (1655 cm “f ) and lipid (2845 cm") distributions. Scale bars, 20 p.m.
  • FIG, 1 shows an exemplary synthesis route for a bond-edited compound.
  • FIG. 20 shows in FIG. 20a another exemplary synthesis route tor a bond-edited compound (alkyne-D-glucose) and in FIG. 20b the spectroscopic characterization of the bond-edited compound in P BS buffer and in mammalian cells.
  • FIG. 21 shows time-dependent alkyne-D-glucose (32 mM) uptake in live HeLa ceils at lOrnin, 30 miu, l b, 2h, 3h and 4h time points.
  • the glucose signal inside mammalian cells is increasing over time.
  • FIG. 22 shows the results of a competition experiment to confirm the uptake of alkyne-D-glucose.
  • Regular D-glucose is added into cell medium for HeLa cells to compete with the uptake of alkyne-D-glucose.
  • the alkyne-D-glucose signal decreases (as shown both in images and bar diagrams).
  • L-ghicose which cells clo not uptake
  • alkyne-D-glucose signal is retained.
  • FIG. 23 shows aik ne-ghicose uptake in both neuronal culture FIG, 23a and brain slices FIG. 23b.
  • FIG. 24 shows multicolor imaging of DMA synthesis with EdU ( 1), EdU- 3 *C (2) and EdU- C2 (3).
  • FIG. 25 shows pulse-chase imaging ofD A synthesis (EdU ( 1) for pulse
  • FIG. 26 shows simultaneous three-color chemical imaging using alkyne probes for DNA synthesis (EdU- 5 i C (2) at 2077 crn-i ) and A synthesis (EU ⁇ U C2 (13) at 2053 cm-1 and 17-ODYA (12) at 2125 cm-1).
  • FIG, 27 shows images of subcutaneous colon cancer.
  • Subcutaneous colon cancer was grown for 15 days in mice, dissected out and cultured ex vivo in deuteraied amino acids containing medium for 47h (400um thick). Live image of the tumors shows intensive protein synthesis activity.
  • FIG. 28 shows active glucose metabolism in HeLa cells cultured m deuterated glucose medium.
  • FIG. 28a illustrates images after culturing in 0.1% D7-Giucose in EMEM for 48 hrs.
  • FIG. 28b illustrates HeLa cell images after culturing 0.2% D7-Glocose in EMEM for 48 hrs.
  • FIG, 29 shows active glucose metabolism in tumor cell line U87MG cultured in deuterated glucose medium (0.1% D7-GJucose in EMEM) for 48 hrs,
  • FIG. 30 shows the detection of D20 as a labeling reagent of the metabolism with stimulated Raman scattering.
  • FIG. 31 shows SRS imaging of C-D formation using D20 as a metabolic reagent for various of live organisms.
  • FIG. 32 shows imaging of C-phenylalanine labeled proteins for protein turnover.
  • FIG. 32 a shows an illustration of a spectroscopic characterization of Raman shift from 3004 cm ⁇ l to 968 cm" with the labeling of ' *C-phenylalanine
  • FIGS. 32(b) and 32c illustrate a time dependent ' ⁇ -phenylalanine labeling
  • FIG. 32b shows a spectrum
  • FIG. 32c illustrates SRS images, where the 968 cm-1 signal for ! , C labeled proteins are increasing while the 1004 cm" signal of old ! 3 ⁇ 4 -proteins are decreasing.
  • FIG. 33 is a set of exemplary images based on SR S imaging of newly synthesized proteins by metabolic incorporation of deuterium- labeled all amino acids in live HeLa cells.
  • FIG. 33a illustrates Spontaneous Raman spectrum of HeLa cells incubated with a medium containing deuterium-labeled all amino acids for 20 hrs, showing a -5 times stronger peak at 2133 cm-! tha the spectrum in FIG. 2
  • FIG. 33b illustrates SRS image targeting the central 2133 cra-i vibrational peak of C-D shows a high-contrast image representing newly synthesized proteins.
  • the same intensit scale bar is used here as in FIG. 2.
  • FIG. 33 c illustrates SRS image of the same cells as in FIG. 33b at off-resonance frequency 2000 cm-j is background-free.
  • FIGS, 33d-33f illustrate SRS images of same ceils as in FIG. 33b at frequency 1655 cm-t (amide I stretching attributed primarily to proteins); 2845 cm-- (CH2 stretching attributed mainly to lipids) and 2940 cm s (CH stretching attributed mainly to proteins) show the intrinsic distributions of total cellular lipids and proteins.
  • FIG. 34 is a set of exemplary images based on SRS imaging of time-dependent de novo protein synthesis and drug-induced protein synthesis inhibition effect in live HeLa cells incubated in deuterium-labeled all amino acid medium.
  • FIG. 34a ⁇ 34f SRS image targeting the DCitrai 2133 crn-f vibrational peak of C-D displays a time-dependent signal increase (5 1MS ⁇ FIG. 34a, 2 hrs ⁇ FIG. 34b, 20 hrs - FIG. 34c) of the newly synthesized proteins, with nucleoli being gradually highlighted.
  • the amide I (1655 crn-i) signal remains at a steady state over time (5 hrs - ⁇ FIG.
  • FIGS. 34g-34i illustrate ratio images between the S RS image at 2133 cm-i (newly synthesized protei ns) and the SRS image at 1655 cm-! (the amide 1 band from total proteins), representing the relative new protein fraction with subcellular resolution at each time point (5 hrs - FIG. 34g, 12 hrs - FIG. 34h, 20 hrs - FIG. 34i).
  • the bar represents the ratio ranging from low to high.
  • FIG. 34j shows time-lapse SRS images of a live dividing HeLa cell during a 25 roin time-course after 20-hour incubation with deuterated all amino acids medium.
  • FIG. 34j shows time-lapse SRS images of a live dividing HeLa cell during a 25 roin time-course after 20-hour incubation with deuterated all amino acids medium.
  • 34k illustrates a spontaneous Raman spectrum of HeLa ceils incubated with both deuterium-labeled all amino acids and a protein synthesis inhibitor anisomycin (5 ⁇ ) for 12 hrs shows the drastic attenuation of the C-D Raman peak at 21 3 cra-i.
  • FIG. 341 shows an exemplary SRS image of the same sample displays near vanishing signal throughout the whole field of view.
  • FIG, 34m shows, as a control, the image of the same cells at 2940 cm-i confirms that anisomycin does not influence the total protein level.
  • FIG. 35 is a set of exemplary images based on SRS imaging of newly synthesized proteins by metabolic incorporation of deuterium-labeled all amino acids in live human embryonic kidney ( ⁇ 293 ⁇ ) ceils.
  • FIG. 35a illustrates the spontaneous Raman spectrum of HEK293T cells incubated with deuterium-labeled all amino acids for 12 hrs shows a. 21.33 cn -t C-D • peak nearly as high as the Amide I (1655 cm-) ) peak.
  • FIG. 35a illustrates the spontaneous Raman spectrum of HEK293T cells incubated with deuterium-labeled all amino acids for 12 hrs shows a. 21.33 cn -t C-D • peak nearly as high as the Amide I (1655 cm-) ) peak.
  • FIG. 35b shows an exemplary SRS image targeting the central 2133 cm-i vibrational peak of C-D shows newl synthesized proteins in live HEK293T cells displaying a similar signal level as HeLa cells at 12 hrs (FIG. 4b).
  • FIG. 35c shows, as a comparison, the off-resonant image is still background-free.
  • FIGS. 35d and 35 (e) illustrate multicolor SRS images of intrinsic cell molecules: total proteins (1655 cra-i (FIG. 35d) and lipids (2845 cm-i (FIG. 35e).
  • FIG. 35f illustrates the ratio image between new proteins (2133 cra-i) and total proteins ( 1655 cra-i) illustrates a spatial map tor nascent protein distribution.
  • FIG. 36 is a set of exemplary images based on SRS imaging of newly synthesized proteins in both ceil bodies and newly grown neurites of neuron-like differentiable mouse neuroblastoma (N2 A) cells.
  • N2 A neuron-like differentiable mouse neuroblastoma
  • FIG. 36a illustrates SRS images targeting the 2133 cm-i peak of C-D show newly synthesized proteins.
  • FIG. 36 b illustrates SRS images targeting the 2940 cm-! CH:> show total proteins.
  • FIGS. 36c and 36d illustrate zoomed-in images as indicated in the white dashed squares in FIG.
  • FIG. 36e illustrates a ratio image between new protein FIG. 36c and total proteins FIG. 36d. While the starred neurites show high percentage of new proteins, the arro w s indicate neitrites displaying very low new protein percentage.
  • FIG. 36f Merged image between new protein c (channel 3605) and total proteins in FIG. 36d (channel 3610). Similarly, starred regions show obvious new proteins; while arrows indicate regions that have undetectable new protein signal.
  • FIG. 37a is a prior art recipe for a mammalian cell culture
  • FIG. 37b is an exemplary deuterium-labeled recipe based on the ceil culture of FIG.
  • FIG. 38 is an illustration of an exemplary block diagram of an exemplary system in accordance with certai exemplary embodiments of the present disclosure.
  • Raman scattering refers to a spectroscopic technique used to observe vibrational, rotational, and other low-frequency modes in a system. It relies on inelastic scattering, or Raman scattering, of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with molecular vibrations, phonons or other excitations in the system, resulting in the energy of the laser photons being shifted up or down. The shift in energy gives information about the vibrationa l modes in the system.
  • a variety of optical processes, both linear and nonlinear in light intensity dependence, are fundamentally related to Raman scattering.
  • Raman scattering includes, but is not limited to, l .> “stimulated Raman scattering” (SRS), “spontaneous Raman scattering", “coherent anti-Stakes Raman scattering” (CARS), “surface-enhanced Raman scattering” (SERS), “Tip-enhanced Raman scattering” (TERS) or "vibrational photoacoustic tomography”.
  • SRS single Raman scattering
  • CARS coherent anti-Stakes Raman scattering
  • SERS surface-enhanced Raman scattering
  • TERS Tip-enhanced Raman scattering
  • the exemplary system, method and computer accessible medium can use alkyne as a vibrational tag coupled with narrow-band sdmuiaied Raman scattering microscopy ("SRS") for the detection of small molecules inside biological systems.
  • SRS narrow-band sdmuiaied Raman scattering microscopy
  • the use of alkyne as a vibrational tag offers a large Raman cross-section enabling sensitive detection (See, e.g., References 13; 14). Additionally, the alkyne Raman peak caa exhibit a narrow spectral width for the specific detection, which can reduce the probability of overlapping with other tags.
  • the Raman peak of alkyne can lay exactly in the cell-silent region in the cell spontaneous Raman spectrum, bypassing the complex interference from vast pool of bioraolecules in the fingerprint region. (See, e.g. , FIG. l b).
  • the exemplary SRS can be a sensitive vibrational imaging microscopy.
  • the exemplary SRS can employ two-laser excitation (e.g., temporally and spatially overlapped Pump and Stokes lasers), boosting up the transition rate about 7 orders of magnitude as compared to the traditional spontaneous Raman microscopy, the transition process of which can be intrinsically weak (e.g., 1.0 to 12 orders of magnitude slower than fluorescence).
  • two-laser excitation e.g., temporally and spatially overlapped Pump and Stokes lasers
  • the exemplary SRS can be a bond-selec tive procedure with high specificity, in contrast with the spontaneous Raman imaging which can be a spectrum-based me thod, instead of spreading the energy to the whole spectrum as in the spontaneous Raman imaging, the exemplary narrow-band SRS can focus its energy to the vibrational transition of a specific bond.
  • a 6-ps pulse width can be chose for both SRS pump and stokes lasers to achieve a spectral resolution of 5 cm " ' for the detection of alkyne.
  • the spectral width of the excitation profile from two combined lasers can be calculated to be 8 cm '1 , which can fit. well within the spectral width of alkyne Raman peak that can be M em " ' .
  • the exemplary laser poise width can be long enough that all the laser energy can be used to specifically detect alkyne without energy waste, but short enough that the two-photon efficiency can be maintained since the exemplary SRS can depend on a nonlinear process.
  • the exemplary SRS signal can offer linear concentration dependence to the analyte without non-specific background.
  • CARS coherent anti-Stakes Raman scattering
  • SRS can also .have its own distinctive characters as an imaging procedure. For example,
  • SRS can be immune to fluorescence background as compared to spontaneous Raman microscopy that can suffer from large fluorescence background.
  • SRS as a nonlinear process, can offer intrinsic 3D sectioning capability.
  • SRS can offer deeper penetration depth and less phototoxicity, which can be well suited for imaging live cells, tissues and animals.
  • .narrow-band SRS has achieved unprecedented sensitivity down to approximately 1000 retinoic acid molecules and up to video rate imaging speed in vivo. (See, e.g., Reference 17).
  • A!kyne can be a metabolic labeling tag in fluorescence microscopy utilizing
  • One aspect of the present disclosure relates to a method for obtaining biological information in a jiving cell or a living organism with bond-edited compounds using Raman, scattering.
  • the method comprises the steps of introducing an effecti ve amount of one or more bond-edited compounds into a live cell or a living organism, and detecting a vibrational tag in the cell or organism with Raman scattering.
  • the Raman scattering is SRS.
  • biological information refers to spatial distribution of the targeted molecules, such as one-dimensional line, or two-dimensional or three-dimensional images, and non-imaging information, such as a simple signal intensity or local spectrum on a single location or its time dependence.
  • bond-edited compounds refers to compounds having one or more chemical bond that may serve as a vibrational tag for detection by Raman scattering.
  • Examples of chemical bond that may serve as a vibrational tag include, b t are not limi ted to, carbon-carbon triple bond, carbon-nitrogen triple bond, azide bond, carbon-deuterium bond, phenol ring, ' ' (. ' modified carbon-carbon triple bond, C modified carbon-nitrogen triple bond, > C modified azide bond, 1 J C modified carbon-deuterium bond, L X modified phenol sing and combinations thereof.
  • the term "effective amount” refers to an amount that, when introduced into a live cell or organism, is sufficient to reach a working concentration needed for SRS imaging.
  • the “effective amount” would vary based on the type of bond-edited compound, as well as the cells or organisms that the bond-edited compound is introduced into.
  • an "effective amount" of a bond-edited compound is the amount that is sufficient to reach an in vivo concentration of 1 uM to 100 m , 3 ⁇ to 30 mM, 10 ⁇ to 10 mM, 100 ⁇ to 1 mM, 10 ⁇ to 1 iaM or 10 ⁇ . ⁇ to 100 ⁇ in a target cell or organ, in some embodiments, an "effec tive amount" of a bond-edited compound comprising a triple bond is the amount that is sufficient to reach an in vivo concentration of 1 ⁇ to 10 mM, 3 ⁇ to 3 mM, 1 ⁇ to 1 mM or 30 ⁇ to 300 ⁇ .
  • an "effective amount" of a bond-edited compound comprising a triple bond is the amount that is sufficient to reach an in vivo concentration of about 100 ⁇ .
  • an "effecti ve amount" of a bond-edited compound comprising a C-D bond is the amount that is sufficient to reach an i vivo concentration of 10 ⁇ to 100 mM, 30 ⁇ to 30 mM, 100 ⁇ to 10 mM or 300 ⁇ . ⁇ to 3 mM.
  • an "effective amount" of a bond-edited compound comprising a C-D bond is the amount mat is sufficient to reach an in vivo concentration of about I mM,
  • the bond-edited compounds are small molecules.
  • small molecules refers to low molecular weight organic compound having a molecular weight of 1000 daltons or less. In some exemplary embodiments, the small molecules have a size on the order of 10 9 ra.
  • small molecules include, but are not limited to, water, rihonucleosides, ribonucleotides, deoxyrtbonucleoside, deoxyribonucleotide, amino acids, peptides, choline, monosaccharides, disaccharides, fatty acids, glucose, adenosine triphosphate, adenosine diphosphate, cholesterol neurotransmitters, secondary messengers, and chemical drugs,
  • said bond-edited compound contains one, two, three, four, five, six, seven, eight, nine, ten or more vibrational tags.
  • the vibrational tags may be the same type of tags or a mixture of one or more different tags.
  • said vibrational tag is an alkyne tag. in other exemplary embodiments, said vibrational tag is an a ide tag. in still other exemplary embodiments, said vibrational tag is an isotope label, in a further exemplary embodiment, said isotope label is a carbon-deuterium tag. In yet still other exemplary embodiments, said vibrational tag is a combination of an alkyne tag and a carbon-deuterium tag,
  • the vibrational comprises at least one L' C atom or one deuterium atom.
  • the bond-edited compound is an amino acid
  • the amino acid is an essential amino acid.
  • the essential amino acid is selected from the group consisting of hisiidine,, isoleueine, leucine, lysing, methionine, phenylalanine, threonine, tryptophan and valine.
  • the bond-edited compound is a nucleoside or a nucleotide.
  • the bond-edited compound is a fatty acid.
  • the bond-edited compound is a
  • the bond-edited compound is glucose, a glucose derivative or proparg l. glucose.
  • the bond-edited compound is a pharmaceutical agent, such as an anti-cancer agent, anti-inflammatory agent, anti-bacterial agent, anti-fungal agent and anti-viral agent.
  • the bond-edited compound is a cytokine or chemokioe.
  • the bond-edited compound is EU- ⁇ Cs having a molecular structure of formula 13:
  • the bond-edited compound is EdU-' ⁇ C-s. having a molecular structure of fomiular 3:
  • the bond-edited compound is EdU- C having a molecular structure of formula 2:
  • the bond-edited compound is EdU- ⁇ C '5 having a molecular structure of formula 14:
  • the bond-edited compound is aikyne-D-giucose having a molecular structure of formula S3;
  • the bond-edited compound is metabolized in the living cell or organism aod the vibrational tag is transfeiTed from the bond-edited compound to a down-stream metabolite of the bond-edited compound (See, e.g.. Figures 28-31).
  • the method comprises introducing into a live cell a mixture of bond-edited compounds that imaging with Raman scattering at two or more different wavelengths, in some relaxed exemplary embodiments, the Raman scattering is SRS,
  • the method comprises introducing into a live cell a mixture of different bond-edited compounds that allow multiple color imaging with Raman scattering.
  • the Raman scattering is SRS.
  • the mixture of different bond-edited compounds comprises ⁇ - 1 ⁇ 4, EdU- ,3 C and 17-ODYA.
  • the two or more bond-edited compounds target the same cellular component but at different time period (See, e.g.. Figure 25).
  • the method comprises introducing into a living cell a mixture of different bond-edited compounds that target different cellular components,
  • the method comprises introducing into a living organism a mixture of different bond-edited compounds that target different types of cells in the living organism.
  • the method comprises introducing into a li ving organism a mixture of different bond-edited compounds carrying different vibrational tags, and detecting the different vibrational tags with Raman scattering using a linear combination algorithm.
  • the Raman scattering is SRS.
  • Another exemplary aspect of the present, disclosure relates to a method for making a bond-edited compound.
  • the bond-edited compound is synthesized by the route illustrated in FIG. 1 . In another exemplary embodiment, the bond-edited compound is synthesized by the route illustrated in FIG. 20.
  • Another exemplary aspect of the present disclosure relates to a method for detecting a disease condition in a subject, comprising: administering to said subject a composition comprising a bond-edited compound targeting a disease tissue or pathogen, and detecting said bond-edited compound by Raman scattering.
  • the subject is a mammal
  • mammal subjects for use in accordance with the methods described herein include humans, monkeys, gorillas, baboons, 200 animals and domesticated animals, such as cows, pigs, horses, rabbits, dogs, cats, goats and the like.
  • the disease condition is cancer.
  • the disease condition is a neurodegenerative disease.
  • the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's, Alzheimer's and Huntington's.
  • the disease condition is an inflammatory disease.
  • the disease condition is a microbial infection.
  • the disease condition is a bacterial infection.
  • the disease condition is a viral infection.
  • the disease condition is a fungal infection.
  • the pathogen comprises bacteria.
  • Another exemplary aspect of the present disclosure relates to a method for monitoring treatment for a disease condition.
  • the method comprises administering to said subject a compositicm comprising a bond-edited compound and detecting said bond-edited compound by SRS at a first time point, further administering to said subject said composition comprising a bond-edited compound and detecting said bond-edi ted compound by Raman scattering at a second time point, and comparing images obtained at the two time points.
  • the first time point is a time point that is about or prior to the initiation of a treatment and the second time point is a time point that is after the initiation of the treatment
  • the first time point and the second time point are two time points dining the course of a treatment.
  • the treatment is a treatment for cancer
  • the treatment is a treatment for an inflammatory disease.
  • the treatment is a treatment for a neurodegenerative disease.
  • Another exemplary aspect of the present di sclosure relates to a method for screening an agent.
  • the method comprises administering said agent and at least one bond-edited compound to a live cell or organism, detecting the bond-edited compound in the live cell or organism using Raman scattering, and selecting a candidate agent based on one or more predetermined criteria, such as the uptake, accumulation, trafficking, or degradation of the said bond-edited compound in the said live cell or organism.
  • the candidate agent is an anti-cancer drag
  • the bond-edited compound is selected from the group consisting of amino acid, nucleic acid, ribonucleic acid and glucose derivatives.
  • the candidate agent is a skin regenerating agent.
  • the candidate agent is a cosmetic agent.
  • Another exemplary aspect of the present disclosure relates to a method for tracing a cellular process in a live cell with Raman scattering.
  • the method comprises introducing into a live cell a bond-edited compound, and following the physical movement or the chemical reaction or the biological interaction of the bond-edited compound within the cell by SRS.
  • the cellular processes are selected from the group consisting of DNA replication, RNA synthesis, protein synthesis, protein degradation, glucose uptake and drug uptake.
  • Composition for labeling cells with bond-edited compounds are selected from the group consisting of DNA replication, RNA synthesis, protein synthesis, protein degradation, glucose uptake and drug uptake.
  • compositions for labeling a target cell with at least one bond-edited compound are a culture medium comprising at least one bond-edited compound containing at least one vibrational tag.
  • the at least one bond-edited compound may be selected based on the type of the target cell ot a target components) within the target ceil.
  • the culture medium comprises a plurality of amino acids, wherein over 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the amino acids are tagged with one or more vibrational tag.
  • the culture medium comprises a plurality of ami no acids, wherein ali amino acids are tagged with one or more vibrational tag.
  • the culture medium comprises two, three, four, five, six, seven, eight, nine, ten or more different bond-edited compounds.
  • Another exemplary aspect of the present disclosure relates to a device for imaging bond-edited compounds by Raman scattering.
  • the device comprises a first
  • single- wa velength laser source that produces a pulse laser beam of a first wavelength
  • a second single-wavelength laser source that produces a pulse laser beam of a second wavelength
  • a modulator that modulates either the intensity or the frequency or the phase or the- olarization or the
  • a photodetector that is capable of detecting SRS or C ARS or spontaneous Raman scattering or the combination of the abo ve from a biosampie, and a computer.
  • the energy difference between the photons produced by the first laser adiation and the photon produced by the second laser radiation matches with the energy of the vibrational transitions of the targeted vibrational tags.
  • Photodetector of SRS detects part o all of the first laser beam or the second laser beam.
  • the output of the photodetector (which could be a photodiode) is further processed by a Lock-in amplifier or a resonant circuit.
  • Another exemplary aspect of the present disclosure relates to an apparatus for providing radiation to at least one structure, comprising: a radiation providing arrangement which is configured to provide a pump radiation and a stokes radiation, each at a fixed wa velength, whose energy difference is between about 2000 and 2500 wavenumbers.
  • the radiation providing arrangement is a laser source.
  • Example 1 In vitro and in vivo labeling with deuterium tags
  • An integrated laser (picoEMERALD with custom modification, Applied Physics & Electronics, Inc.) was used as the light source for both Pump and Stokes beams. Briefly. picoEMERALD provides an output poise train at ⁇ 064 am w th 6 psnoie width and 80 MHz repetition rate, which serves as the Stokes beam. The frequency-doubled beam at 532 am is used to synchronously Seed a picosecond optical parametric oscillator (OPO) to produce a mode-lockedmodule train (the idler beam of the OPO is blocked with an tnterferometric filter) with 5- ps pulse width.
  • OPO optical parametric oscillator
  • the wavelength of the OPO is tunable from 720 to 990 nra, which serves as the Pump beam.
  • the intensity of the 1 64 rim Stokes beam is modulated smusoidaliy by a built-in electro-optic modulator (EOM) at 8 MHz with a modulation depth of more than 95%.
  • EOM electro-optic modulator
  • the Pump beam is spatially overlapped with the Stokes beam with a dichroic mirror inside picoEMERALD, The temporal overlap between Pump and Stokes pulse trains is ensured with a built-in delay stage and optimized by the SRS signal of pure dodecane liquid.
  • a high O.D. bandpass filter (890/220 CARS, Chroma Technology) is used to block the Stokes beam completely and transmit the Pump beam only.
  • the output current of the photodiode is electronicall pre-filtered by an 8-MHz band-pass filter ( R 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low- frequency contribution due to laser scanning across the scattering sample. It is then fed into a radio frequency lock-in amplifier (HF2L1, Zurich instrument) terminated with 50 ⁇ to demodulate the stimulated Raman loss signal experienced by the Pump beam.
  • the R-output of the lock-in ampl ifier is fed back into the analog i nterface box
  • the time constant of the lock-in amplifier is set for 8 ps, and the images are acquired by a 12.5 ps pixel dwell time, corresponding to 3.3 s for a 512-by-SJ 2 pixel frame.
  • the time constant is set to be 20 ⁇ , ⁇ , and the images are acquired by a 40 ps of pixel dwell time, corresponding to 10.5 s for a 5 ! 2-by-512 pixel frame.
  • Laser powers after 60X !R objective used for cell imaging are: 100 raW for modulated Stokes beam and 1 12 raW for the Pump beam at 2133 cm * ', 2000 cm “5 and 1655 cm * ' channels; 50 roW for modulated Stokes beam and 56 raW for Pump beam at 2940 cm 4 and 2845 cm " ' channels.
  • Laser powers after 25X objective used for tissue and in vivo imaging are: 134 niW for modulated Stokes beam: 120 niW
  • Htt-mEos2 piasmid construct and transection were constructed by replacing CFP gene sequence in pTreTight-Hrt94Q-CFP piasmid (Addgene, 23966) with mEos2 gene sequence from pRSETa-mEos2 piasmid (Addgene, 20341).
  • mHtt-mEos2 piasmid in HeLa ceils 4pg mHtt94Q-niEos2 piasmid was transfected using Transfection Reagent (FuGene, Promega).
  • D-AA medium for HeLa cells; adapted from regular recipe of DMEM medium (1. 1965, Invitrogen).
  • the D-AA culture medium for HeLa cells was made with 90% CD-DMEM, 10% FBS (10082, invitrogen) and IX penicillin/streptomycin ( 15140, invitrogen).
  • D-AA medium (CD- etirobasal. A) for hippocampal neuron culture and organotypic brain slices: adapted from regular recipe of Ne urobasal A medium (10888, Invitrogen).
  • the D ⁇ A s culture medium for hippocampal neurons was made of CD-Neurobasal A Medium, Ix B27 serum free supplement (17504, Invitrogen) and 0.5 mM glntamrae-d? (DLM-l 826, Cambridge isotope).
  • the CD ⁇ Neurobasal A culture medium for organotypic brain slices was made of CD -Neurobasal A Medium, IX B27 serum free supplement ( 17504. Invitrogen), 0.5% glucose ( 5023, invitrogen), 2 mM giutamine-ds (DLM-l 826 ' , Cambridge isotope) and IX
  • Neorobasal A medium (10888, fevi.trogen).
  • Group 1 D-AA medium for HeLa cells The group I D-AA culture medium for HeLa cells was made with 90% group I D-AA medium, 10% FBS (10082, mvifrogen) and IX penicillin/streptomycin (15140, mvnrogen).
  • Group M D-AA medium for HeLa cells The group li D-AA culture medium for HeLa cells was made with 90% group 0 D-AA medium, 10% FBS (1.0082, invitxogen) and I X penicilHn streptomycin (15140, invftrogen).
  • D-AA medium (CD-MEM) for organotypic brain slice adapted from regular recipe of MEM medium ( 11095, Iirvi trogen).
  • the CD-MEM culture medium for orgaaotypi. brain slice was made ith 90% CD-MEM, 10% FBS (10082, invitrogen), 0.5% glucose (15023, invitrogen) and I X penicillin/streptomycin (15140, invi trogen).
  • mice For mice: 1. Oral administration; 3-week-old mice were fed with D-AA containing drinking water for 12 days before harvesting the liver and intestine tissues. The drinking water was made of 500 mg uniformly deuterium- labeled amino acid mix (20 aa) (DLM-6819, Cambridge Isotope) dissolved in 200 ml PBS, with subsequent filtration using illipore sterile syringe Filters (0.22 ⁇ , SLGV033RS). 2. Intraperitoneal injection: 3 -week-old mice were injected with 500 ⁇ D-AAs solution at the 0 th h, 12 ( " h and 24 te h. The tissues were then harvested at the 36* h after the first injection.
  • D-AA uniformly deuterium- labeled amino acid mix
  • D-AA solution was made of 500 mg uniformly deuterium-labeled amino acid mi (20 aa) (DLM-6819, Cambridge Isotope) dissol ved in 2 ml PBS solutions, with subsequent filtration using Millipore sterile syringe Filters (0.22 pm, SLGV033RS).
  • Example la Sensitivity optimization and time-lapse imaging of the de nam proteome synthesis dynamics.
  • the cell culture medium reported previously was prepared by supplying uniformly deuterium- labeled whole set of amino acids to a commercially available medium that is deficient of leucine, lysine and arginine (Wei L, Yu Y, Shen Y, Wang MC, Min W (2013)
  • non-trivial instrumentation optimizations are also carried out to furthe improve SRS detection sensiti ity and acquisition speed, including increasing the laser output and microscope system throughput for near-IR wa elengths, replacing the aconsto-optic modulator (AOM) with an electro-optic modulator (BOM) for a 30% higher modulation depth, and employing a high-speed lock-in amplifier for faster image acquisition.
  • AOM aconsto-optic modulator
  • BOM electro-optic modulator
  • FIG. 2 presents time-lapse SRS imaging of a same set of live HeLa cells gradually synthesizing new proteins over time from 10 min to 5 h incubation in optimized D-AA medium. The obvious cell migration and division prove their viability, supporting high bio-compatibility of our technique. To our best knowledge, this is the first time that long-term time-lapse imaging of proieome synthesis dynami s is demonstrated on single live mamian cells.
  • xample lb SRS imaging of protein degradation in live HeLa cells
  • FIG. 3a shows time -dependent SRS images of old protein distributions f CH3) in live HeLa cells when incubated with D-AAs from ⁇ ) h to 96 h.
  • the old protein pool is degrading, as shown by the decay of its average intensity.
  • the total lipid images display no obvious intensity change (FIG. 3b).
  • the spatial patterns of old proteins reveal a faster decay in the nucleoli than the cytoplasm.
  • Example lc Two-color pulse-chase SRS imaging of two sets of temporally defined proteins.
  • Group I contains three amino acids, leuiue-dlO, isoleuc.i»e ⁇ dl0 and valine ⁇ d8, structurally known, as branched-chain amino acids (FIG. 4a), All members of group I exhibits multiple distinct R m n peaks with the first one around 2067 cm- 1.
  • pure signals of proteins labeled by group 1 D-AAs and by group II D-AAs can be suscesfuSly separated and quantitatively visualized (e.g. FIG. 7).
  • the images are obtained with a hyper-spectral imaging approach using broadband femtosecond lasers.
  • HeLa cells were first transfected with mHtt94Q-mEos2 plasmid in regular medium for 4 h, and then replaced with group H D-AA medium for 22 h before changing to group 1 D-AA medium for another 20 h.
  • SRS images are acquired at 2067 cm-1 and 2133 cm- ' l channels, respectively, and subsequently processed with linear combination.
  • the measured SRS signals at 2067 cm-1 and 2133 cm-1 channels can be written as the following, with linear relationship to group 1 D-AA and group II D-AA concentrations c rou 1 and ' jgroup II) :
  • '.iLijofB ' 1 ⁇ are the average pixel intensity recorded inside cells in FIG. 7a and FIG. 7b.
  • Example lit SRS imaging of newly synthesized protons i» live mouse brain tissues.
  • s 5a, C-D of a live mouse organotypic brain slices cultured in D-AA medium for 30 h, reveals active protein synthesis from both the soma and the neurites of individual neurons in dentate gyrus, in addition, the old protein (CP13) and total lipids (CH2) images are presented sibmtitaneous!y for multichannel analysis (FIG. 5a).
  • Example le SRS imaging of newly synthesized proteins in vivo.
  • FIG. 8 shows raw C-D on-resonance (2133 cm-l) and off-resonance (2000 cm-l.) SRS images of newly synthesized proteins in vivo in FIG. 6.
  • FIG. 8a SRS C-D on-resonance and off-resonance images of a 24 hpf embryonic zebrafish. The difference image between C-D on-resonance and off-resonance (pixel-by-pixel subtraction) shows pure C-D labeled protein distribution in the somites of an embryonic zebrafish tail, as in FIG. 6a.
  • FIGS. 8b-8c SRS C-D on-resonance and off-resonance images of live mouse liver FIG. 8b and intestine FIG.
  • FIG. 9 shows SRS imaging for newly synthesized proteins in viv with intraperitoneal injection of mice with D-AA solutions.
  • FIGS. 9a-9b SRS images of live mouse liver FIG- 9a and intestine 9b tissues harvested from mice after intraperitoneal injection injected with D-AAs solutions for 36 h. 21 3 cm-l channel shows newly synthesized proteins (off-resonance image subtracted) thai resemble the distribution of total proteins as shown in the 1655 cm-l image (Amide I), (c-d) Corresponding raw C-D on-resonance (2133 cm- 1 ) and off-resonance (2000 cm- 1 ) images are shewn as references for liver c and intestine d tissues. Scale bar, 10 ⁇ ,
  • SRS microscopy can be a molecular-contrast, highly sensitive, imaging procedure with intrinsic 3D sectioning capability. It selectively images the distribution of molecules that carry a given type of chemical bonds through resonating with the specific vibrational frequency of the targeted bonds. (See, e.g., References 47, 54 and 65).
  • FIG. 5a illustrates, by focusing both temporally and spatially overlapped Pump and Stokes laser pulse trains into samples, the rate of vibrational transition can be greatly amplified by about. 107 times when the energy difference of the two laser beams matches the particular chemical bond vibration, Ovih, (See, e.g.. Reference 65).
  • FIG. 5b shows a high-frequency modulation procedure, where the intensity of the Stokes beam can be turned on and off at 10 MHz, and can be employed to achieve shot-noise-limited detection sensitivity by suppressing laser intensity fluctuations occurring at low frequencies.
  • the transmitted Pump beam after the sample can be detected by a.
  • the vibrational signal of C-D can be detected as an indicator for newly synthesized proteins that metaholically incorporate deuterium-labeled amino acids. (See, e.g., FIG. 5b).
  • the € ⁇ D stretching motion can display a distinct vibrational frequency from all the other vibrations ofbiolosical molecules inside live cells.
  • the reduced mass of the C-D oscillator can be increased by two folds when hydrogen can be replaced by deuterium. Based on the above Equation, Ovib can be reduced by a factor of 2.
  • the experimentally measured stretching frequency can be shifted from -2950 cm- 1 o C-H to -2100 cm- 1 of C-D.
  • the vibrational frequency of 2100 cm-1 can be located in a cell-silent spectral window in which no other Raman peaks exist, thus enabling detection of exogenous C- D with both high specificity and sensitivity.
  • T he corresponding SRS image at 2133 cm-i can show a significantly more pronounced signal than that in FIG. 2 under the same Intensity scale.
  • nucleoli e.g., indicated by arrows in FIG . 33b and verified by DIC visualization
  • Nucleoli the active sites for ribosomai biogenesis, have been reported to involve rapid nucleolar assembly and proteomic exchange (See, e.g.. Reference 68-70), Such fast proteio turnover can be reflected by the spatial enrichment of newly synthesized protein signals in those subcellular areas. (See, e.g.. FIG. 33b).
  • SRS imaging here can be directly performed on live cells and hence free from potential complications due to fixation and dye conjugation.
  • the off-resonant image at 2000 cm-1 can be clean and dark (e.g., FIG. 33c), proving the specificity of SRS imaging o C-D at 2133 cm-1.
  • SRS can readily image intrinsic bioraolecules in a label-free manner. By simply adjusting the energ difference between the Pump and the Stokes beams to match the vibrational frequency of amide I, lipids and total proteins respectively, FIGS.
  • 33d-f show the SRS images of amide i band at 1655 cm-1 primarily attributed to proteins, CH2 stretching at 2845 era-?, predominantly for lipids and CH3 stretching at 2940 cm-1 mainly from proteins with minor contribution from lipids.
  • Tinie-Bependent lie Novo Protein Synthesis And Protein Synthesis Inhibition
  • SRS contrast can be well suited for quantification, of de novo protein synthesis in live cells.
  • the time -dependent protein synthesis images can be shown under the same intensity scale.
  • the new protein signal eg,, 2133 cm- 1
  • the amide I e.g., 1655 cm-1
  • FIGS. 34d-f See, e.g., FIGS. 34d-f.
  • FIGS. 34g-i depict the fraction of newly synthesized proteins ⁇ e.g., 2133 cm-1) among the total proteome (e.g., 1655 cm-1) and its spatial distribution.
  • the t action of newly synthesized proteins growing with time from 5 hours to 20 hours can highlight, nucleoli as the subcellular compartments with fast protein turnover. (See, e.g.. Reference 68-70).
  • Such quantitative ratio imaging of new versus old proteomes can be very difficult to obtain using BONCAT or mass spectroscopy without the destruction of cells.
  • FIG. 34j shows time-lapse SRS images of a live dividing HeLa cell after 20- hour incubation in deuterium-labeled all amino acids medium, clearly proving the viabilit of cel ls under the imaging condition.
  • the corresponding 2940 cm-1 image e.g.. FIG. 34m
  • the detected C-D SRS signal can originate from deuterium-labeled nascent proteins, which can vanish upon adding the protein synthesis inhibitor.
  • Two additional mammalian cell lines can be chosen for further demonstration: human embryonic kidney HEK293T cells, and neuron-like neuroblastoma mouse N2A cells, which can be induced to differentiate with the growt of neurites (e.g., axons and dendrites).
  • the spontaneous Raman spectrum e.g., FIG. 35a
  • HEK293T ceils incubated with deuterium-labeled all amino acids for 12 hours can exhibit, a 2133 cm-1 C ⁇ D channel signal nearly as high as the 1655 era-! amide channel signal.
  • He resulting SRS image can show a bright signal tor new proteins with an interne pattern residing in nucleoli, (See, e.g., FIG.
  • the off-resonant image (e.g., 2000 cm-.!) can display vanishing background (e.g. , FIG. 35c); the amide I channel (e.g. , 1655 cm-1 ) image (e.g., FIG. 35d) can exhibit consistent overall proteome distributions similar to that in HeLa cells; CH2 channel (e.g., 2845 cm-1) image (e.g., FIG. 35e) depicts a more diffusive lipid distribution in cytoplasm compared to that in HeLa cells. Consistent with the results obtained in HeLa ceils above, the ratio image (e.g., FIG. 35f) between the newly synthesized proteins (e.g., FIG. 35b) and the total proteins (e.g., FIG. 35d) highlight nucleoli for active protein turnover in HEK293T cells as well, (See, e.g.. References 44-46),
  • the exemplary SRS can also be applied to tackle more complex problems, such as de novo protein synthesis in neuronal systems. (See, e.g.. Reference 26-28). Under differentiation condition,
  • FIG. 10a shows the image of newly synthesized proteins after induction for differentiation, by simultaneously differentiating the N2.A ceils and supplying with the deuterium labeled all amino acids for 24 hours. Similar to HeLa and HEK293I ceils, N2 A ceil bodies can be observed to display high-level protein synthesis. Newly synthesized proteins can also be observed in a subset of, but not all neurites (e.g., Figs 36a and 36b), which can imply that the observed neurites in FIG. 360a can be newly grown under the differentiation condition.
  • FIGS, 36c and lOd show the zoomed-in regions in the dashed squares in FIGS. 36a and 36b respectively.
  • both the ratio image and the merged image highlight the neurites with higher percentage of new proteins (e.g., indicated by stars), implying these neurites can be newly grown.
  • neurites e.g., indicated by arrows
  • obvious signals in the green channel e.g., total proteins
  • red channel e.g., new proteins
  • the neurites indicated by arrows can be most likely older than their starred counterparts, in addition, the transition f ra 3610 to 3605 in the merged image (e.g., FIG. 360f) can imply the growth direction by which new neurites form and grow.
  • a more relevant system to study de novo protein synthesis and neuronal activities can be hippocampal neurons, which can be known to ' be involved in long-term memory formation (See, e.g., Reference 26-28).
  • SRS image e.g., 2133 cm- !
  • hippocampal neuron cells incubated with deuterium- labeled all amino acids ca show a newly synthesized protein pattern in the neurites.
  • Example 2 In vitro an in vivo labeling with a!kyne tags
  • FIG. 37b shows details of the microscopy setup.
  • An integrated laser system (picoEMERALD, Applied Physics & Electronics, Inc.) was chosen as the light source for both pomp and Stokes beams.
  • picoEMERALD provides an output pulse train at 1064 nm with 6 ps pulse width and 80 MHz repetition rate, which serves as the Stokes beam.
  • the frequency doubled beam at 532 nm is used to synchronously Seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train with 5-6 ps pulse width (the idler beam of the OPO is blocked with an raterferoraetric filter).
  • OPO optical parametric oscillator
  • the output wavelength of the OPO is tunable from 720 to 990 nm, which serves as the pump beam.
  • the intensity of the 1064 nm Siokes beam is modulated simisoidally by a built-in electro-optic modulator at 8 MHz with a modulation depth of more than 95%.
  • the pump beam is then spatially overlapped with the Stokes beam by using a dtchroic mirror inside picoEMERALD.
  • the temporal overlap between pump and Stokes pulse trains is ensured with a built-in delay stage and optimized by the SRS signal of pure dodeeane liquid at the microscope.
  • Pump and Stokes beams are coupled into an inverted multiphoton laser-scanning microscope (FVT200MPE, Olympus) optimized for oear-IR throughput.
  • FVT200MPE inverted multiphoton laser-scanning microscope
  • a 60 water objective (UPkmAPO/lR, 1.2 N.A., Olympus) with high tiear-lR. transmission is used for all cell imaging.
  • the pump/Stokes beam size is matched to fill the back-aperture of the objective.
  • the forward going pump and Stokes beams after passing through the sample are collected in transmission with a high N.A. condenser lens (oil immersion, 1.4 N.A., Olympus) which is aligned following K ihler illumination.
  • a telescope is then used to image the scanning mirrors onto a large area (10 by 10 mm) Si photodiode (FDS 1.010. Thorlabs)to desca beam motion during laser scanning.
  • the photodiode is reverse biased by 64 V from a DC power supply to increase both the saturation threshold and response bandwidth.
  • a high O. D. bandpass filter (890/220 CARS, Chroma Technology) is placed in front of the photodiode to block the Stokes beam completely and to transmit the pomp beam only .
  • the output current of the photodiode is electronically pre- filtered by an 8-MHz band-pass filter (KR 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low-frequency fluctuations due to laser scanning cross the scattering sample. It is then fed into a radio frequency lock-in amplifier (SR844, Stanford Research Systems) terminated with 50 ⁇ to demodulate the stimulated Raman loss signal experienced by the pump beam. The m-phase X-outpn of the lock-in amplifier is fed back into the analog interface box (F V 10- ANALOG) of the microscope. The time constant is set for 10 us (the shortest available with no additional filter applied).
  • the current SRS imaging speed is limited by the shortest time constant available from the lock-in amplifier (SR844). For all imaging, 512 by 512 pixels are acquired for one frame with a 100 ps of pixel dwell time (26 s per frame) for laser scanning and 10 ps of time constant from the lock-in amplifier.
  • Laser powers after 60X IR objective used for imaging are; 130 itiW for modulated Stokes beam; 1.20 mW for the pump beam in 2133 cm "1 , 2142 cm ' *, 2000 cm *1 and 1655 cm " ' channels, 85 mW for the pump beam in 2230 cm '1 and 2300 cm “1 channels, and 50 mW for pump beam in 2845 cnT ! channels.
  • 10170 ⁇ DMEM culture medium was made by adding 10% (vol/vol) FBS and 1% (vol/vol) per «cilIiu3 ⁇ 4treptomycin to the DMEM medium.
  • Methiomne-deficient culture medium was made by supplying 4 mM L-glutamine, 0.2 mM L-cystine, 1 % FBS and 1% penicillin/streptomycin to the DMEM medium without L-methionine, L-cystine and L-glufamine.
  • RPMI-1640 culture medium was made of supplying the RPMI-1640 medium with 10% FBS, 1% petucillin streptomyciti and 50 ⁇ 2-Mercaptoethanol.
  • Neuron culture medium was made of Neurobasai A Medium adding with IX B27 supplement and 0.5 mM glutamine.
  • Culture medium for NIH3T3 cells was made by adding 10% (vol/vol) BCS and 1 % (vol/vol) penicillin/streptomycin to the DMEM medium.
  • Propargyritiiine synthesis Propargylcholine was synthesized according to Jao, C, Y., Roth, M., Welti, R. & Salic, A. Free, Nad. Acad Set USA 106, 15332-15337 (2009). 3 mL propargyl bromide (80 wt. % solution in toluene) were added dropwise to 3 g 2-dimethylaminoeth.anol in 10 mL anhydrous THF on ice under argon gas protection and stirring. The ice bath was removed and the mixture was kept stirring at room temperature overnight.
  • EU 100 mM stock in PBS
  • DMEM culture medium directly to a concentration of 2 mM for 7 h.
  • DMEM culture medium was then changed to methionme-deficient culture medium for 1 h, followed by supplying 2 mM Hpg (200 mM stock in PBS) in the medium for 24 h.
  • DMEM culture medium was changed to DMEM medium (FBS-free) for synchronization.
  • FBS-free DMEM medium
  • syitchrot 2ation 5
  • medium was replaced back to DMEM culture, medium by simultaneously adding both propargylcholine (25 mM stock in PBS) and EdU (10 mM stock in PBS) to the culture medium to a concentration of 1 mM and 100 ⁇ , respectively, fo 24 h.
  • ⁇ - ⁇ cells were first Seeded on coversiips at a density of 2 l05/mL in 2 ml RPMi-1640 culture medium for 24 fa, followed by 72 fa induction of differentiation to macrophages by incubating with 100 og/rai PhorboJ 12-rayristate 1.3-acetate (PMA) in the medium. Medium was then replaced with RPMI-1640 culture medium containing 400 ⁇ . ⁇ 17-ODYA (6: 1 complexed to BSA) for 15 h.
  • PMA og/rai PhorboJ 12-rayristate 1.3-acetate
  • N1H 3T3 cells cultured with 0.5 DM propargykholine for 48 hours were fixed with 4 % PFA for 15 minutes, .rinsed with 1 niL TBS buffer twice and incubated with 1 mL 1 rag/mL BSA in TBS buffer for I hour at 37 °C 5 with or without 0.02 U/mL phosphohpase C (Type XIV from Clostridium fxsrfrmgens, Sigma), in the presence of 10 mM CaCi?
  • FIG. 10b shows linear dependence of stimulated Raman loss signals (2125 cm") with EdlJ concentrations under a 100 . ⁇ acquisition time.
  • FIG. 10c shows the metabolic incorporation scheme for a broad spectrum of alkyne-tagged small precursors. a.o. arbitrary units.
  • Alkynes possess desirable chemical and spectroscopic features. Chemically, they are small (only two atoms), exogenous (nearly non-existent inside cells), and bioorthogonal (inert to reactions with endogenous biomoiecules). These properties render alkynes key players in bioorthogonal chemistry, in which precursors labeled with alkyne tags form covended bonds with azides fused to probes such as fiuorophores for detection.
  • 1 1 is an illustration showing a Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second) are both temporally and spatially synchronized before focused onto cells that have been metaboHcally labeled with alkyne-tagged small molecules of interest.
  • the energy difference between the Pump photon and the Stokes photon matches the vibrational f equency (Ovib) of alkyne bonds
  • alkyne bonds are efficiently driven from their vibrational ground state to their vibrational excited state, passing through a virtual state.
  • a photon in the Pump beam is annihilated (Raman loss) and a photon in the Stokes beam is created (Raman gain).
  • the detected pump laser intensity changes through a lock-in amplifier targeted at the same frequency as the modulation of Stokes beam serve as the contrast for alkyne distributions.
  • SRS microscopy offers a number of advantages.
  • SRS boosts vibrational excitation by a factor of 107, rendering a quantum leap of sensitivity (i.e., detectabi!ity and speed) over spontaneous Raman.
  • CARS coherent anti-stokes Raman scattering
  • the detection limit of alkynes in mammalian cells approaches 200 ⁇ (with 100 ps pixel, dwell, time) based on an average signal-to-noise ratio of 2 as we obtained in HeLa cells.
  • the Hpg signal is well retained in fixed cells (e.g. FIG. 1.4), indicating little contribution from freely diffusing Hpg.
  • the alkyne-on image displays the Hpg distribution for the newly synthesized proteins.
  • the off-resonant (alkyne-off) image shows vanishing signal
  • the amide image shows total protein distribution. This result confirms that the detected signal is not from freely diffusive precursor Hpg itself (which is eliminated during the fixatio process).
  • FIG. 12f Note that we verified the spatial patterns of EdU, EU and Hpg incorporation in live cells by performing click chemistry on fixed cells, with FIGS. 1.5A-C showing the fluorescence images of HeLa cells incorporated with a, EdU (for DMA); b, EU (for RNA); c, Hpg (for protein). Scale bars, 10 pm.
  • Lipid metabolism is critical for many functions in healthy and diseased tissues, but few non-per turbative tags are available to monitor lipids in the ceil. We thus moni tored the metabolic incorporation of alkyne-tagged choline and fatty acids. HippocampaS neurons grown on propargylcholine present a clear 21 2 cm- 1 Raman peak (e.g. FIG. 12a). Such a frequency shift from 2125 cm- 1 is due to the positive charge on the nitrogen near the alkyne (FIG. 10c). As revealed by enzymatic assays (e.g. FIGS. 16a- 1.6c), the alkyne-on signal (FIG.
  • FIG. 12g mainly originates from newly synthesized choline phospholipids at membranes.
  • 17-octadecynoic acid 17-ODYA
  • THP-l macrophages which actively scavenge cholesterol and .fatty acids.
  • FIG. 1 a fixed M1H3T3 cells are seen after culturing with 0.5 mM propargylcholme for 48 hours.
  • the alkyne-on image shows alkyne-tagged choline distribution
  • FIG, 16b treatment of fixed IH3T3 cells with phospholipase C, which removes Choline head groups of phospholipids only in the presence of calcium.
  • the alkyne-on image shows the strong decrease of incorporated propargylcholine signal, supporting its main incorporation into membrane phospholipids.
  • FIG. 16c treatment of fixed NIM3T3 cells with phospholipase C in the presence of EDTA (chelating calcium). Propargylcholine signal is retained in the alkyne-on image.
  • FIGS . 1.6a-c in the same set of cells as in alkyne-on images, the alkyn.e-o.iT images show a clear background.
  • the amide images display total protein distribtuion. Scale bars, 10 ⁇ .
  • the alkyne-on image (FIG.
  • ⁇ 2h depicts the formation of numerous lipid droplets that indicates transtormation into foam cells, a hallmark of early atherosclerosis.
  • Multicellular organisms are also capable of taking up 17-ODYA for lipid imaging.
  • New fatty acids in C. elegans appeared mainly inside lipid droplets upon SRS imaging, known to exist largely in the form of triglycerides (e.g. FIG. 12t).
  • Such a fat accumulation process could serve as a useful model for studying obesity and diabetes.
  • FIG. 17a depicts Rama spectra of a drug cream. Lamisil, containing 1% TH and mouse ear skin tissue.
  • FIGS. 17b-e illustrate SRS imaging of tissue layers from stra tum corneum (z ⁇ 4 pm) to viable epidermis (z ⁇ 24 ⁇ ), sebaceous gland (z ⁇ 48 ⁇ ) and subcutaneous fat (z ⁇ 88 ⁇ ).
  • DM SO solution containing I 3 ⁇ 4 TH was applied onto the ears of an anesthetized live mouse for 30 mm and the dissected ears are imaged afterwards. For all 4 layers shown in FIGS.
  • alkyne-on images display TH penetration; alkyne-off images show off-resonant background (The bright spots in d are due to two-photon absorption of red blood cells).
  • the composite images show protein ( 1655 cm- 1) and lipid (2845 cm-!) distributions. Scale bars, 20 p.m. a.u, :::: arbitrar units.
  • FIGS, 1 Sa- show SRS imaging of the viable epidermis layer (z :::: 20 pm) and the sebaceous gland layer (sr. :::: 40 prn).
  • the alkyne-on images display the TH penetration into mouse ear tissues through lipid phase.
  • the composite images show both protein (1655 cm- 1) and lipid (2845 cm-i) distributions. Scale bars, 20 pm.
  • alkvnes Unlike bulky ftuorophores, alkvnes have little perturbation to pharmacokinetics and are common moieties in many pharmaceuticals.
  • TH terbkiafine hydrochloride
  • aUcyne-bearing antifungal skin drug a US Federal Drug Administration approved aUcyne-bearing antifungal skin drug, and imaged its drug delivery pathways inside mouse ear tissue to a depth of about 100 pan by targeting its internal alkyne at 2230 cm-1.
  • TH images captured at various depths ail exhibit patterns that highly resemble lipid distributions but not protein distributions, suggesting that TH penetrates into tissues through the lipid phase, consistent with its lipophilic nature.
  • Our technique should be applicable to tracking other drags after proper alkyne derivatization,
  • catalyst 8 (36,5 ⁇ , 5 eq.) was prepared in 0.5 mL dry CCU in situ according to the procedure documented by Jyothish and Zhang (Ang w. Chem. fat Ed. Engl 50, 3435-8 (201.1 )). To the solution of catalyst 8 in CCL ⁇ was added 9 (267 mg, 0.73 mmol) and a solution of Compound 5 (3.0 mg, 7.3 ⁇ ⁇ ⁇ ) in 0.5 mL dry CCU, The mixture was heated to 70 °C for 8 h before concentrated in vacuo.
  • FIG. 38 shows a block diagram of an exemplary embodiment of a system according to the present disclosure.
  • exemplary procedures in accordance with the present disclosure described herein can be performed by a processing arrangement and/or a computing arrangement 3802.
  • Such processing/computing arrangement 3802 can be, for example entirely or a part of, or include, but not limited to, a computer/processor 3804 that can include, for example one or more microprocessors, and use instructions stored on a computer-accessible medium (e.g., RAM, ROM, hard drive, or other storage device).
  • a computer-accessible medium e.g., RAM, ROM, hard drive, or other storage device.
  • a computer-accessible medium 3806 e.g., as described herein above, a storage device such as a hard disk, floppy disk, memory stick, CD-ROM, RAM, ROM, etc., or a collection, thereof
  • the computer-accessible medium 3806 can contain executable instructions 3808 thereon, in addition or alternatively, a storage arrangement 3810 can be provided separately from the computer-accessible medium 3806, which can provide the instructions to the processing arrangement 3802 so as to configure the processing arrangement to execute certain exemplary procedures, processes and methods, as described herein above, for example.
  • the exemplary processing arrangement 3802 can be provided with or include an input/output arrangement 3814, which can include, for example a wired network, a wireless network, the internet, an intranet, a data collection probe, a sensor, etc.
  • the exemplary processing arrangement 3802 can be in communication with an exemplary display arrangement 3812, which, according to certain exemplary embodiments of the present disclosure, can be a touch-screen configured for inputting information to the processing arrangement in addition to outputting information from the processing arrangement, for example.
  • the exemplary display 3812 and/or a storage arrangement 3810 can be used to display and/or store data in a user-accessible format and/or user-readable format.
  • Knoll B Keilmann F (1 99) Near- field probing of vibrational absorption for chemical microscopy. Na lure 39 : 7 -10.
  • Bloembergen N (1 67) The Stimulated Raman Effect. Am. J. Phys. 35:989-1023. 64. Min W, Freudiger CW, Lu S, Xie XS (2011 ) Coherent nonlinear optical imaging: beyond fluorescence microscopy. Anna Rev Phys Chem. 62:507-530.

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Abstract

Methods, systems and computer-accessible medium for imaging a living cell or a living organism with bond-edited compounds using stimulated Raman scattering are disclosed. The method comprises the steps of introducing one or more bond-edited compounds into a live cell or a living organism, and detecting a vibrational tag in the cell or organism with stimulated Raman scattering. Also disclosed are methods for detecting a disease condition in a subject methods for monitoring treatment for a disease condition, methods for screening an agent, methods for tracing a cellular process in a live cell using bond-edited compounds in combination with stimulated Raman scattering. Also disclosed are a composition for labeling a target cell with at least one bond-edited compound and devices for imaging bond-edited compounds by stimulated Raman scattering.

Description

TITLE
DEVICES, COMPOSITIONS AND METHODS FOR IMAGING WITH RAMAN
SCATTERING
CROSS-REFERENCE TO RELATED APPLICATION
100011 This application claims the priority of U .S. Provisional Application Serial Nos. 61/836,235, filed June 58, 2013, and 61/946,296, filed February 28. 2014, which are incorporated herein in their entirety.
FIELD OF TH DISCLOSURE
[0002 ί The present disclosure relates generally to imaging technology, and in particular to vibrational microscopy and spectroscopy with Raman scattering technology.
BACKGROUND INFORMATION
[0003 j Innovations in light microscopy have expanded the know ledge of biological processes at the microscopic level. Tn particular, fluorescence microscopy, utilizing versatile fluorescent probes (e.g. genetic label ing of fluorescent proteins, organic dyes and quantum dots) (See, e.g.. References 1-3 ), can facilitate specific detection of molecules of interest in biological systems, facilitating people to actually visualize and understand fundamental processes. Taking advantage of the development of fluorescent probes (e.g. brighter, more photostable, multicolor etc.) (See, e.g., Reference 4), fluorescent microscopy such as confocal microscopy, t o-phoion microscopy, single molecule microscopy and superresolution microscopy have enabled detection, of structures that can be much deeper and finer than before. However, according to quantum mechanics (e.g. particle in the box), the chromopSiore within a fluorophore have to be a large conjugation, system in order for the efficient absorption i the visible spectrum. Thus, in spite of the significance in various applications such as in cell biology, fluorescent tags intrinsically cannot be properly used for tagging small molecules such as glucose, nucleosides, amino acids, choline, fatty acids and small molecule drags, for their relatively large size perturbs with the small molecule dynamics.
[O0O4 | An opposite strategy for visualizing these important building block small molecules in biological systems can be label-free imaging. Representative imaging procedures of the kind can include vibration microscopies based on infrared absorption and Raman scattering detecting the characteristic vibrational mode of specific chemical bond from the molecules themselves (See, e.g., References 5-9). Other label-free procedures can be second harmonic generation ("SHG"), imaging special. «on-cetUrosym.m.eirk structures, third harmonic generation. ("THG"), sensing interfaces and optical heterogeneities and optical coherence tomography ("OC '), measuring the backscattered light from tissues through low-coherence ioterfero etry. However, label-free imaging can suffer from two fundamental problems; first, there can be insufficient specificity because small molecules usually do not have unique spectroscopic signature in the vast pool of other biomolecuSes; second, there can be unsatisfying sensitivity due to usually low concentration of the small molecules in the biological systems.
[0005] As an imaging tag, alkyne (e.g. carbon-carbon triple bond) can offer three advantages over others. First, alkyne is only a chemical bond, second alkyne can enable background-free detection, and third, alkyne can be inert to react with any intrinsic bio-molecules in the biological systems, in fact, a!kyne can be widely used in the powerful hioorthogonai chemistry utilizing ajkyne-azide specific click-chemistry reaction for various purposes (See, e.g.. References 10-12). For example, using alkyne tagged molecule of interest followed by aztde tagged detection reagent (e.g. affinity probes or fluorescent tag) can enable detection 'using mass spectrometry or fluorescence microscopy.
[O0O6j The proteome of a cell can be highly dynamic in nature and tightly regulated by both protein synthesis and degradation to actively maintain homeostasis. Many intricate biological processes, such as ceil growth, differentiation, diseases and response to environment stimuli, can require protein synthesis and translatk ai control (See, e.g., Reference 24). In particular, long-lasting forms of synaptic plasticity, such as those underlying long-term memory, can need new protein synthesis in a space- and time- dependent, manner (See, e.g., References 26-30). Therefore, direct visualization and quantification of newly synthesized proteins at a global level can be indispensable to unraveling the spatial-temporal characteristics of the proteomes in live cells.
[0007] Extensive efforts have been devoted to probing protein synthesis via fluorescence contrast. The inherent fluorescence of green fluorescent protein ("GFP") and its genetic
encodability, can the following of a gi ven protein of interest inside living cells with high spatial and temporal resolution (See, e.g., References 29 and 30). However, GFP tagging through genetic manipulation works only on individual proteins, and not at the whole proteome level. To probe newly synthesized proteins at the proteome level, a powerful procedure named bioorthogonal noncanonical amino acid tagging (BONCAT) was developed by metabolic incorporation of unnatural amino acids containing reactive chemical groups such as azide or alkyne. (See, e.g., References 31-37). A .related labeling method was recently demonstrated using an alkyne analog of puromycin. (See, e.g., Reference 28). Newly synthesized proteins ca then be visualized through subsequent conjugation of the reactive amino acids to .fluorescent tags via click chemistry. (See, e.g.. Reference 29). Unfortunately, these fluorescence-based methods generally use non-physiological fixation and subsequent dye staining and washing.
(0008] i addition to fluorescence tagging, radioisotope or stable isotope, labeling can be another powerful tool to trace and quantify proteome dynamics. Classical radioisotope-labeled amino acids (e.g., 35S-meth.ion.ine) can provide vigorous analysis of global protein synthesis.
However, samples must be fixed and then exposed to film for autoradiography. For stable isotopes, the discovery of deuterium by Urey in 1932 immediately led to the pioneer work of Schoenheimer and Rittenberg studying intermediary metabolism. (See, e.g., References 40 and 51), To study proteome changes between different cells or under different conditions, stable isotope labeling by amino acids in cell culture ("SILAC") coupled with mass spectrometry ("MS") has matured into a popular method for quantitative proteoroics (See, e.g., References 42-45). However, S!LAC-MS does not usually provide spatial information down to sub-cellular level and its invasive nature limits its application for live cell imaging. The same limitation applies to the recent ribosome profiling study using deep sequencing procedure (See, e.g., Reference 46).
[0009 j Spontaneous Raman microscopy has been used for label-free molecular and biomedical imaging (See, e.g., References 8, 13, 17, 59 and 73-77). However, this technology suffers from low sensitivity and slow imaging speed.
(OOlOj Thus, it may be beneficial to have an imaging strategy that makes up for the gap between fluorescence microscopy and label-free imaging for the sensitive and specific detection of small molecules while offering minimum perturbation to the biological systems (e.g. to have small tags with distinct spectroscopic characteristics), and which can overcome at least some of the deficiencies described herein above.
SUMMARY OF EXEMPLARY EMBODIMENTS
(0011] One exemplary aspect of the present disclosure relates to a method for obtaining biological information in a living cell or a living organism with bond-edited compounds using stimulated Raman scattering. The method comprises the steps of introducing one or more bond-edited compounds into a li e cell or a living organism, and detecting a vibrational tag in the cell or organism with stimulated Raman scattering,
(001 ) Another exemplary aspect of the present disclosure relates to methods for making a bond-edited compound. 0013] Another exemplary aspect of the present disclosure relates io a method for detecting a disease condition in a subject, comprising: administering to said subject a composition comprising a bond-edited compound targeting a disease tissue or pathogen, and detecting said bond-edited compound by stimulated Raman scattering.
[0014] Another exemplary aspect of the present disclosure relates to a method for monitoring a treatment for a disease condition. The method comprises administering to the subject
composition comprising a bond-edited compound and detecting the bond-edited compound by stimulated Raman scattering at a first time point, performing the treatment after the first time point, further administering to said subject the composition comprising a bond-edited compound, and detecting the bond-edited compound by stimulated Raman scattering at a second time point, and comparing images obtained at the two time points,
[0015] Another exemplary aspect of the present disclosure relates to a method for screening an agent. The method comprises administering the agent and at least one bond-edited compound to a live cell or organism, detecting the bond-edited compound in the live cell or organism using stimulated Raman scattering, and selecting a candidate agent based on one or more predetermined criteria, such as the uptake, accumulation, trafficking or degradation of the bond-edited compound by the said live cell or organism.
[0016] Another exemplary aspect of the present disclosure relates to a device for imaging bond-edited compounds by stimulated Raman scattering. The device comprises a first
single-wavelength laser source that produces a pulse laser beam of a first wavelength,, a second single- wavelength laser source that produces a pulse laser beam of a second wavelength, a modulator that modulates the pulse laser beam of one of the first or second laser source, a pbotodetector that is capable of or configured to detect stimulated Raman scattering from a biosample, and a computer.
[0017] Another exemplary aspect of the present disclosure relates to a non-transitory computer-accessible medium having stored thereon computer-executable instructions for determining data associated with at least one tissue, wherein, when a computer hardware
arrangement executes the instructions, the computer arrangement is configured to perform procedures comprising; receiving first information related to at least one bond between at least two atoms attached to a metabolite; and determining the data based on the at least one bond,
[00] 8[ Another exemplary aspect of the present disclosure relates to a method for determining data associated with at least one tissue, comprising: receiving first information related io at least one bond between at least two atoms attached to a metabolite; and using a computer hardware arrangement, determining the data based on the at least one bond. [0019] Another exemplary aspect of the present disclosure relates to a system for determining data associated with at least one tissue, comprising: a computer processing arrangement configured to receive first information related to at least one bond between at least two atoms attached to a metabolite; and determine the data based on the at least one bond.
[0020] Another exemplary aspect of the present disclosure relates to a pre-mixed essential amino acid combination, comprising: at least one non-denierated essential amino acid: and at least 5 deuterated essential amino acids.
[0021 J These and other objects, features and advantages of the exemplary embodiments of the present disclosure will become apparent upon reading the following detailed description of the exemplary embodiments of the present disclosure, as also exemplified by the appended claims.
BRIEF DESCRIPTION OF THE BRA WINGS
[0022} Further objects, features and advantages of the present disclosure will become apparent from the following detailed descriptio take in conjunction with the accompanying FIGS, showing illustrative exemplary embodiments of the present disclosure, in wh ich:
[0023] FIG. 1 shows imaging complex protein metabolism by stimulated Raman scattering (SRS) microscopy in live cells, tissues and animals. For example, FIG. la illustrates a cartoon for SRS imaging following metabolic labeling of deuterated amino ac ids (D-AAs) in live organisms (e.g. mice), which are first administered with D-A As for certain period of time and then imaged by SRS to probe protein metabolism. FIG. lb illustrates spontaneous Raman spectra from HeLa cells incubated with medium containing either regular amino acids (gray, dashed) or D-AAs illustrate three distinct ways to probe complex protein metabolism: imaging newly synthesized proteins by targeting 2133 cnvi from carbon-deuterium bonds (C-D), imaging degradation of pre-existing proteins by targeting the pure methyl group (Clh) distribution, and two-color pulse-chase protein imaging by labeling with two sub-groups of D-AAs (i.e., group I and group II).
[0024) FIG. 2 depicts sensiti vity optimization and time-lapse imaging of the de novo proteome synthesis dynamics. For example. FIG. 2a illustrates spontaneous Raman spectra of C-D peaks in HeLa cells incubated in optimized deuteration medium display a 50% increase when compared to the previously reported partial deuteration medium, and about 8 times higher than using leucine-djft only. FIG. 2b illustrates SRS images of newly synthesized proteins in live HeLa cells confirm a 50% average signal increase. FIG. 2c illustrates SRS images of newly synthesized proteins in live neurons in optimized deuteration medium for 20 h. The zoom-in image highlights the fine dendritic structures (likely dendritic spines, arrow-headed). FIG. 2d illustrates SRS image of newly
3 synthesized proteins in live HeLa cells with 1. h incubation of optimized deuteration medium.
Control image with protein synthesis inhibition deprives most of the signal. FIG. 2e illustrates time-lapse SRS images of protein synthesis dynamics in a same set of live HeLa cells with
continuous incubation in optimized deuteration medium. Scale bar. 10 μηι.
[0025 Ϊ FIG. 3 shows time-dependent SRS images of protein degradation. For example, FIG. 3a illustrates adopting linear combination algorithm between 2940 cm" and 2845 cm' channels, the obtained SRS image exclusi vely from 0¾ vibration dispiay gradual degradation of pre-existing proteins in live HeLa cells cultured in optimized deuteration medium for 0 h, 24 h, 48 h and 96 h, FIG, 3b illustrates SRS images exclusively from (¾ vibratio dispiay the total lipid distribution at the corresponding time point. FIG. 3c illustrates a single exponential decay fitting from averaged image intensities from pre-existing protein in FIG. 3a, yielding a protein degradation time constant of 45 ± 4 h. Error bars., standard deviation. Scale bar, 10 μηι.
[0026 J FIG. 4 depicts pulse-chase SRS imaging of temporally defined proteins. For example, FIG. 4a illustrates structures and spontaneous Raman spectra of group I D-AAs (i.e. the branched chain amino acids). FIG. 4b illustrates structures and spontaneous Raman spectra of three examples of group II non-branched D~AAs. FIG. 4c illustrates spontaneous Raman spectra of HeLa cells cultured with group I D-AAs { element 405), showing multiple peaks with the first around 2067 cm'1, and with group 11 D-AAs ( element 410), showing a common peak around 2133 cm'1. FIG, 4d illustrates two-color pulse-chase imaging by sequential labeling of group and group I D-AAs in time with simultaneous expression of mutant himtingtin (mHtt94Q-m£os2) proteins. Cartoon displays experimental timeline of plasn.iid transaction and D-AA medium exchanges. The fluorescence image (overlaid with bright field) indicates the formation of a large aggregate
(arrow-headed) of roHtt94Q~mBos2, The retrieved signals from linear combination of the original images at 2067 and 2.133 channels display a large aggregation of mBtt proteins solely labeled by group II D-AAs during the first 22 h (pulse 415) and mHtt only labeled by group 1 D-AAs during the following 20 h (chase 420). The merged image, as well as the intensity profile, from, the pulsed (element 415) and chased (element 420) images confirms with a yellow core and a green shell. Scale bar, 10 um.
j 0027| FIG . 5 shows SR S imaging of newly synthesized proteins in live mouse brain tissues. For example, FIG. 5a illustrates SRS images at dentate gyrus of a. live organotypic brain slice (400 pin thick, from a ΡΊ0 mouse) after culturing in D-AA medium for 30 hr. 2133 cm"1 (C-D) image presents the distribution of newly synthesized proteins. The C¾ and CI L images show the old protein pools and total lipids, respectively. FIG. 5b illustrates a 4-by-3 ram large field view overla image of new proteins (C-D, element 505), old proteins (€¾, element 510) and total lipids CC¾, element 515) for a brain slice (400 μηι thick, from a PI 2 mouse) cultured in D-AA medium for 30 h. Scale bar, 100 μη ,
[0028] FIG. 6 shows SRS imaging of newly synthesized proteins in viva. For example, FIG. 6a illustrates SRS images of a 24-hpf (hpf: hours post fertilization) zebrafish. Wild-t pe zebrafish embryos were injected at I -cell stage with I nL D-AA solution and allowed to develop normally for another 24 h before imaging. Bright field image shows the gross morphology of embryonic zebrafish at 24 hpf (dashed boxes ). 2133 cm'1 (C-D) image presents the distribution of newly sy nthesized proteins (Supplemental FIG. 2a) in the somites of an embryonic zebrafish tail. The C¾ image shows the old protein pool while the C¾ image depicts total lipid in the same fish. FIG. 6b and 6c SRS images of live mouse liver are shown in FIG. 6b and. intestine tissues FIG. 6c harvested from mice after administered with D-AA containing drinking water for 12 days, 2133 cm*1 (C-D) channel shows newly synthesized proteins (Supplemental FIG. 2b~2c that resemble the distribution of total protein as shown in the 1 55 cm4 image (Amide Ϊ). Scale bar, 10 μχη.
JO029] FIG, 7 depicts SR images at 2067 cm ! and 2133 cm"1 channels of proteins labeled with grou 1 D-AA only shown in FIG, 7a and group 0. D-AA only shown in FIG. 7b.
[00301 FIG. 8 shows raw C-D on-resonance (2133 cm*5) and off-resonance (2000 cm*1) SRS images of newly synthesized proteins in vivo in FIG. 6a, which illustrates SRS C-D on-resonance and off-resonance images of a 24 hpf embryonic zebrafish. The difference image between C-D
on-resonance and off-resonance (pixel -b -pixel subtraction) shows pure C-D labeled protein distribution i the somites of an embryonic zebrafish tail, as in FIG. 6, FIG, 8 b and 8c illustrate SRS C-D on-resonance and off-resonance images of live mouse liver FIG. 6b and intestine FIG. 6c tissues harvested from the mice after administering with D-AA containing drinking water for 12 days. The difference image between C-D on-resonance and off-resonance (pixel -by-pixel subtraction) shows pure C-D labeled protein distribution in the liver and intestine tissues, shown in FIG. 6b and FIG. 6c, respectively. The residual signal presented m the off-resonance images mainly comes from
cross-phase modulation induced by highly scattering tissue structures,
j'O031J FIG. shows SRS imaging for newly synthesized proteins in vivo with
intraperitoneal injection of mice with D-AA solutions. For example, FIGS. 9a and 9b illustrate SRS images of live mouse liver FIG, 9a and intestine FIG. 9b tissues harvested from mice after
intra peritoneal injection injected with D-AAs solutions for 36 h. 2133 cm'1 channel shows newly synthesized proteins (off-resonance image subtracted) that resemble the distribution of total proteins as shown in the 1655 cm"1 image ( Amide I). FIG . 9c and 9d illustrate corresponding raw C-D on-resonance (2133 cm*1) and off-resonance (2000 cm") images are shown as references for liver FIG. 9c and intestine FIG. 9d tissues. Scale bar, 10 μηι.
j0032] FIG, 10 depicts bond-selecti ve SRS imaging of alkynes as nonlinear vibrational tags. For example, FIG. 1 a illustrates Spontaneous Raman spectra of HeLa ceils and 10 mM EdU solution. Inset: the calculated SRS excitation profile (FWHM 6 cm-') is well fitted within the 2125 cm" alkyne peak (FWHM 14 era ', magenta). FIG. 10b illustrates linear dependence of stimulated 'Raman loss signals (2125 cm'*) with EdU concentrations under a 100 ps acquisition time. F.1G. 10c illustrates the metabolic incorporation scheme for a broad spectrum of Jkyne-tagged small precursors. a.u. arbitrary units.
[ΘΘ33] FIG, 1 1 illustrates the working mechanism of a stimulated Raman scattering with A Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second). The Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second) are both temporally and spatially synchronized before focused onto cells that have been metabolically labeled with alkyne-tagge small raolecoies of interest. When the energy difference between the Pump photon and the Stokes photon matches the vibrational .frequency (-¾&) of alkyne bonds, alkyne bonds are efficiently driven from their vibrational ground state to their vibrational excited state, passing through a virtual state. For each excited alkyne bond, a photon in the Pump beam is annihilated (Raman loss) and a photon in the Stokes beam is created (Raman gain). The detected pump laser intensity changes through a lock-in amplifier targeted at the same frequency as the modulation of Stokes beam serve as the contrast for alkyne distributions,
[0034] FIG. 12 shows live SRS imaging of de novo synthesis of DNA, RNA, proteomes,
•phospholipids and triglycerides by metabolic incorporation of alkyne-tagged small precursors. For example,
FIG. 12a illustrates Raman spectra of cells incubated with EdU, EU, Hpg, propargylehoiine and i 7-Qctadecynoic acid ( 17-ODYA). FIG, 12b illustrates live HeLa cells incubated with 100 uM EdU alone
(alkyne-on) and with .10 mM hydroxyurea (Control.). FIG. 12c illustrates time-lapse images of a dividing ceil incubated with 100 μΜ EdU. FIG. 12d illustrates live HeLa cells incubated with 2 mM EU alone
(alkyne-on) and with 200 aM actinomyein D (Control). FIG. 12e iilosiraies pulse-chase imaging of RNA turnover in HeLa cells incubated with 2 mM EU for 12 h followed by EU-frce medium. FIG. 12f illustrates live HeLa cells incubated with 2 raM Hpg alone (alkyne-on) and with 2 mM methionine (Control). FIG. 12g illustrates live neurons incubated with i mM propargylehoiine (alkyne-on). FIG. I h illustrates live macrophages incubated with 400 μΜ 17-ODYA (alkyne-on). FIG. 12i, illustrates C. egctns fed with
17-ODYA (alkyne-on). FIG. 12j, illustrates dual-color images of simultaneous EdU' (2125 cm "1) and propargylehoiine (2142 cm") incorporation. For FIGS. 12b, FIG, d, and FIG, 12f, alkyne-off and amide images display the same set of cells as the alkyne-on images; lipid images capture the same cells as controi images. Scale bars, .10 um. Representative images of 10-15 trials, a.o, - arbitrary units. [0035] FIG. 13 shows SRS imaging of distal mitotic region of e!egam gerrnlrae incorporated with EdU. The composite image shows both the protein derived 1655 cm"1 (amide) signal, from all the germ cells, and the direct visualization of alkynes (2125 cm"1 (EdU)) highlighting the proliferating germ cei ls. White circles show examples of EdU positive gertn cells in the mitotic region of C iegcms gerailine. Scale bar, 5 pm,
[0036} FIG. .1 shows SRS imaging of fixed HeLa cells after incorporating with.2 rnM Hpg. The alkyne-on image displays the Hpg distribution for the newly synthesized proteins. For the same set of ceils, the off-resonant (alkyne-ofT image shows vanishing signal, and the amide image shows total protein distribution. This result confirms that the detected signal is not from freely diffusive precursor Hpg itself (which is eliminated during the fixation process). Scale bar, 10 pin,
10037] FIG. 15 shows click-chemistry based fluorescence staining of fixed HeLa ceils. Fluorescence images of HeLa cells incorporated with FIG, 15a, EdU (for D' A); FIG. 15b Ell (for R A): FIG, 15c Hpg (for protein). Scale bars, 10 μηι.
10038] FIG, 16 shows SRS imaging of propargylcholine incorporation in NIH3T3 cells and control experiments. For example, FIG. 16a illustrates fixed NIH3T3 cells after culturing with 0.5 x»M propargylcholine for 48 hours. The alkyne-on image shows alkyne-tagged choline distribution. FIG. 1 b illustrates treatment of fixed IH3T3 cells with phospholipase C, which removes Choline head groups of phospholipids onl in the presence of calcium. The alkyne-on image shows the strong decrease of incorporated propargylcholine signal, supporting its main incorporation into membrane phospholipids. FIG. 16c illustrates treatment of fixed ΝΪΗ3Τ3 cells with phospholipase C in the presence of EDTA (chelating calcium). Propargylcholine signal is retained in the alkyne-on image. FIGS, 16a-16c illustrate images in the same set of cells as in alkyne-on images, the alkyne-ofT images show a clear background. The amide images display total protein distribution. Scale bars, 10 μηι,
[0039] FIG. 17 shows m vim delivery of an alkyne-bearin drug (TH in DM SO) into mouse ear. For example, FIG. 1 a illustrates Raman spectra of a drug cream, Lamisil, containing 1% TH and mouse ear skin tissue. FIGS. I 8b-18e illustrate SRS imaging of tissue layers from stratum corneum (z :::: 4 pm) to viable epidermis (z - 24 pm), sebaceous gland (z ~ 48 μηι) and subcutaneous fat (r - 88 μτα). To facilitate tissue penetration, DMSO solution containing 1 % TH wa applied onto the ears of an anesthetized live mouse for 30 mm and the dissected ears are imaged afterwards. For all 4 layers: alkyne-on images display TH penetration; alkyne-off images show off-resonant background (The bright spots in FIG. 18d are due to two-photon absorption of red blood cells). The composite images show protein (1 55 cm*5) and lipid (2845 cm*1) distributions. Scale bars, 20 pm, a.u. arbitrary units. 0040] FIG. 18 shows in vivo delivery of an. alkyne-bearing drag (TH m Lamisli cream, a FDA approved drug cream) into mouse ear. For example, FIGS. I S(a-b) illustrates SRS imaging of the viable epidermis layer (z - 20 pm) and the sebaceous gland layer (z - 40 μη ). For both F IG. 18a and FIG. 18b; illustrates the alkyne-on images display the TH penetration into mouse ear tissues through lipid phase. The composite images show both protein (1655 cm"f) and lipid (2845 cm") distributions. Scale bars, 20 p.m.
[0041] FIG, 1 shows an exemplary synthesis route for a bond-edited compound.
[00421 FIG. 20 shows in FIG. 20a another exemplary synthesis route tor a bond-edited compound (alkyne-D-glucose) and in FIG. 20b the spectroscopic characterization of the bond-edited compound in P BS buffer and in mammalian cells.
[0043] FIG. 21 shows time-dependent alkyne-D-glucose (32 mM) uptake in live HeLa ceils at lOrnin, 30 miu, l b, 2h, 3h and 4h time points. The glucose signal inside mammalian cells is increasing over time.
JO044] FIG. 22 shows the results of a competition experiment to confirm the uptake of alkyne-D-glucose. Regular D-glucose is added into cell medium for HeLa cells to compete with the uptake of alkyne-D-glucose. With the increasing concentration of regular D-giucose (l OraM, 50mM and QOrnM h the alkyne-D-glucose signal decreases (as shown both in images and bar diagrams). When using L-ghicose (which cells clo not uptake) as competition for aikyne-giucose, the
alkyne-D-glucose signal is retained.
[0045] FIG. 23 shows aik ne-ghicose uptake in both neuronal culture FIG, 23a and brain slices FIG. 23b.
10046 FIG. 24 shows multicolor imaging of DMA synthesis with EdU ( 1), EdU-3 *C (2) and EdU- C2 (3).
(0047J FIG. 25 shows pulse-chase imaging ofD A synthesis (EdU ( 1) for pulse and
EdU-i 5C2 (3) for chase). The merged images show that the two compoimds can label two temporally different cells populations for DNA synthesis.
[0048] FIG. 26 shows simultaneous three-color chemical imaging using alkyne probes for DNA synthesis (EdU-5 iC (2) at 2077 crn-i ) and A synthesis (EU~UC2 (13) at 2053 cm-1 and 17-ODYA (12) at 2125 cm-1).
[0049] FIG, 27 shows images of subcutaneous colon cancer. Subcutaneous colon cancer was grown for 15 days in mice, dissected out and cultured ex vivo in deuteraied amino acids containing medium for 47h (400um thick). Live image of the tumors shows intensive protein synthesis activity. [0050] FIG. 28 shows active glucose metabolism in HeLa cells cultured m deuterated glucose medium. For example, FIG. 28a illustrates images after culturing in 0.1% D7-Giucose in EMEM for 48 hrs. FIG. 28b illustrates HeLa cell images after culturing 0.2% D7-Glocose in EMEM for 48 hrs.
[0051 J FIG, 29 shows active glucose metabolism in tumor cell line U87MG cultured in deuterated glucose medium (0.1% D7-GJucose in EMEM) for 48 hrs,
[0052] FIG. 30 shows the detection of D20 as a labeling reagent of the metabolism with stimulated Raman scattering.
[0053] FIG. 31 shows SRS imaging of C-D formation using D20 as a metabolic reagent for various of live organisms.
[O054i FIG. 32 shows imaging of C-phenylalanine labeled proteins for protein turnover. For example, FIG. 32 a shows an illustration of a spectroscopic characterization of Raman shift from 3004 cm~l to 968 cm" with the labeling of ' *C-phenylalanine, and FIGS. 32(b) and 32c illustrate a time dependent '^-phenylalanine labeling, whereas FIG. 32b shows a spectrum and FIG. 32c illustrates SRS images, where the 968 cm-1 signal for ! ,C labeled proteins are increasing while the 1004 cm" signal of old !¾ -proteins are decreasing.
[0055J FIG. 33 is a set of exemplary images based on SR S imaging of newly synthesized proteins by metabolic incorporation of deuterium- labeled all amino acids in live HeLa cells. For example, FIG. 33a illustrates Spontaneous Raman spectrum of HeLa cells incubated with a medium containing deuterium-labeled all amino acids for 20 hrs, showing a -5 times stronger peak at 2133 cm-! tha the spectrum in FIG. 2, FIG. 33b illustrates SRS image targeting the central 2133 cra-i vibrational peak of C-D shows a high-contrast image representing newly synthesized proteins. The same intensit scale bar is used here as in FIG. 2. Consistent with previous reports, nascent proteins are distributed with a higher percentage in nucleoli (indicated by arrows) which are the active sites for ribosorae biogenesis involving rapid import and degradation of proteins. FIG. 33 c illustrates SRS image of the same cells as in FIG. 33b at off-resonance frequency 2000 cm-j is background-free. FIGS, 33d-33f illustrate SRS images of same ceils as in FIG. 33b at frequency 1655 cm-t (amide I stretching attributed primarily to proteins); 2845 cm-- (CH2 stretching attributed mainly to lipids) and 2940 cm s (CH stretching attributed mainly to proteins) show the intrinsic distributions of total cellular lipids and proteins.
[0056] FIG. 34 is a set of exemplary images based on SRS imaging of time-dependent de novo protein synthesis and drug-induced protein synthesis inhibition effect in live HeLa cells incubated in deuterium-labeled all amino acid medium. For example, FIG. 34a~34f SRS image targeting the ceiitrai 2133 crn-f vibrational peak of C-D displays a time-dependent signal increase (5 1MS ···· FIG. 34a, 2 hrs ··· FIG. 34b, 20 hrs - FIG. 34c) of the newly synthesized proteins, with nucleoli being gradually highlighted. As a control, the amide I (1655 crn-i) signal remains at a steady state over time (5 hrs -· FIG. 34ds 12 his - FIG, 34e, 20 hrs - FIG. 34i). FIGS. 34g-34i illustrate ratio images between the S RS image at 2133 cm-i (newly synthesized protei ns) and the SRS image at 1655 cm-! (the amide 1 band from total proteins), representing the relative new protein fraction with subcellular resolution at each time point (5 hrs - FIG. 34g, 12 hrs - FIG. 34h, 20 hrs - FIG. 34i). The bar represents the ratio ranging from low to high. FIG. 34j shows time-lapse SRS images of a live dividing HeLa cell during a 25 roin time-course after 20-hour incubation with deuterated all amino acids medium. FIG. 34k illustrates a spontaneous Raman spectrum of HeLa ceils incubated with both deuterium-labeled all amino acids and a protein synthesis inhibitor anisomycin (5 μ ) for 12 hrs shows the drastic attenuation of the C-D Raman peak at 21 3 cra-i. FIG. 341 shows an exemplary SRS image of the same sample displays near vanishing signal throughout the whole field of view. FIG, 34m shows, as a control, the image of the same cells at 2940 cm-i confirms that anisomycin does not influence the total protein level.
[00S7| FIG. 35 is a set of exemplary images based on SRS imaging of newly synthesized proteins by metabolic incorporation of deuterium-labeled all amino acids in live human embryonic kidney (ΗΕΚ293Ϊ) ceils. For example, FIG. 35a illustrates the spontaneous Raman spectrum of HEK293T cells incubated with deuterium-labeled all amino acids for 12 hrs shows a. 21.33 cn -t C-D •peak nearly as high as the Amide I (1655 cm-) ) peak. FIG. 35b shows an exemplary SRS image targeting the central 2133 cm-i vibrational peak of C-D shows newl synthesized proteins in live HEK293T cells displaying a similar signal level as HeLa cells at 12 hrs (FIG. 4b). FIG. 35c shows, as a comparison, the off-resonant image is still background-free. FIGS. 35d and 35 (e) illustrate multicolor SRS images of intrinsic cell molecules: total proteins (1655 cra-i (FIG. 35d) and lipids (2845 cm-i (FIG. 35e). FIG. 35f illustrates the ratio image between new proteins (2133 cra-i) and total proteins ( 1655 cra-i) illustrates a spatial map tor nascent protein distribution.
J0058J FIG. 36 is a set of exemplary images based on SRS imaging of newly synthesized proteins in both ceil bodies and newly grown neurites of neuron-like differentiable mouse neuroblastoma (N2 A) cells. During the cell differentiation, process by sertun-depriyation and 1. μΜ retinoic acid, deuterium- labeled all amino acids medium is also supplied for 24 hrs. For example, FIG . 36a illustrates SRS images targeting the 2133 cm-i peak of C-D show newly synthesized proteins. FIG. 36 b illustrates SRS images targeting the 2940 cm-! CH:> show total proteins. FIGS. 36c and 36d illustrate zoomed-in images as indicated in the white dashed squares in FIG. 36a and FIG. 36b. FIG. 36e illustrates a ratio image between new protein FIG. 36c and total proteins FIG. 36d. While the starred neurites show high percentage of new proteins, the arro w s indicate neitrites displaying very low new protein percentage. FIG. 36f Merged image between new protein c (channel 3605) and total proteins in FIG. 36d (channel 3610). Similarly, starred regions show obvious new proteins; while arrows indicate regions that have undetectable new protein signal.
10059) FIG. 37a is a prior art recipe for a mammalian cell culture,
[0060) FIG. 37b is an exemplary deuterium-labeled recipe based on the ceil culture of FIG.
3 ; and
)O06.l] FIG. 38 is an illustration of an exemplary block diagram of an exemplary system in accordance with certai exemplary embodiments of the present disclosure.
[0062) Throughout the drawings, the same reference numerals and characters, unless otherwise stated, are used to denote l ike features, elements, components or portions of the illustrated exemplar}'' embodiments. Moreover, while the present disclosure will now be described in detail with reference to the figures it. is done so in connection with the illustrati e exemplary embodiments and is not limited by the particular exemplary embodiments illustrated in the figures, and provided in the appended claims.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS ΘΘ63] The following detailed description is presented to enable any person skilled in the art to make and use the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present invention. However, it will, be apparent to one skilled in the art that these specific details are not required to practice the invention- Descriptions of specific applications are provided only as representative examples. The present disclosure is not intended to be limited to the exemplary embodiments shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.
[0064) As used herein, the term "Raman scattering" refers to a spectroscopic technique used to observe vibrational, rotational, and other low-frequency modes in a system. It relies on inelastic scattering, or Raman scattering, of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with molecular vibrations, phonons or other excitations in the system, resulting in the energy of the laser photons being shifted up or down.. The shift in energy gives information about the vibrationa l modes in the system. A variety of optical processes, both linear and nonlinear in light intensity dependence, are fundamentally related to Raman scattering. As used herein, the term Raman scattering" includes, but is not limited to, l .> "stimulated Raman scattering" (SRS), "spontaneous Raman scattering", "coherent anti-Stakes Raman scattering" (CARS), "surface-enhanced Raman scattering" (SERS), "Tip-enhanced Raman scattering" (TERS) or "vibrational photoacoustic tomography".
}O 651 The exemplary system, method and computer accessible medium, according to an exemplary embodiment of the present disclosure, can use alkyne as a vibrational tag coupled with narrow-band sdmuiaied Raman scattering microscopy ("SRS") for the detection of small molecules inside biological systems. The use of alkyne as a vibrational tag (e.g. a Raman tag) offers a large Raman cross-section enabling sensitive detection (See, e.g., References 13; 14). Additionally, the alkyne Raman peak caa exhibit a narrow spectral width for the specific detection, which can reduce the probability of overlapping with other tags. Furthermore, the Raman peak of alkyne can lay exactly in the cell-silent region in the cell spontaneous Raman spectrum, bypassing the complex interference from vast pool of bioraolecules in the fingerprint region. (See, e.g. , FIG. l b).
(0066| The exemplary SRS can be a sensitive vibrational imaging microscopy. By harnessing Einstein's stimulated emission process, 'the exemplary SRS can employ two-laser excitation (e.g., temporally and spatially overlapped Pump and Stokes lasers), boosting up the transition rate about 7 orders of magnitude as compared to the traditional spontaneous Raman microscopy, the transition process of which can be intrinsically weak (e.g., 1.0 to 12 orders of magnitude slower than fluorescence). (See, e.g., References 6; 8; 15). The exemplary SRS can be a bond-selec tive procedure with high specificity, in contrast with the spontaneous Raman imaging which can be a spectrum-based me thod, instead of spreading the energy to the whole spectrum as in the spontaneous Raman imaging, the exemplary narrow-band SRS can focus its energy to the vibrational transition of a specific bond. A 6-ps pulse width can be chose for both SRS pump and stokes lasers to achieve a spectral resolution of 5 cm"' for the detection of alkyne. The spectral width of the excitation profile from two combined lasers can be calculated to be 8 cm'1, which can fit. well within the spectral width of alkyne Raman peak that can be M em"' . (See, e.g., FIG. lb). Hence, the exemplary laser poise width can be long enough that all the laser energy can be used to specifically detect alkyne without energy waste, but short enough that the two-photon efficiency can be maintained since the exemplary SRS can depend on a nonlinear process.
[ 067| The exemplary SRS signal can offer linear concentration dependence to the analyte without non-specific background. Compared to a previously known nonlinear vibrational imaging procedure such coherent anti-Stakes Raman scattering ("CARS") microscopy, which suffers from spectral distortion, unwanted non-resonant background, non-straightforward concentration dependence and coherent image artifact, the exemplary SRS can exhibit straightforward image interpretation and quantification without complications from non-resonant background and
•phase-matching conditions {See, e.g., References 7; 8; 16). Besides the above-mentioned
advantages, SRS can also .have its own distinctive characters as an imaging procedure. For example,
SRS can be immune to fluorescence background as compared to spontaneous Raman microscopy that can suffer from large fluorescence background. In addition, SRS, as a nonlinear process, can offer intrinsic 3D sectioning capability. Moreover, by adopting near-infrared excitation, SRS can offer deeper penetration depth and less phototoxicity, which can be well suited for imaging live cells, tissues and animals. Recently, .narrow-band SRS has achieved unprecedented sensitivity down to approximately 1000 retinoic acid molecules and up to video rate imaging speed in vivo. (See, e.g., Reference 17).
[00681 A!kyne can be a metabolic labeling tag in fluorescence microscopy utilizing
click-chemistry with azide-lraked fluorescent tags (See. e.g., References 18-23). Unfortunately, this type of click-chemistry based fluorescence detection usually requires non-physiological fixation and subsequent dye staining and washing. The exemplary Raman detection, in contrast, does not. have such requirements, since it can directly image vibrational modes ofalkyne, bypassing the subsequent additional processes.
[0069] All of the above applications can show the uni versal and distinct ad vantage of the exemplary SRS coupled with alkyne tags to image the small molecule metabolites dynamics and drug distributions in the live cells, organisms and animals with minimum perturbation and high specificity and sensitivity, extending the repertoire of reporters for biological imaging beyond fiuorophores.
Method tor obtaining biological information in a living cell or a living organism with bond-edited compounds
[0070] One aspect of the present disclosure relates to a method for obtaining biological information in a jiving cell or a living organism with bond-edited compounds using Raman, scattering. The method comprises the steps of introducing an effecti ve amount of one or more bond-edited compounds into a live cell or a living organism, and detecting a vibrational tag in the cell or organism with Raman scattering. In some exemplary embodiments, the Raman scattering is SRS.
[0071 [ The term "biological information" as used herein, refers to spatial distribution of the targeted molecules, such as one-dimensional line, or two-dimensional or three-dimensional images, and non-imaging information, such as a simple signal intensity or local spectrum on a single location or its time dependence. [0072] As used herein, the term "bond-edited compounds" refers to compounds having one or more chemical bond that may serve as a vibrational tag for detection by Raman scattering.
Examples of chemical bond that may serve as a vibrational tag include, b t are not limi ted to, carbon-carbon triple bond, carbon-nitrogen triple bond, azide bond, carbon-deuterium bond, phenol ring, ' '(.' modified carbon-carbon triple bond, C modified carbon-nitrogen triple bond, >C modified azide bond, 1 JC modified carbon-deuterium bond, LX modified phenol sing and combinations thereof.
[0073] As used herein, the term "effective amount" refers to an amount that, when introduced into a live cell or organism, is sufficient to reach a working concentration needed for SRS imaging. The "effective amount" would vary based on the type of bond-edited compound, as well as the cells or organisms that the bond-edited compound is introduced into. In some embodiments, an "effective amount" of a bond-edited compound is the amount that is sufficient to reach an in vivo concentration of 1 uM to 100 m , 3 μΜ to 30 mM, 10 μΜ to 10 mM, 100 μΜ to 1 mM, 10 μΜ to 1 iaM or 10 μ.Μ to 100 μ in a target cell or organ, in some embodiments, an "effec tive amount" of a bond-edited compound comprising a triple bond is the amount that is sufficient to reach an in vivo concentration of 1 μΜ to 10 mM, 3 μΜ to 3 mM, 1 μΜ to 1 mM or 30 μΜ to 300 μΜ. In some embodiments, an "effective amount" of a bond-edited compound comprising a triple bond is the amount that is sufficient to reach an in vivo concentration of about 100 μΜ. h other embodiments, an "effecti ve amount" of a bond-edited compound comprising a C-D bond is the amount that is sufficient to reach an i vivo concentration of 10 Μ to 100 mM, 30 μΜ to 30 mM, 100 μΜ to 10 mM or 300 μ.Μ to 3 mM. in some embodiments, an "effective amount" of a bond-edited compound comprising a C-D bond is the amount mat is sufficient to reach an in vivo concentration of about I mM,
[0074] In some exemplary embodiments, the bond-edited compounds are small molecules. As used herein, the term "small molecules" refers to low molecular weight organic compound having a molecular weight of 1000 daltons or less. In some exemplary embodiments, the small molecules have a size on the order of 10 9 ra. Examples of small molecules include, but are not limited to, water, rihonucleosides, ribonucleotides, deoxyrtbonucleoside, deoxyribonucleotide, amino acids, peptides, choline, monosaccharides, disaccharides, fatty acids, glucose, adenosine triphosphate, adenosine diphosphate, cholesterol neurotransmitters, secondary messengers, and chemical drugs,
[0075j In some exemplary embodiments, said bond-edited compound contains one, two, three, four, five, six, seven, eight, nine, ten or more vibrational tags. The vibrational tags may be the same type of tags or a mixture of one or more different tags. [0076] in some exemplary embodiments, said vibrational tag is an alkyne tag. in other exemplary embodiments, said vibrational tag is an a ide tag. in still other exemplary embodiments, said vibrational tag is an isotope label, in a further exemplary embodiment, said isotope label is a carbon-deuterium tag. In yet still other exemplary embodiments, said vibrational tag is a combination of an alkyne tag and a carbon-deuterium tag,
[0077] in particular exemplary embodiments, said at least one vibrational tag comprises at least one vibrational tag selected from the group consisting of -C¾C-, -C=N, -N-N-N, -€=€-€=€-, -OC-ON, -C~D, and -C-C-D.
]0078] In a further exemplary embodiment, the vibrational comprises at least one L'C atom or one deuterium atom.
[0079] in some exemplary embodiments, the bond-edited compound is an amino acid,
[0080] In further exemplary embodiments, the amino acid is an essential amino acid.
[0081] In a still further exemplary embodiment, the essential amino acid is selected from the group consisting of hisiidine,, isoleueine, leucine, lysing, methionine, phenylalanine, threonine, tryptophan and valine.
[0082] In other exemplary embodiments, the bond-edited compound is a nucleoside or a nucleotide.
[0083] In still other exemplary embodiments, the bond-edited compound is a fatty acid.
[0084] in still other exemplary embodiments, the bond-edited compound is a
monosaccharide or a disaccharide. In a further exemplary embodiment, the bond-edited compound is glucose, a glucose derivative or proparg l. glucose.
[0085] In slil! other exemplary embodiments, the bond-edited compound is a pharmaceutical agent, such as an anti-cancer agent, anti-inflammatory agent, anti-bacterial agent, anti-fungal agent and anti-viral agent.
[0086] In still other exemplary embodiments, the bond-edited compound is a cytokine or chemokioe.
[0087] In some exemplary embodiments, the bond-edited compound is EU-^Cs having a molecular structure of formula 13:
Figure imgf000019_0001
10 88] In some exemplary enibodimeiits, the bond-edited compound is EdU-'^C-s. having a molecular structure of fomiular 3:
Figure imgf000020_0001
[0089] In some exemplary enibodimeiits, the bond-edited compound is EdU- C having a molecular structure of formula 2:
Figure imgf000020_0002
2
[00901 In some exemplary embodiments, the bond-edited compound is EdU-^C'5 having a molecular structure of formula 14:
Figure imgf000020_0003
[0091 j in some exemplary embodiments, the bond-edited compound is aikyne-D-giucose having a molecular structure of formula S3;
Figure imgf000021_0001
[0092] in some exemplary embodiments, the bond-edited compound is metabolized in the living cell or organism aod the vibrational tag is transfeiTed from the bond-edited compound to a down-stream metabolite of the bond-edited compound (See, e.g.. Figures 28-31).
10093] In still other exemplary embodiments, the method comprises introducing into a live cell a mixture of bond-edited compounds that imaging with Raman scattering at two or more different wavelengths, in some relaxed exemplary embodiments, the Raman scattering is SRS,
[0094) in still other exemplary embodiments, the method comprises introducing into a live cell a mixture of different bond-edited compounds that allow multiple color imaging with Raman scattering. In some related exemplary embodiments, the Raman scattering is SRS. in a particular exemplary embodiment, the mixture of different bond-edited compounds comprises Ευ- ¼, EdU-,3C and 17-ODYA.
[0095] In some exemplary embodiments, the two or more bond-edited compounds target the same cellular component but at different time period (See, e.g.. Figure 25).
[0096] In still other exemplar}' embodiments, the method comprises introducing into a living cell a mixture of different bond-edited compounds that target different cellular components,
[0097) In still other exemplary embodiments, the method comprises introducing into a living organism a mixture of different bond-edited compounds that target different types of cells in the living organism.
[0098] In still other exemplary embodiments, the method comprises introducing into a li ving organism a mixture of different bond-edited compounds carrying different vibrational tags, and detecting the different vibrational tags with Raman scattering using a linear combination algorithm. In some related exemplary embodiments, the Raman scattering is SRS.
Method tor making bond-edited compounds
[0099) Another exemplary aspect of the present, disclosure relates to a method for making a bond-edited compound.
| 00100] In one exemplary embodiment, the bond-edited compound is synthesized by the route illustrated in FIG. 1 . In another exemplary embodiment, the bond-edited compound is synthesized by the route illustrated in FIG. 20. Method of deiecting disease conditions
[00101] Another exemplary aspect of the present disclosure relates to a method for detecting a disease condition in a subject, comprising: administering to said subject a composition comprising a bond-edited compound targeting a disease tissue or pathogen, and detecting said bond-edited compound by Raman scattering.
[00102] la some exemplary embodiments, the subject is a mammal Exemplary mammal subjects for use in accordance with the methods described herein include humans, monkeys, gorillas, baboons, 200 animals and domesticated animals, such as cows, pigs, horses, rabbits, dogs, cats, goats and the like.
[001031 hi some exemplary embodiments, the disease condition is cancer.
[00104] In some exemplary embodiments, the disease condition is a neurodegenerative disease. In further exemplary embodiments, the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis, Parkinson's, Alzheimer's and Huntington's.
[00105) In some exemplary embodiments, the disease condition is an inflammatory disease.
[00106] In some exemplar>; embodiments, the disease condition is a microbial infection.
[00107) In some exemplary embodiments, the disease condition is a bacterial infection.
[00108] In some exemplary embodiments, the disease condition is a viral infection.
[00109] In some exemplary embodiments., the disease condition is a fungal infection.
[00110] In some exemplary embodiments, the pathogen comprises bacteria.
Method for monitoring treatment for a disease condition
[001.11] Another exemplary aspect of the present disclosure relates to a method for monitoring treatment for a disease condition. The method comprises administering to said subject a compositicm comprising a bond-edited compound and detecting said bond-edited compound by SRS at a first time point, further administering to said subject said composition comprising a bond-edited compound and detecting said bond-edi ted compound by Raman scattering at a second time point, and comparing images obtained at the two time points. [00112] In some exemplary embodiments, the first time point is a time point that is about or prior to the initiation of a treatment and the second time point is a time point that is after the initiation of the treatment
|00l 13] In other exemplary embodiments, the first time point and the second time point are two time points dining the course of a treatment.
S 0011 j In some exemplary embodiments, the treatment is a treatment for cancer,
[00115] In other exemplary embodiments, the treatment: is a treatment for an inflammatory disease.
j 0011 ] In other exemplary embodiments, the treatment is a treatment for a neurodegenerative disease.
Method for screening an agent
[00117] Another exemplary aspect of the present di sclosure relates to a method for screening an agent. The method comprises administering said agent and at least one bond-edited compound to a live cell or organism, detecting the bond-edited compound in the live cell or organism using Raman scattering, and selecting a candidate agent based on one or more predetermined criteria, such as the uptake, accumulation, trafficking, or degradation of the said bond-edited compound in the said live cell or organism.
[00118] In some exemplary embodiments, the candidate agent is an anti-cancer drag,
[00119} In some exemplary embodiments, the bond-edited compound is selected from the group consisting of amino acid, nucleic acid, ribonucleic acid and glucose derivatives.
[001 0] In some exemplary embodiments, the candidate agent is a skin regenerating agent.
[00121] In some exemplary embodiments, the candidate agent is a cosmetic agent.
Method for tracing a cellular process in a live cell with Raman scattering
[00122] Another exemplary aspect of the present disclosure relates to a method for tracing a cellular process in a live cell with Raman scattering. The method comprises introducing into a live cell a bond-edited compound, and following the physical movement or the chemical reaction or the biological interaction of the bond-edited compound within the cell by SRS.
[001 3] In some exemplary embodiments, the cellular processes are selected from the group consisting of DNA replication, RNA synthesis, protein synthesis, protein degradation, glucose uptake and drug uptake. Composition for labeling cells with bond-edited compounds
[00124] Another exemplary aspect of the present disclosure relates to a composition for labeling a target cell with at least one bond-edited compound. In some exemplary embodiments, the composition is a culture medium comprising at least one bond-edited compound containing at least one vibrational tag. The at least one bond-edited compound may be selected based on the type of the target cell ot a target components) within the target ceil.
[00125] in some exemplary embodiments, the culture medium comprises a plurality of amino acids, wherein over 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the amino acids are tagged with one or more vibrational tag. in other exemplary embodiments, the culture medium comprises a plurality of ami no acids, wherein ali amino acids are tagged with one or more vibrational tag.
[001.26] In some exemplary embodiments, the culture medium comprises two, three, four, five, six, seven, eight, nine, ten or more different bond-edited compounds.
Device for imaging a living cell or a living organism with bond-edited compounds
[00127] Another exemplary aspect of the present disclosure relates to a device for imaging bond-edited compounds by Raman scattering. The device comprises a first
single- wa velength laser source that produces a pulse laser beam of a first wavelength, a second single-wavelength laser source that produces a pulse laser beam of a second wavelength, a modulator that modulates either the intensity or the frequency or the phase or the- olarization or the
combination of the above of the pulse laser beam of one of the first or second laser source, a photodetector that is capable of detecting SRS or C ARS or spontaneous Raman scattering or the combination of the abo ve from a biosampie, and a computer.
[00128] In some exemplary embodiments, the energy difference between the photons produced by the first laser adiation and the photon produced by the second laser radiation matches with the energy of the vibrational transitions of the targeted vibrational tags. Photodetector of SRS detects part o all of the first laser beam or the second laser beam. The output of the photodetector (which could be a photodiode) is further processed by a Lock-in amplifier or a resonant circuit.
[00129] Another exemplary aspect of the present disclosure relates to an apparatus for providing radiation to at least one structure, comprising: a radiation providing arrangement which is configured to provide a pump radiation and a stokes radiation, each at a fixed wa velength, whose energy difference is between about 2000 and 2500 wavenumbers. 00130] In some exemplary embodiments, the radiation providing arrangement is a laser source.
[00131] The foll owing examples are put forth so as to prov ide those of ordinary skill in the art with a complete disclosure and description of how to carry out the method of the present disclosure and is not intended to limit the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade and pressure is at or near atmospheric.
EXAMPLES
Example 1: In vitro and in vivo labeling with deuterium tags
(ΘΘ132] In the examples described below, three major technical advances are being implemented, to gether with a series of biological, applications on complex tissues and mode! animals in vivo (FIG. l ). First, we optimized the chemical composition of the deuterated culture medium that achieved much higher deuterium labeling efficiency, and improved imaging sensitivity and speed of our SRS instrumentation. These optimizations allow us to demonstrate time-lapse imaging of protein synthesis dynamics within single live cells. Second, we successfully imaged protein degradation in live HeLa cells by targeting Raman peak of methyl group (C¾) for the pre-existing protein pools and then employing a recently developed linear combination algorithm on measured SRS images at 2940 cm'" and 2845 cm*1 channels. Third, inspired by the classic pulse-chase analysis of complex protein dynamics, two-color pulse-chase imaging was accomplished by rational ly dividing D-AAs into two structurally different sub-sets that exhibit resoivabie vibrational modes, a demonstra ted by tracki ng aggrega te forma tion of mutant hundngtin (mHtt) proteins. Finally, going beyond the cellular level to visualizing more complex tissues and animals in vivo, we imaged the spatial distribution of newly synthesized proteins inside live brain tissue slices and in both developmental embryonic zebrailsh and mice (FIG, lb). Taken together, these technical advances and biological applications demonstrate SRS microscopy coupled with metabolic labeling of D-AAs as a comprehensive and generally applicable imaging platform to evaluate complex protein metabolism with high sensitivity, resolution and biocompadbil y in a broad spectrum of live ceils, tissues and animals.
Exemplary Materials and Methods 00133] Stimulated aman scattering microscopy. An integrated laser (picoEMERALD with custom modification, Applied Physics & Electronics, Inc.) was used as the light source for both Pump and Stokes beams. Briefly. picoEMERALD provides an output poise train at Ϊ 064 am w th 6 ps puise width and 80 MHz repetition rate, which serves as the Stokes beam. The frequency-doubled beam at 532 am is used to synchronously Seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked puise train (the idler beam of the OPO is blocked with an tnterferometric filter) with 5- ps pulse width. The wavelength of the OPO is tunable from 720 to 990 nra, which serves as the Pump beam. The intensity of the 1 64 rim Stokes beam is modulated smusoidaliy by a built-in electro-optic modulator (EOM) at 8 MHz with a modulation depth of more than 95%. The Pump beam is spatially overlapped with the Stokes beam with a dichroic mirror inside picoEMERALD, The temporal overlap between Pump and Stokes pulse trains is ensured with a built-in delay stage and optimized by the SRS signal of pure dodecane liquid.
[00134] Pump and Stokes beams are coupled into an inverted laser-scanning microscope (FV120G PE, Olympus) optimized for near 1R throughput A 60X water objective
(UPlanAPO/lR, 1.2 N.A., Olympus) with high near 1R transmission is used for all cellular level imaging, and a 25X water objective (XLPlan N, 1.05 N.A., MP, Olympus) with both hig near IR transmission and large field of view is used for brain tissue and in vivo imaging. The Pump/Stokes beam size is matched to fill the back-aperture of the objective. The forward going Pump and Stokes beams after passing through the sample are collected in transmission with a high 'NLA. condenser lens (ail immersion, I..4 N.A., Olympus), which is aligned following Kdhl.er illumination. A telescope is then used to image the scanning mirrors onto a large area ( 10 mm by 10 mm) Si photodiode (FDS I OIO, Thoriabs) to descan beam motion during laser scanning. The photodiode is
reverse-biased by 64 V from a DC power supply to increase both the saturation threshold and response bandwidth, A high O.D. bandpass filter (890/220 CARS, Chroma Technology) is used to block the Stokes beam completely and transmit the Pump beam only. The output current of the photodiode is electronicall pre-filtered by an 8-MHz band-pass filter ( R 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low- frequency contribution due to laser scanning across the scattering sample. It is then fed into a radio frequency lock-in amplifier (HF2L1, Zurich instrument) terminated with 50 Π to demodulate the stimulated Raman loss signal experienced by the Pump beam. The R-output of the lock-in ampl ifier is fed back into the analog i nterface box
(FV 10- ANALOG) of the microscope. [00135] For HeLa ceil imaging and! brain tissue imaging, the time constant of the lock-in amplifier is set for 8 ps, and the images are acquired by a 12.5 ps pixel dwell time, corresponding to 3.3 s for a 512-by-SJ 2 pixel frame. For neurons and in vivo imaging of embryonic zebrafish and mice livers and intestines, the time constant is set to be 20 μ,δ, and the images are acquired by a 40 ps of pixel dwell time, corresponding to 10.5 s for a 5 ! 2-by-512 pixel frame. Laser powers after 60X !R objective used for cell imaging are: 100 raW for modulated Stokes beam and 1 12 raW for the Pump beam at 2133 cm*', 2000 cm"5 and 1655 cm*' channels; 50 roW for modulated Stokes beam and 56 raW for Pump beam at 2940 cm4 and 2845 cm"' channels. Laser powers after 25X objective used for tissue and in vivo imaging are: 134 niW for modulated Stokes beam: 120 niW
i l l
for the Pomp beam of 2133 cm" , 2000 cm" and 1655 era" channels; 67 mW for modulated Stokes beam and 60 mW for Pump beam at 2940 cm"' and 2845 cm"5 channels.
[00136] Metabolic incorporation of eeterated amino acids. For HeLa ceils; cells are Seeded on a covers'! ip in a petri-dish with 2 raL of regular medium for 20 h, and then replaced with D-AA .medium (or group I and group ΙΪ D-AA media) for designated amount of time. The coversltp is taken out to make an imaging chamber filled with. PBS for S S imaging. For hippocampal neurons, the dissociated neurons from newborn mice are Seeded for 1.0 days in regular Neurobasal A medium, and the replaced with the corresponding D-AA medium for designated amount of time before Imaging. For organotypic brain slice, 400 μ.ιη. thick, P10 mouse brain slices are cultured on Millicell-CM inserts (PICM03050, miUipore) in 1 mL CD-MEM culture medium for 2 h, and then change to in I mL CD-neurobasal a culture medium for another 28 h before imaging. For detailed recipe of D-AA media and in vivo labeling procedure in zebrafish and mice. (See Supporting .information). The experimental protocol or in vivo mice experiments (AC-AAAG2702) and zebrafish experiments (AC-AAAD6300) were approved by Institutional Animal Care and Use Committee at Columbia University.
[00137] Spontaneous Raman spectroscopy. The spontaneous Raman, spectra were acquired using a laser Raman spectrometer (inVia Raman microscope, anishaw) at room temperature. A.27 mW (after objective) 532 nm diode laser was used to excite the sample through a 5ΘΧ, N.A. 0.75 objective ( FLAN EPL Leica). The total data acquisition was performed during 60 seconds using the WIRE software, AO the spontaneous Raman spectra have subtracted the PBS solution as background.
(00138] Image progressing. Images are acquired with FiuoView scanning software and assigned color or overlaid by ImageJ. Linear combination was processed with Matiab. Graphs were assembled with Adobe Illustrator, [00139] Culture medium. Regular HeLa cells medium was made of 90% DM EM medium (1 1965, invitrogen), 10% FBS (10082, invitrogen) and SX penicillin strepioraycin (15140, invitrogen); regular hippocampal neuron medium was made of Neurobasal A Medium ( 10888, invitrogen), I X B27 serum free supplement (17504, Invitrogen) and 0.5 mM glutamine (25030, Invitrogen).
100140] Htt-mEos2 piasmid construct and transection. i«Htt94Q-mEos2 piasmid was constructed by replacing CFP gene sequence in pTreTight-Hrt94Q-CFP piasmid (Addgene, 23966) with mEos2 gene sequence from pRSETa-mEos2 piasmid (Addgene, 20341). For transfection ofmHtt-mEos2 piasmid in HeLa ceils, 4pg mHtt94Q-niEos2 piasmid was transfected using Transfection Reagent (FuGene, Promega).
(00141] Optimized Deuterium-Labeling Media 1) D-AA medium (CD-DMEM) for HeLa cells; adapted from regular recipe of DMEM medium (1. 1965, Invitrogen). The D-AA culture medium for HeLa cells was made with 90% CD-DMEM, 10% FBS (10082, invitrogen) and IX penicillin/streptomycin ( 15140, invitrogen).
J00142J
Amino acids components Concentration Product company
(mM) and catalo number
Giycine-dj 0.4 DLM-280, Cambridge isotope
L-Argmme-HCI-d- 0.398 DLM-54L Cambridge isotope
? . \- «i-:- h UK:-. ϊ ί ·<"..-Α: 0.2 C6727, SIGMA (regular)*
L-Gkuaminc-d; 4.0 DLM-l 826, Cambridge isotope
L-Hisiidiiie-HC!-¾0 0.2 H5659, SIGMA (regular)*
i. - isi)kuc:jne-d:!: 0...802 DIAL i 41. Cambridge isotope
L-Leociae-dso 0.802 DLM-567, Cambridge isotope
L-Lysise'HC!-ds 0.798 616214, ALD'RICH (Isotech)
L-Memionine-d;i 0,201 DLM-43 ! , Cambridge isotope
L-Phenylalanine-ds 0.4 DLM-372, Cambridge isotope
.t.:".;} i ίί.ίν- U;i 0 4
L- Threonine 0.798 T8441 , SIGMA (regular)*
L-Tryptophan 0.078 T894L SIGMA (regular)*
L-Tyrosme-di 0.398 DLM-2317, Cambridge isotope
L-Valine-ds 0.803 DLM-488, Cambridge isotope
Other components (vitamins, Inorganic Saks and glucose) are exactly the same as in the
regular DM EM medium (11965, invitrogen).
JO0143] *The reasons these 4 amino acids are remai in their regular forms are because: first, their deuterated forms have limited number of side chain deuterium and are also relatively expensive; second, their occurrence (percentage) in mammalian cell proteins are sraall. Thus the lack of the deuterated version for these 4 amino acids would not influence the general deuterium labeling efficiency for CD-DMEM, Same reason applies to below media.
[00144] 2) D-AA medium (CD- etirobasal. A) for hippocampal neuron culture and organotypic brain slices: adapted from regular recipe of Ne urobasal A medium (10888, Invitrogen). The D~A s culture medium for hippocampal neurons was made of CD-Neurobasal A Medium, Ix B27 serum free supplement (17504, Invitrogen) and 0.5 mM glntamrae-d? (DLM-l 826, Cambridge isotope). The CD~Neurobasal A culture medium for organotypic brain slices was made of CD -Neurobasal A Medium, IX B27 serum free supplement ( 17504. Invitrogen), 0.5% glucose ( 5023, invitrogen), 2 mM giutamine-ds (DLM-l 826', Cambridge isotope) and IX
penicillin/streptomycin (15140, invitrogen),
Amino acids components Concentration Product company (mM) and catalog number
Giycirte-ds 0.4 DLM-280, Cambridge isotope
L-Aianine-d.} 0.022 DLM-250. Cambridge isotope
L-Arginii 'HCi-d- 0398 DLM-541, Cambridge isotope
L-Asparagitie-da 0.006 672947 ALDRICH (Isotecb)
L~Cystein.e-2HC} 0.26 C6727, SIGMA (regular)*
..^iistidme-MCi-M-O 0.2 H5659, SIGMA (regular)*
L-isoieucifie-djo 0.802 DL - 1 1, Cambridge isotope
L-LeifCfi e-d srt 0.802 DLM-567, Cambridge isotope
L-Lysme-HCMs 0.798 616214, ALDRICH (Isotecb)
L-Methionine-dj 0.-2 1 DLM-431. Cambridge isotope
L-P heny ! a 1st. ύ ne-ds 0.4 DL -372, Cambridge isotope
\ . - Proline -ύ■ 0.067 DLM-487, Cambridge isotope
I.-Serine-d;; 0.4 DLM-582, Cambridge isotope
L-Threonme 0.798 T8441, SIGMA (regular)*
L-Ti-ypiophaa 0.078 T8941 , SIGMA (regular)*
L-TytOsme-d? 0.398 D'L -2317, Cambridge isotope
L-Va!ine-dx 0.803 ilLM-488, Cambridge isotope
Other components (vitamins, Inorganic Salts and glucose) are exactly the same as in the
regular Neorobasal A medium (10888, fevi.trogen).
[001451 3) Group 1 D-AA medium for HeLa cells. The group I D-AA culture medium for HeLa cells was made with 90% group I D-AA medium, 10% FBS (10082, mvifrogen) and IX penicillin/streptomycin (15140, mvnrogen).
Amino acids components Concentration Product company
(itiM) and catalo number
Glycine 0.4 50046, SIGMA (regular)
L-Arginiiie iiG 0.398 A6969, SIGMA (regular)
L-Cysteme-2RC1 0.2 C6727, SIGMA (regular)
L-Giutamiue 4.0 G8540, SIGMA (regular)
L iistidine-HClBj.O 0.2 H5659, SIGMA (regular)
L-isokiicine-dje 0.802 DLM-I41 , Cambridge isotope
L-Leucine-dto 0.S02 DLM-567, Cambridge isotope
L-Lysine-HCl 0.798 L8662 SIGMA (regular)
L- Methionine 0.201 M5308 SIGMA (regular)
L-Phenylalanme 0.4 P5482 SIG A (regular)
L~Serine 0.4 S43I.1 SIGMA (regular)
L- Threonine 0,798 T8441, SIGMA (regular)
L-Tryptophan 0.078 T8941 , SIGMA (regular)
L-T rosHic
L 'a!me-d¾ 0.803 DLM-488, Cambridge isotope
Other components (vitamins, inorganic Saks and glucose) are exactly the same as in the
regular DM EM medium ("1 1965. irrvitrogen).
[00146| 4) Group M D-AA medium for HeLa cells. The group li D-AA culture medium for HeLa cells was made with 90% group 0 D-AA medium, 10% FBS (1.0082, invitxogen) and I X penicilHn streptomycin (15140, invftrogen).
Ammo acids components Concentration Product company
(mM) and catalo number
Giycine-dj 0.4 DLM-280, Cambridge isotope
L-Argmme-HCl-d? 0.398 DLM-54L Cambridge isotope
L-Cysteme-2HO 0.2 C6727. SIGMA (regular)
i.-Gi t.am;ne-d 4.0 DLM-1826, Cambridge isotope
L>Histidwe-HClHj.O 0.2 H5659, SIGMA (regular)
l.-Isoleifcise 0.802 17403 SIGMA (regular)
L~Leucine 0.S02 LS 12 SIGMA (regular)
L-Lysiae-HCI-ds 0.798 616214, ALD 1CH (isotech)
L~Mefhio«ine~d:; 0.201 DLM-431 , Cam ridge isotope
L-Phenylala ne~ds 0.4 DLM-372, Cambridge isotope
L-Serine-d;; 0.4 DLM-582, Cambridge isotope
L-Threonme 0.798 T8441, S!G M A (regular)
L-Trypiophan 0.078 T89 1, SIGMA (regular)
L-Tyrosme-di 0.398 DLM-2317, Cambridge isotope
L~Vaiinc 0.803 V0513 SIGM A (regular)
Other components (vitamins, inorganic Saks and glucose) are exactly the same as in the
regular DMEM medium (1 1 65. invitrogea).
[00147| 5) D-AA medium (CD-MEM) for organotypic brain slice: adapted from regular recipe of MEM medium ( 11095, Iirvi trogen). The CD-MEM culture medium for orgaaotypi. brain slice was made ith 90% CD-MEM, 10% FBS (10082, invitrogen), 0.5% glucose (15023, invitrogen) and I X penicillin/streptomycin (15140, invi trogen).
Amino acids components Concentration Product company
(ittM) and catalo number
L-Arginme-HCl-d? 0.59? DLM-54 i , Cambridge isotope
I,-C sieioe-2HC! 0.1 C6727, SIGMA (regular)*
L-GItiiaraine-dj 2.0 DLM- i 826, Cambridge isotope
L-Histidme-HCi-H20 0.2 H5659, SIGMA (regular)*
L-isoteucine-diy 0.397 DLM-141, Cambridge isotope
L-Leucine-dic 0.397 DLM-567, Cambridge isotope
L-Lysioe-HCl-ds 0.399 616214, ALDRICH (isotech)
L-Methionine-d:i 0.1 DLM-43 L Cambridge isotope
L-Phenylalanine-ds 0.19 DLM-372, Cambridge isotope
L-Threomne 0,403 T84 1 , SIGMA (regular)*
I.,- Tryptophan 0.049 T8941 , SIGMA (regular)*
L-Tyros.rae-d2 0.1 9 DLM-23I7, Cambridge isotope
L-Valine-ds 0.393 DLM-488, Cambridge isotope
Other components (vitamins, Inorganic Salts and glucose) are exactly the same as in the
regular MEM medium (1 1 95, invitrogen).
(00148] 6) For xebrafish: Wild-type zebratlsh embryos at the l-ceii stage were irijected with 1 »L O-AA solution and allowed to develop normally for another 24 It. The zebratlsh embryos at 24 hpf were manually deehorionated before imaging. D-AA solution was made of 150 mg uniformly deuteriism-iabeied amino acid mix (20 aa) (DLM-6819, Cambridge isotope) dissolved in I niL PBS, with subsequent filtration using Mil!ipore sterile syringe Filters (0.22 μτη,
SLGV033 S).
[00149] 7) For mice: 1. Oral administration; 3-week-old mice were fed with D-AA containing drinking water for 12 days before harvesting the liver and intestine tissues. The drinking water was made of 500 mg uniformly deuterium- labeled amino acid mix (20 aa) (DLM-6819, Cambridge Isotope) dissolved in 200 ml PBS, with subsequent filtration using illipore sterile syringe Filters (0.22 μτη, SLGV033RS). 2. Intraperitoneal injection: 3 -week-old mice were injected with 500 μΐ D-AAs solution at the 0th h, 12(" h and 24te h. The tissues were then harvested at the 36* h after the first injection. D-AA solution was made of 500 mg uniformly deuterium-labeled amino acid mi (20 aa) (DLM-6819, Cambridge Isotope) dissol ved in 2 ml PBS solutions, with subsequent filtration using Millipore sterile syringe Filters (0.22 pm, SLGV033RS). Example la: Sensitivity optimization and time-lapse imaging of the de nam proteome synthesis dynamics.
[00150] The cell culture medium reported previously was prepared by supplying uniformly deuterium- labeled whole set of amino acids to a commercially available medium that is deficient of leucine, lysine and arginine (Wei L, Yu Y, Shen Y, Wang MC, Min W (2013)
Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy. Proc. Natl. Acad Set. USA, 1 10: 1 1226-1 123 i ). Due to the presence of other regular amino acids already in the commercial medium, the resulting partiaily deuteraied medium has only about 60% deuteration efficiency. In the present paper, we custom prepared new media that replace nearly all the regular amino acids by the D-AA counterparts (details in Supporting .information). As shown in the spontaneous Raman spectr (FIG. 2a), the optimized medium (spectrum 205) displays a 50% signal increase compared with the partially deuteraied medium (spectrum 210). indeed, SRS images targeting C-D vibrational peak at 2133 cm'1 confirms a 50% average intensity boost in live HeLa cells (FIG. 2 b). The use of optimized D-AA medium now leads to an about 8 times higher signal than when using a single ieucine-dw (FIG. 2a, spectrum 205 vs. spectrum 2.10). In addition to improving labeling strategy, non-trivial instrumentation optimizations are also carried out to furthe improve SRS detection sensiti ity and acquisition speed, including increasing the laser output and microscope system throughput for near-IR wa elengths, replacing the aconsto-optic modulator (AOM) with an electro-optic modulator (BOM) for a 30% higher modulation depth, and employing a high-speed lock-in amplifier for faster image acquisition.
[00151] With much-improved sensitivity, protein synthesis can now be imaged with superb spatial and temporal resolutions. Spatially, we visualized newly synthezied proteins from fine structures (likely dendritic spines, indicated by arrow heads) of live neurons (FIG. 2c). Temporally, we could readily image newly synthesized proteins in l ve HeLa cells in less tha one-hour incubation with the optimized deuteration medium (FIG. 2d). Control image with protein synthesis inhibitors only displays vague cell outlines which presumably come from the free D-AA pool
(sub-mM concentration). Moreover, using fast lock-in amplifier (details in Meth ds), our current imaging speed can be as fast as 3 s per frame (512 x 512 pixels), nearly 10 times faster than before, which enables time-lapse imaging in live ceils with minimum photo-toxicity to- cell viabilities. FIG. 2€ presents time-lapse SRS imaging of a same set of live HeLa cells gradually synthesizing new proteins over time from 10 min to 5 h incubation in optimized D-AA medium. The obvious cell migration and division prove their viability, supporting high bio-compatibility of our technique. To our best knowledge, this is the first time that long-term time-lapse imaging of proieome synthesis dynami s is demonstrated on single live mamamian cells. xample lb: SRS imaging of protein degradation in live HeLa cells,
|00152| Besides imaging protein synthesis, our imaging platform offers the abilit ' to probe protein degradation simultaneously. Experimentally, we intend, to probe the pre-existing protein pool by targeting the CH3 showing a strong peak at 2940 era- 1 , as newly synthesized proteins will be mostly carrying C-D peaked around 21.33 c.m-1. However, the 2940 cm~ l CH3 protein channel is known to suffer from undesired crosstalk from the CH2 lipid signal peaked at 2845 era- J . To obtain a clean protein component, we adopt two-color SRS imaging at both 2940 cm-l and 2845 cm-! channels followed by a linear combination algorithm which has been effectively applied in cells, tissues and animals. The subsequently obtained images show the pure distribution of old protein pools (exclusively from CHS) and the distribution of lipids (exclusively from CH2), respectively. Hence protein degradation could be tracked by imaging the old protein distributions over time when cells are growing in the D-AA medium.
[001 S3] FIG. 3a shows time -dependent SRS images of old protein distributions f CH3) in live HeLa cells when incubated with D-AAs from {) h to 96 h. Clearly, the old protein pool is degrading, as shown by the decay of its average intensity. As a contrast, the total lipid images display no obvious intensity change (FIG. 3b). In addition, the spatial patterns of old proteins (FIG. 3a) reveal a faster decay in the nucleoli than the cytoplasm. This observation is consistent with the fact that neue!eoli have active protein turnover and aslo with our previous report that C-D labeled newly synthesized proteins are more prominent in nucleoli (Wei L, Yu Y, Sheit Y, Wang MC, Mm. W (2013) Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy. Proc. Natl. Acad. Set USA. 1 10: 1.1226 -1123.1). Single exponential decay fitting of the average intensities in FIG. 3a yields a decay time constant of 45 ± 4 h (FIG. 3c), corresponding to a proteome half-live of 31 ± 3 h which is very close to the data reported by mass spectrometry (35 h) (Cambridge SB et al. (201 1) Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover. J. Proieome Res. 10:5275-5284). Therefore, our imaging platform is capabie of observing both protein synthesis and degradation b imaging at C-D channel and. C¾ channel, respectively, thus capturing proteomic metabolism dynamics in full-scope.
Retrieval of pure C l and CI¾ signals by linear combination between 2940 cm'5 and 2845 cm" ! channels was conducted employing equations follow Lu F-K et al. (2012) Multicolor stimulated Raman scattering microscopy, MoL Phys. 1 10: 1927 -1 32; and Yu Z et al. (2012) Label-free chemical imaging in vivo; three-dimensional noninvasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS). Chem. ScL 3:2646-2654. Pure CH.? signal can be retrieved as [c]priJKUS»- 5.2* (2940 cm"1 signal) -· 4.16*(2845 cm'1 signal); Pure€¾ signal can be retrieved as [c^a** l.2*(2845 cm" signal) - 0.3*(2940 cm*1 signal). This algorithm was tested with skin tissue samples, yielding similar results as reported in Lu F- et al. (2012) Multicolor stimulated Raman scattering microscopy. Mot Phys. 1 10:1927-1932; and Yu Z et al. (2012) Label-tree chemical imaging in vivo: three-dimensional non-invasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS). Chem. Set. 3:2646-2654,
Example lc: Two-color pulse-chase SRS imaging of two sets of temporally defined proteins.
[0139] inspried by the popular pulse-chase analysis in classic autoradiography techniques and recent two-color BONCAT imaging (Beatty KB, Tirrell DA (2008) Two-color labeling of temporally defined protein populations in mammalian cells. Bioorg. Med. Chem. Lett. 18:5995-5999), we aim to exploit another dimension of probing dynamic protein metabolism with two-color pulse-chase imaging of proteins labeled at different times. To do so, we need to rationally divide total D-AAs into two sub-sets with distinct Raman spectra. We reasoned thai Raman peaks of C-D stretching are closely related to their chemical environments, thus the structural difference between D-AAs should lead to diverse Raman peak positions and shapes. We then examined the spontanesous Raman spectra of each D-AA sequentially, and subsequently identified two subgroups. Group I contains three amino acids, leuiue-dlO, isoleuc.i»e~dl0 and valine~d8, structurally known, as branched-chain amino acids (FIG. 4a), All members of group I exhibits multiple distinct R m n peaks with the first one around 2067 cm- 1.
(O'I40j The rest of D-AAs without branched chains are then categorized into group II, all of which show a prominent Raman peak around 2133 cm-l (three examples shown in FIG. 4b). To test inside cells, Raman spectra of ReLa cells cultured in either group Ϊ D-AA medium only (element 405) or group S I D-AA medium only ( element 410) are shown in FIG, 4c, Based on the spectra, we choose to acquire two-color narrow-band SRS images at 2067 cm- 1 and at 21.33 cm.-! . By constructing and utilizing a linear combination algorithm,, similar to the one used for CH3 and CH2 above, pure signals of proteins labeled by group 1 D-AAs and by group II D-AAs can be suscesfuSly separated and quantitatively visualized ( e.g. FIG. 7). In other exemplary embodiments, the images are obtained with a hyper-spectral imaging approach using broadband femtosecond lasers.
(014! I We now chose the mutant huntingtin ( mi it } protein in Huntington's disease as our model system for pulse-chase imaging demonstration. It is believed that Huntington's disease is caused by a mutation from normal huntingtin gene to mHtt gene expressing aggregation-prone mHtt proteins with poly-glirtamine (polyQ) expansion (Walker FO (2007) Huntington's Disease. The Lancet. 369: 218-228). For easy visualization by fluorescence, we tagged mHtt (with 94Q) with a fluorescent protein marker, mEos2. As illustrated by the cartoon in FIG. 9d, HeLa cells were first transfected with mHtt94Q-mEos2 plasmid in regular medium for 4 h, and then replaced with group H D-AA medium for 22 h before changing to group 1 D-AA medium for another 20 h. SRS images are acquired at 2067 cm-1 and 2133 cm-'l channels, respectively, and subsequently processed with linear combination.
[0142] Fluorescence overlaid with bright field image informs us the formation of a large aggregate triggered by aggregation-prone polyQ expansion in niHtt94Q-mEos2 (FIG. 4d, fluorescence). Interestingly, proteins labeled with group H D-AAs during the initial pulse period ma inly concentrate within the core of the aggregate (FIG. 4d, element 415), whereas proteins labeled with group I D-AAs during the subsequent chase period occupy the entire volume of the aggregate (FIG. 4d, element 420). The merged image between group I and group H images, as well a the intensity profiles across the aggregate, further confirm the observation of a yellow core inside and a green shell outside ( FIG. 4d, merged). This two-color pulse-chase result suggests that the core is aggregated earlier in time and the later produced mHtt proteins are then recroited to and percolate through the aggregate to increases its overall size, in agreement with recently reported results by fluorescence (Schipper-Krom S et al. (2014)) Dynamic recruitment of active proteasomes into polyglutamrne initiated inclusion bodies. F.EBS Lett. .388; 1.51-159). The demonstration, here thus illustrates that our imaging platform using the two subgroups of D-AAs is readily applicable for performing pulse-chase imaging to probe the complex and dynamic aspects ofproteome metabolism.
10.143] in order to achieve SRS imaging of pure group i D-AA labeled protein distribution and pure group 11 D-AA labeled protein distribution simultaneously, we construct a robust linear combination algorithm to retrieve the underlying pure concentration information for two-color pulse-chasing imaging similar to the one presented above from Lu F-K et al. (2012) Multicolor stimulated Rama scattering microscopy. MoLPhys.l 10:1927-1932; and Yu Z et al, (2012) Label-tree chemical imaging in vivo: three-dimensional non-invasive microscopic observation of amphioxus notochord through stimulated Raman scattering (SRS). Chem. Sci, 3:2646-2654. Since SRS signals exhibit linear concentration dependence with analyte concentrations, two chemical species with different Raman spectra can be retrieved quantitatively with two-color SRS imaging. Hence, based on the spectra shown in FIG. 4c, we choose to acquire narrow-band SRS images at 2067 cm-1 and 21 3 cm-! channels, respectively, and perform subsequent linear combination algorithm to remove the spectral cross-talk..
0144J The proper algorithm with the corresponding cross-talk coefficients is constructed with SRS images of standard reference samples, i.e., pure group I D-AA labeled protein and pure group II D-AA labeled protein. To do so, we labeled HeLa cells with only group 1 D-AA medium FIG, 7a and only group 0 D-AA medium FIG. 7b, respectively, and acquired a set of image pairs at 2067 cm- 1 and 2133 cm-1 channels for each cell samples ( e.g. FIG. 7).
[0145J For any sample labeled with both groups of D-AAs, the measured SRS signals at 2067 cm-1 and 2133 cm-1 channels can be written as the following, with linear relationship to group 1 D-AA and group II D-AA concentrations c rou 1 and 'jgroup II) :
067 col ' signal
33 cm. ! signal
Figure imgf000038_0001
[01461 where 5
'.iLijofB' 1 ί are the average pixel intensity recorded inside cells in FIG. 7a and FIG. 7b.
[0147] Thus group I D-AA and group ΪΪ D-AA concentrations can then be easily solved as:
Figure imgf000038_0002
i „, (2133 cm"1 sisnal )~i (2067 cm" ' signal)
½a>up}l i . ~i .i
[014SJ Taking the average pixel intensity recording in FIG. 7a and FIG. 7b into the above equations, the final linear combination algorithm reads as;
|0 i49| [c'jgroup 1∞ t .06*(2067 cm- 1 signal) - G.004?*(2.!33 cm-1 signal), (1)
[0150] [cjgroup a <* (2133 cm"1 signal) - 1.., 15* (2067 cm" ' signal). (2)
Example lit: SRS imaging of newly synthesized protons i» live mouse brain tissues.
j015'i I Going above the cellular level , we now apply our imaging platform to a more complex level, organotypical brain tissues. In our study, we focus on the hippocampus because it is the key region in brains that involves extensive protein synthesis. As expected, active protein synthesis is found in the hippocampal region, particularly in the dentate gyrus, which is known for its significant role in both long-term .memory formation and adult neurogenesis. SRS image at 2133 cm~ l (FIG. s 5a, C-D) of a live mouse organotypic brain slices cultured in D-AA medium for 30 h, reveals active protein synthesis from both the soma and the neurites of individual neurons in dentate gyrus, in addition, the old protein (CP13) and total lipids (CH2) images are presented sibmtitaneous!y for multichannel analysis (FIG. 5a).
10152| In order to investigate spatial pattern of protein synthesi on a larger scale, we imaged the entire brain slice by acquiring large-area image mosaics. A 4-by-3 mm image (FIG. 5b) of another organotypic slice displays overlayed patterns from new proteins (2133 cm~ l, element 505), old proteins (CB3, element 510) and lipids (CH2, element 515), Intriguing spatial variation is observed: while the distribution of old proteins are relatively homogenous across the field of view, newly synthesized proteins are either concentrated in dentate gyrus or scattered within individual neurons throughout the cortex, suggesting high activities in these two regions. Thus, we have demonstrated the ability to directly image protein synthesis dynamics on living brain tissues with subcellular resolution and multi-channel analysis, which was difficult to achieve with other existing methods. The intricate relationship between protein synthesis and neuronal plasticity is currently under investigation on this platform.
Example le: SRS imaging of newly synthesized proteins in vivo.
[0153 J One prominent advantage of our labeling strateg is its non-toxichy and minimal invasiveness to animals. We thus mo e up to the physiological level to image protein metabolism in embryonic zebratish and mice, Zebrafish are popular model organisms due to their well-understood genetics and transparent embryos, amenable to optical imaging. We injected 1 nL D-AA solution into zebratish embryos at the t -cell stage (150 ng D-AAs per embryo), and then allowed them to develop normally for 24 h (FIG. 6a, bright field) before imaging the whole animal. We found a high signal of newly synthesized proteins (FIG, 6a, 2.133 cm- 1) in the somites at the embiyonic zebrafish tail, consistent with the earlier BONCAT result (Hinz FI, Dieterieh DC, Tirrell DA, Schuraan EM (2012) Non-canonical amino acid labeling in vivo to visualize and affinity purif newly synthesized proteins in larval zebratish. ACS Chem, Neuroses . 3:40-49), The spatial pattern of this signal appears similar to that of the old protein distribution (FIG. 6a,€113), but almost complementary to the lipid distribution (FIG. 6a, CH2).
10154] Finally we demonstrate on mammals - mice. We administered the drinking water containing D-AAs to 3- week-old mice for 1.2 days, and then harvested liver and intestine tissues for subsequent imaging. No toxicity was observed tor the fed mice. The SR.S images from both live liver tissues (FIG. 6b) and live intestine tissues (FIG. 6c) illustrate the distributions of newly synthesized proteins (2133 cm-l , C-D) during the feeding period, which resemble the total protein distribution (1655 cm-l. Amide I). On a faster incorporation timescab, live liver and intestine tissues obtained after intraperitoneal injection of D-AAs into mice for 36 h reveal spatial patterns (FIGS. 6b-6c) similar to the feeding results above as well as the click-chemistry based fluorescence staining (Liu J, Xu Y , Stoleru D, Salic A (2012) Imaging protein synthesis in cel ls and tissues with an alkyne analog of poromycin. Proc, Natl Acad. Sci. USA 09:413 -4 IS). All these results support our imaging platform as a highly suitable technique for in vivo interrogation.
[0155] FIG. 8 shows raw C-D on-resonance (2133 cm-l) and off-resonance (2000 cm-l.) SRS images of newly synthesized proteins in vivo in FIG. 6. FIG. 8a SRS C-D on-resonance and off-resonance images of a 24 hpf embryonic zebrafish. The difference image between C-D on-resonance and off-resonance (pixel-by-pixel subtraction) shows pure C-D labeled protein distribution in the somites of an embryonic zebrafish tail, as in FIG. 6a. FIGS. 8b-8c SRS C-D on-resonance and off-resonance images of live mouse liver FIG. 8b and intestine FIG. 8c tissues harvested from the mice after administering with D-AA containing drinking water for 12 days. The difference image between C-D on-resonance and off-resonance (pixel-by-pixel subtraction) shows pure C-D labeled protein distribution in the liver and intestine tissues, shown in FIG. 6b and FIG. 6c, respectively. The residual signal presented in the off-resonance images mainly comes from cross-phase modulation induced by highly scattering tissue structures.
(0156} FIG. 9 shows SRS imaging for newly synthesized proteins in viv with intraperitoneal injection of mice with D-AA solutions. FIGS. 9a-9b SRS images of live mouse liver FIG- 9a and intestine 9b tissues harvested from mice after intraperitoneal injection injected with D-AAs solutions for 36 h. 21 3 cm-l channel shows newly synthesized proteins (off-resonance image subtracted) thai resemble the distribution of total proteins as shown in the 1655 cm-l image (Amide I), (c-d) Corresponding raw C-D on-resonance (2133 cm- 1 ) and off-resonance (2000 cm- 1 ) images are shewn as references for liver c and intestine d tissues. Scale bar, 10 μη ,
Exemplary Physical Principle Of 'isotope-Based SRS Imaging
jOI57] SRS microscopy can be a molecular-contrast, highly sensitive, imaging procedure with intrinsic 3D sectioning capability. It selectively images the distribution of molecules that carry a given type of chemical bonds through resonating with the specific vibrational frequency of the targeted bonds. (See, e.g., References 47, 54 and 65). As FIG. 5a illustrates, by focusing both temporally and spatially overlapped Pump and Stokes laser pulse trains into samples, the rate of vibrational transition can be greatly amplified by about. 107 times when the energy difference of the two laser beams matches the particular chemical bond vibration, Ovih, (See, e.g.. Reference 65). Accompanying such stimulated activation of one vibrational mode, one photon can be created into the Stokes beam, and simultaneously another photon can be annihilated from the Pump beam, a process called stimulated Raman, gain and stimulated Raman loss, respectively. The energy difference between the Pump photon and the Stokes photon can be used to excite the vibrational mode, fulfilling energy conservation. FIG. 5b shows a high-frequency modulation procedure, where the intensity of the Stokes beam can be turned on and off at 10 MHz, and can be employed to achieve shot-noise-limited detection sensitivity by suppressing laser intensity fluctuations occurring at low frequencies. The transmitted Pump beam after the sample can be detected by a. large-area photodiode, and the corresponding stimulated Raman loss signal, which also occurs at 10 MHz, can be demodulated by a lock-in amplifier. B scanning across the sample with a laser-scanning microscope, a quantitative map with chemical contrast can be produced from the targeted vibrating chemical bonds. As the SRS signal ca be dependent on both Pump and Stokes laser beams, the nonlinear nature can provide a 3D optical sectioning ability.
liiiSSJ The vibrational signal of C-D can be detected as an indicator for newly synthesized proteins that metaholically incorporate deuterium-labeled amino acids. (See, e.g., FIG. 5b). When hydrogen atoms can be replaced by deuterium, the chemical and biological activities of biomolecules remain largel unmodified. The€~D stretching motion, can display a distinct vibrational frequency from all the other vibrations ofbiolosical molecules inside live cells. The reduced mass of the C-D oscillator can be increased by two folds when hydrogen can be replaced by deuterium. Based on the above Equation, Ovib can be reduced by a factor of 2. The experimentally measured stretching frequency can be shifted from -2950 cm- 1 o C-H to -2100 cm- 1 of C-D. The vibrational frequency of 2100 cm-1 can be located in a cell-silent spectral window in which no other Raman peaks exist,, thus enabling detection of exogenous C- D with both high specificity and sensitivity.
10159] Imaging optimization by metabolic incorporation of deuterium-labeled all amino acids in live HeLa cells with multicolor SRS imaging. Although leucine can he the most abundant essential amino acid, it only accounts for a small fraction of amino acids in proteins. Thus, the deuterium labeling of all the amino acids can lead to a substantial signal enhancement. Indeed, the spontaneous Raman spectrum {e.g., FIG. 33a) of HeLa cells incubated with deuterium- labeled all 20 amino acids (e.g., prepared by supplying uniformly deuterium- labeled whole set of amino acids to leucine, lysine and arginine deficient DMEM medium) can exhibit C~D vibrational peaks about five times higher than shown in FIG. 2 under the same condition. T he corresponding SRS image at 2133 cm-i (e.g., FIG. 33b) can show a significantly more pronounced signal than that in FIG. 2 under the same Intensity scale. In particular, nucleoli (e.g., indicated by arrows in FIG . 33b and verified by DIC visualization) can exhibit the highest signal. Nucleoli, the active sites for ribosomai biogenesis, have been reported to involve rapid nucleolar assembly and proteomic exchange (See, e.g.. Reference 68-70), Such fast proteio turnover can be reflected by the spatial enrichment of newly synthesized protein signals in those subcellular areas. (See, e.g.. FIG. 33b). Note that SRS imaging here can be directly performed on live cells and hence free from potential complications due to fixation and dye conjugation. Again, the off-resonant image at 2000 cm-1 can be clean and dark (e.g., FIG. 33c), proving the specificity of SRS imaging o C-D at 2133 cm-1. In addition to imaging newly synthesized proteins, SRS can readily image intrinsic bioraolecules in a label-free manner. By simply adjusting the energ difference between the Pump and the Stokes beams to match the vibrational frequency of amide I, lipids and total proteins respectively, FIGS. 33d-f show the SRS images of amide i band at 1655 cm-1 primarily attributed to proteins, CH2 stretching at 2845 era-?, predominantly for lipids and CH3 stretching at 2940 cm-1 mainly from proteins with minor contribution from lipids.
Exemplary Tinie-Bependent lie Novo Protein Synthesis And Protein Synthesis Inhibition
[0160] Being linearly dependent on analyte concentration, SRS contrast can be well suited for quantification, of de novo protein synthesis in live cells. Here the time -dependent protein synthesis images can be shown under the same intensity scale. (See, e.g., FIGS. 348a~c). As expected, the new protein signal (eg,, 2133 cm- 1 ) from 5-hour, 12-hotir and 20-hotir incubation can increase substantially over time {e.g., FIGS. 34a-c) while the amide I (e.g., 1655 cm-1 ) signal can remain at a steady state. (See, e.g., FIGS. 34d-f). Since protein distribution can often be heterogeneous in biological systems, a more quantitative representation by acquiring ratio images can be shown between the newly sy thesized proteins and the total proteome {e.g., from either amide I or CH3). FIGS. 34g-i depict the fraction of newly synthesized proteins {e.g., 2133 cm-1) among the total proteome (e.g., 1655 cm-1) and its spatial distribution. The t action of newly synthesized proteins growing with time from 5 hours to 20 hours can highlight, nucleoli as the subcellular compartments with fast protein turnover. (See, e.g.. Reference 68-70). Such quantitative ratio imaging of new versus old proteomes can be very difficult to obtain using BONCAT or mass spectroscopy without the destruction of cells. Moreover, FIG. 34j shows time-lapse SRS images of a live dividing HeLa cell after 20- hour incubation in deuterium-labeled all amino acids medium, clearly proving the viabilit of cel ls under the imaging condition.
[0161 j The effect of protein synthesis inhibition by chemical drugs can be further tested to validate that the detected C-D signal indeed derives from nascent proteins, HeLa cells incubated with deuterium-labeled ail amino acids together with 5 μΜ anisomycin, which can work as a protein synthesis inhibitor by inhibiting peptidyl transferase or the 80S ribosome system, show the absence of the C-D signal in the spontaneous Raman spectrum . (See, e.g.. FIG. 34k). Furthermore, S S imaging of the same samples (e.g., FIG. 34i) can exhibit drastically weaker signal (See, e.g.. Reference 71) when compared to FIG. 34b without the protein synthesis inhibitor. As a control, the corresponding 2940 cm-1 image (e.g.. FIG. 34m) of total proteome remains at a similar level as the non-drug treated counterpart in FIG, 34f. Thus, the detected C-D SRS signal (e.g., FIGS. 34a-c) can originate from deuterium-labeled nascent proteins, which can vanish upon adding the protein synthesis inhibitor.
Exemplary emomtrat m On HEK293T Cells And Neuron-Like Differentiabl Neuroblastoma Ν2Λ Cells
[0162] Two additional mammalian cell lines can be chosen for further demonstration: human embryonic kidney HEK293T cells, and neuron-like neuroblastoma mouse N2A cells, which can be induced to differentiate with the growt of neurites (e.g., axons and dendrites). The spontaneous Raman spectrum (e.g., FIG. 35a) of HEK293T ceils incubated with deuterium-labeled all amino acids for 12 hours can exhibit, a 2133 cm-1 C~D channel signal nearly as high as the 1655 era-! amide channel signal. He resulting SRS image can show a bright signal tor new proteins with an interne pattern residing in nucleoli, (See, e.g., FIG. 35b), As before, the off-resonant image (e.g., 2000 cm-.!) can display vanishing background (e.g. , FIG. 35c); the amide I channel (e.g. , 1655 cm-1 ) image (e.g., FIG. 35d) can exhibit consistent overall proteome distributions similar to that in HeLa cells; CH2 channel (e.g., 2845 cm-1) image (e.g., FIG. 35e) depicts a more diffusive lipid distribution in cytoplasm compared to that in HeLa cells. Consistent with the results obtained in HeLa ceils above, the ratio image (e.g., FIG. 35f) between the newly synthesized proteins (e.g., FIG. 35b) and the total proteins (e.g., FIG. 35d) highlight nucleoli for active protein turnover in HEK293T cells as well, (See, e.g.. References 44-46),
[0163J In addition to showing the ability to image newly synthesized proteins inside cell body, the exemplary SRS can also be applied to tackle more complex problems, such as de novo protein synthesis in neuronal systems. (See, e.g.. Reference 26-28). Under differentiation condition,
4.1 N2A cells massively grow new neurites from cell bodies and form connections with other cells. FIG. 10a shows the image of newly synthesized proteins after induction for differentiation, by simultaneously differentiating the N2.A ceils and supplying with the deuterium labeled all amino acids for 24 hours. Similar to HeLa and HEK293I ceils, N2 A ceil bodies can be observed to display high-level protein synthesis. Newly synthesized proteins can also be observed in a subset of, but not all neurites (e.g., Figs 36a and 36b), which can imply that the observed neurites in FIG. 360a can be newly grown under the differentiation condition. For a detailed visualization, FIGS, 36c and lOd show the zoomed-in regions in the dashed squares in FIGS. 36a and 36b respectively. A. more corapreheosive examination is illustrated by both the ratio image (e.g., FIG. 36e) between FIGS. 36c and 36d and the merged image (e.g., FIG. 36f) with 3605 designating new protein signal from FIG. 36c and 3 10 designating total protein signal from FIG. 36d, On one hand, both the ratio image and the merged image highlight the neurites with higher percentage of new proteins (e.g., indicated by stars), implying these neurites can be newly grown. On the other hand, from the merged image, there can be some neurites (e.g., indicated by arrows) showing obvious signals in the green channel (e.g., total proteins) only but with no detectable signal in the red channel (e.g., new proteins).
[0164] Hence, the neurites indicated by arrows can be most likely older than their starred counterparts, in addition, the transition f ra 3610 to 3605 in the merged image (e.g., FIG. 360f) can imply the growth direction by which new neurites form and grow. A more relevant system to study de novo protein synthesis and neuronal activities can be hippocampal neurons, which can be known to 'be involved in long-term memory formation (See, e.g., Reference 26-28). SRS image (e.g., 2133 cm- ! ) of hippocampal neuron cells incubated with deuterium- labeled all amino acids ca show a newly synthesized protein pattern in the neurites.
Example 2: In vitro an in vivo labeling with a!kyne tags
Exemplary Methods ami materials
[ul65{ Bond-selective stimulated Raman scattering (SRS) microscopy. FIG. 37b shows details of the microscopy setup. An integrated laser system (picoEMERALD, Applied Physics & Electronics, Inc.) was chosen as the light source for both pomp and Stokes beams. Briefly, picoEMERALD provides an output pulse train at 1064 nm with 6 ps pulse width and 80 MHz repetition rate, which serves as the Stokes beam. The frequency doubled beam at 532 nm is used to synchronously Seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train with 5-6 ps pulse width (the idler beam of the OPO is blocked with an raterferoraetric filter). The output wavelength of the OPO is tunable from 720 to 990 nm, which serves as the pump beam. The intensity of the 1064 nm Siokes beam is modulated simisoidally by a built-in electro-optic modulator at 8 MHz with a modulation depth of more than 95%. The pump beam is then spatially overlapped with the Stokes beam by using a dtchroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulse trains is ensured with a built-in delay stage and optimized by the SRS signal of pure dodeeane liquid at the microscope.
[0.166] Pump and Stokes beams are coupled into an inverted multiphoton laser-scanning microscope (FVT200MPE, Olympus) optimized for oear-IR throughput. A 60 water objective (UPkmAPO/lR, 1.2 N.A., Olympus) with high tiear-lR. transmission is used for all cell imaging. The pump/Stokes beam size is matched to fill the back-aperture of the objective. The forward going pump and Stokes beams after passing through the sample are collected in transmission with a high N.A. condenser lens (oil immersion, 1.4 N.A., Olympus) which is aligned following K ihler illumination. A telescope is then used to image the scanning mirrors onto a large area (10 by 10 mm) Si photodiode (FDS 1.010. Thorlabs)to desca beam motion during laser scanning. The photodiode is reverse biased by 64 V from a DC power supply to increase both the saturation threshold and response bandwidth. A high O. D. bandpass filter (890/220 CARS, Chroma Technology) is placed in front of the photodiode to block the Stokes beam completely and to transmit the pomp beam only .
[0167] The output current of the photodiode is electronically pre- filtered by an 8-MHz band-pass filter (KR 2724, KR electronics) to suppress both the 80 MHz laser pulsing and the low-frequency fluctuations due to laser scanning cross the scattering sample. It is then fed into a radio frequency lock-in amplifier (SR844, Stanford Research Systems) terminated with 50 Ω to demodulate the stimulated Raman loss signal experienced by the pump beam. The m-phase X-outpn of the lock-in amplifier is fed back into the analog interface box (F V 10- ANALOG) of the microscope. The time constant is set for 10 us (the shortest available with no additional filter applied). The current SRS imaging speed is limited by the shortest time constant available from the lock-in amplifier (SR844). For all imaging, 512 by 512 pixels are acquired for one frame with a 100 ps of pixel dwell time (26 s per frame) for laser scanning and 10 ps of time constant from the lock-in amplifier. Laser powers after 60X IR objective used for imaging are; 130 itiW for modulated Stokes beam; 1.20 mW for the pump beam in 2133 cm"1, 2142 cm'*, 2000 cm*1 and 1655 cm"' channels, 85 mW for the pump beam in 2230 cm'1 and 2300 cm"1 channels, and 50 mW for pump beam in 2845 cnT! channels.
[0168] Spontaneous Raman Spectroscopy. The spontaneous Raman spectra were acquired using a laser confocal Raman microscope (Xplora, Horiba Jobin Yvon) at room temperature. A 12 in W (after the microscope objective), 532 nm diode laser was used to excite the sample through 50X, N.A.:::0.75 air objective (MPlan N, Olympus). The iota! data acquisition time was 300 s using the LabSpec 6 software. All the spontaneous Raman spectra have subtracted the PBS solution background.
101691 Materials. 5-Ethynyi-2 -deoxyuridme (EdU) (Τ5Π285), 17-Octadecynoic acid (17-ODYA) (08382), DMEM medium without L-methionme, L-cystine and L-glutamme (D0422), L-methionine (M53Q8), L-cystine (C7602), 2-Mereaptoeilmnol (M3 I48) and Phorbol 12-tnyrisiate 13-acetate (PI 585) were purchased from Sigma-Aldrich. 5-Ethynyl Uridine (EU) (B- 10345), eomoproparg [glycine (Hpg) (CIO 186), Alexa Fluor® 488 Azide (A 10266), Click-iT® Cell Reaction Buffer Kit (C 10269), DMEM medium ( 1 1965), FBS (10082), penkillin/streptomycin (15140), L-glutamiae (25030), Neurobasai A Medium (1.0888) and B27 supplement (17504) were purchased front Iiiviirogen. RPMI-1640 Medium (30-2001 ) was purchased from ATCC. BCS (hyclone SH30072) was purchased from Fisher Scientific..
10170} DMEM culture medium was made by adding 10% (vol/vol) FBS and 1% (vol/vol) per«cilIiu¾treptomycin to the DMEM medium. Methiomne-deficient culture medium was made by supplying 4 mM L-glutamine, 0.2 mM L-cystine, 1 % FBS and 1% penicillin/streptomycin to the DMEM medium without L-methionine, L-cystine and L-glufamine. RPMI-1640 culture medium was made of supplying the RPMI-1640 medium with 10% FBS, 1% petucillin streptomyciti and 50 μΜ 2-Mercaptoethanol. Neuron culture medium was made of Neurobasai A Medium adding with IX B27 supplement and 0.5 mM glutamine. Culture medium for NIH3T3 cells was made by adding 10% (vol/vol) BCS and 1 % (vol/vol) penicillin/streptomycin to the DMEM medium.
10171 } Propargykhoiine synthesis. Propargylcholine was synthesized according to Jao, C, Y., Roth, M., Welti, R. & Salic, A. Free, Nad. Acad Set USA 106, 15332-15337 (2009). 3 mL propargyl bromide (80 wt. % solution in toluene) were added dropwise to 3 g 2-dimethylaminoeth.anol in 10 mL anhydrous THF on ice under argon gas protection and stirring. The ice bath was removed and the mixture was kept stirring at room temperature overnight. The white solids were filtered the next day and washed extensively with cold anhydrous THF to obtain 5 g pure propargylcholine bromide. All chemicals here are purchased from Sigma-Aldrich. NMR spectrum was recorded on a Bruker 400 (400MHz) Fourier Transform (FT) NMR spectrometers at the Columbia University Chemistr Department. Ή NMR spectra are tabulated i the following order; multiplicity (s, singlet; ά, doublet; t, triplet; m, multiplet), number of protons. *H NMR (400 MHz, D¾0) δ ppm: 4.37 (d, J 2.4 Hz, 211); 4.10 (m. 211); 3.66 (t, J™ 4.8 Hz, 21 i); 3.28 (s, 6H); MS (APQ+) m/z CaScd. for C?HWNO [M : 128.19. Found: 128.26.
[01721 Sample preparation for SRS imaging of live cells and organisms. For all SRS imaging experiments of HeLa cells ( e.g. FIG. .12), cells were first Seeded on coversiips with a density of 1 x 10'/mL in petri. dishes with 2 mL DMEM culture medium for 20 h at 37 °C and 5% CO?.
1) EdU experiment, DMEM culture medium was then changed to DMEM medium
(FBS-firee) for 24 b for cell cycle synchronization. After synchronization., medium was replaced back to DMEM culture medium and EdU (10 niM stock in PBS) was smiultaneously added to a concentration of 100 μ.Μ for I S h.
2) EU experiment, EU (100 mM stock in PBS) was added to the DMEM culture medium directly to a concentration of 2 mM for 7 h.
3) Hpg experiment, DMEM culture medium was then changed to methionme-deficient culture medium for 1 h, followed by supplying 2 mM Hpg (200 mM stock in PBS) in the medium for 24 h.
4) Propargylcholine and EdU dual-color experiment, DMEM culture medium was changed to DMEM medium (FBS-free) for synchronization.. After syitchrot»2ation5 medium was replaced back to DMEM culture, medium by simultaneously adding both propargylcholine (25 mM stock in PBS) and EdU (10 mM stock in PBS) to the culture medium to a concentration of 1 mM and 100 μΜ, respectively, fo 24 h.
10173] For the propargylcholine experiment in neurons, bippocampal neurons were cultured on coversiips in 1 ml neuron culture medium for 14 d, and then propargylcholine (25 mM stock in PBS) is directly added into the medium to a final concentration of 1 raM for 24 h.
j0!74] For the 17-ODYA experiment in macrophages, ΊΉΡ-Ι cells were first Seeded on coversiips at a density of 2 l05/mL in 2 ml RPMi-1640 culture medium for 24 fa, followed by 72 fa induction of differentiation to macrophages by incubating with 100 og/rai PhorboJ 12-rayristate 1.3-acetate (PMA) in the medium. Medium was then replaced with RPMI-1640 culture medium containing 400 μ.Μ 17-ODYA (6: 1 complexed to BSA) for 15 h.
[0175] For ail of the above experiments, after incubation, the coverslip is taken out to make an imaging chamber filled with PBS for SRS imaging.
[0176] For the 17-ODYA experiment in C. elegans, OP50 bacterial culture was mixed well with 4 mM 1 7-ODYA (from 100 niM etbanol stock solution), and then Seeded onto nematode growth media ( GM) plates. After drying the plates in hood, wild type N2 day 1 adult C. elegans were placed ont the plates and fed for 40 h. C. elegans were then mounted on 2% agarose pads containing 0.1% aSMj as anesthetic on glass microscope slides for SRS imaging.
[0177] SRS imaging of elegans germline after feeding with EdU. MG1 93 (thymidine defective MG 1655) £. C li strain was cultured in 2 ml LB medium at 37 °C overnight, and transferred to 100 mi of M9 medium containing 400 μΜ EdU for farther growth at 37 °C for 24 h. The EdU~lafaeied MGI693 E. Colt was then Seeded on M agar plate. Synchronized day 1 adult worms developed in 20 °C were transferred to EdU-laheled bacterial plate for 3 h, and then were dissected to take out the genu line for imaging (e.g. FIG. 1.3).
[0178J Cell preparation for click chemistry-based fluorescence microscopy. All experiments (e.g. FIG, 5) were carried out following the manufacturer's protocol f om Invitmgen. HeLa cells were first incubated with 10 μΜ EdU in DMEM culture medium for 24 h, or 1 mM EU in DMEM culture medium for 20 h, or 1 mM Hpg in memionine-deficient culture medium for 20h, respectively. Cells were then fixed in 4% PFA. for 15 min, washed twice with 3% BSA in PBS, permeabil ized with 0.5% Triton PBS solution for 20 min, and performed dick chemistry staining using Alexa Fluor 488 Azide in the Click- ΓΓ Cell Reaction Buffer Kit for 30 min. After washing with 3% BSA in PBS for three times, fluorescence images were obtained using an Olympus FV1200 ccmfocai microscope with 488nm laser excitation while the ceils were immersed in PBS solution. j0!79] Enzymatic assays confirming propargykhoSine incorporation into cellular choline phospholipids. We design our control experiments according to the click chemistry based assays reported in Jao, C, Y., Roth, M. , Welti, R. SL Salic, A. Proc. Natl. Acad ScL USA. 1.06, 15332-15337 (2009) (e.g. FIG. 16). N1H 3T3 cells cultured with 0.5 DM propargykholine for 48 hours were fixed with 4 % PFA for 15 minutes, .rinsed with 1 niL TBS buffer twice and incubated with 1 mL 1 rag/mL BSA in TBS buffer for I hour at 37 °C5 with or without 0.02 U/mL phosphohpase C (Type XIV from Clostridium fxsrfrmgens, Sigma), in the presence of 10 mM CaCi? (required for pliosphoiipase C activity .) (e.g. FIG, 16b) or 10 mM ED'FA (e.g. FIG. 16c). The ceils were then washed with TBS buffer and ready for SRS imaging,
[0180] Sample preparation for drug delivery into mouse ear tissues. Either DMSO solution or Drug cream (Lamisii, Novartis) containing 1% (w/w) active terbinafme hydrochloride (Tf'I) was applied to the ears of an anesthetized live mouse (2-3 weeks old white mouse of either sex) for 30 min, and the dissected ears from the sacrificed mouse were then imaged by SRS ( e.g. FIGS. 17 and 18). The amide ( 1 55 cm"1 ) and lipid (2845 cm*1) images have been applied with linear spectral unmixing to eliminate cross talk before composition. The experiments! protocol for drug delivery on mice (AC-AA AG4703) was approved by Institutional. Animal Care and Use Committee at Columbia University,
[0181 image progressing, images are acquired with FluoView scanning software and assigned color or overlaid by Image.!. Graphs were assembled with Adobe Illustrator. Example 2a; A!kyne Tags
[0182] As an effective imaging modality for small biomolecules, we report a general strategy of using stimulated Raman, scattering (SRS) microscopy to image alkynes (i.e., OC) as nonlinear vibrational tags, shown as bond-selective SRS in FIGS. Oa-c. As shown in FIG. 10a, Spontaneous Raman spectra of BeLa cells and 10 mM EdlJ solution, inset: the calculated SRS excitation profile (FWH 6 cm'1, element 905) is well fitted within the 2125 cm"! alkyne peak (FWHM 14 cm"1, magenta). FIG. 10b shows linear dependence of stimulated Raman loss signals (2125 cm") with EdlJ concentrations under a 100 .δ acquisition time. FIG. 10c shows the metabolic incorporation scheme for a broad spectrum of alkyne-tagged small precursors. a.o. arbitrary units. Alkynes possess desirable chemical and spectroscopic features. Chemically, they are small (only two atoms), exogenous (nearly non-existent inside cells), and bioorthogonal (inert to reactions with endogenous biomoiecules). These properties render alkynes key players in bioorthogonal chemistry, in which precursors labeled with alkyne tags form covaient bonds with azides fused to probes such as fiuorophores for detection. However, such a 'click-chemistry' approach prohibits live imaging, as it usually involves a copper-catalyzed reaction that requires cell fixation, while the copper-free version has slow kinetics and high background. Spectroseopically, the C==C stretching motion exhibits a substantial change of poiarizability, displaying a sharp Raman peak around 2125 cm"5, which lies m a desirable cell-silent spectral region* ' (FIG, 10a). Compared to the popular carbon-deuterium (C-D) Raman tag, alkynes produce about 40 times higher peaks. However the signal is still relatively weak and extremely long acquisition times (- 49 min per frame consisting of 1.27x127 pixels) limit dynamic imaging in live systems.
[0183] The coupling of SRS microscopy to alkyne tags that we report offers sensitivity, specificity and biocompatibility for probing complex living systems. When the energy difference between incident photons from two lasers (pump and Stokes) matches with the 2125 cm-1 mode of alkyne vibrations, their joint action wi ll greatly accelerate the vibrational excitation of alkyne bonds. As a result of energy exchange between the input photons and alkynes, the output pump and Stokes beams will experience intensity loss and gain, respectively. Such intensity changes measured by SRS microscopy generate concentration-dependent alkyne distributions in three-dimensions (3D). FIG. 1 1 is an illustration showing a Pump beam (pulsed, pico-second) and an intensity-modulated Stokes beam (pulsed, pico-second) are both temporally and spatially synchronized before focused onto cells that have been metaboHcally labeled with alkyne-tagged small molecules of interest. When the energy difference between the Pump photon and the Stokes photon matches the vibrational f equency (Ovib) of alkyne bonds, alkyne bonds are efficiently driven from their vibrational ground state to their vibrational excited state, passing through a virtual state. For each excited alkyne bond, a photon in the Pump beam is annihilated (Raman loss) and a photon in the Stokes beam is created (Raman gain). The detected pump laser intensity changes through a lock-in amplifier targeted at the same frequency as the modulation of Stokes beam serve as the contrast for alkyne distributions.
[0184] SRS microscopy offers a number of advantages. First, SRS boosts vibrational excitation by a factor of 107, rendering a quantum leap of sensitivity (i.e., detectabi!ity and speed) over spontaneous Raman. Second, we use a 6-ps ulse width to match the excitation profile of alkyne (e.g. PK! 1.0a), assuring efficient and selective nonlinear excitation. Third, SRS is free of background, whereas spontaneous Raman suffers from auto-fluorescence and coherent anti-stokes Raman scattering (CARS) suffers from non-resonant backgrounds. Finally, we employ near-tnfrared laser wavelengths for enhanced tissue penetration, intrinsic 3D sectioning (due to nonlinear excitation) and minimal photo-to.xicity.
[0185) We first detected the aikyne-tagged thymidine analogue 5-ethynyS~2 -deoxyuridine (EdU) in sol tion (e.g. FIG. 10b). Under a fast imaging speed of 100 μ$_ we determined its detection limit to be 200 μΜ, corresponding to 12,000 alkynes within the laser focus. This approaches the shot-noise limit (Δίρ Ιρ - 2x1 -7) of the pump beam, which represents the maximum theoretical sensitivity of the system. To explore the general applicability of our approach, we went on to examine a broad spectrum of small biomolecules including aikyne-tagged deoxyribonucleoside, ribonucleoside, amino acid, choline and fatty acid (FIG. 10c). whose metabolic incorporation has been thoroughl tested in bioorthogonal chemistry studies.
[0186] We imaged the metabolic uptake of EdU during de novo DNA synthesis. HeLa cells grown in media with EdU show a sharp Raman peak at 2125 cm-! in the cell-silent region (e.g. FIG. 12a). Live-cell SRS imaging revealed EdU incorporation into the newly synthesized genomes of dividing cells (e.g. FIG. 12b, a!kyne-on). Off-resonance images of the same cells, taken when the energy difference between pump and Stokes photons does not match vibrational peaks (alkyne-off), are background-free, confirming the purely chemical contrast of SRS. No EdU signal shows up in cells treated with the DNA synthesis inhibitor hydroxyurea, whereas lipids imaged at 2845 cm-! verify that these cells ate normal based on morphology. Moreover, we tracked dividing cells every 5 min during mitosis (e.g. FIG. 12c), demonstrating acquisition speed and compatibility with live dynamics that are nearly impossible with spontaneous Raman. We also showed thai our method is applicable to multicellular organisms. Actively proliferating celis can be clearly distinguished in. C. elegans grown in the presence of EdU as exemplified in FIG. 13, where the composite image shows both the protein derived 1655 cm- 1 (amide) signal from all the germ cells, and. the direct visualization of alkynes (2125 cm-1 (EdU)) highlighting the proliferating germ cells. White circles show examples of EdU positive germ cells in the mitotic region of C, elegans germline. Scale bar, 5 μηι.
I'M 87] Next, we studied RNA transcription and turnover using the alkyne-tagged uridine analogue, 5-ethynyl uridine (EU)8 in HeLa cells (e.g. FIG, .12a). The alkyne-on image (e„g, FIG. 12d) reveals localized EU inside the nucleus with higher abundance in the nucleoli, which are major compartments of .r NA-rich ribosomal assembly, and nearly disappears in the presence of the RNA synthesis inhibitor actinomycm D. Turnover dynamics are further demonstrated by pulse-chase SKS imaging (FIG. I 2e), which indicates a short nuclear RNA lifetime (-3 h) in live HeLa cells.
[0188] Ma y intricate biological processes such as long-term memory require protein synthesis in a spatiotemporal dependent manner. We imaged L-Homopropargylglycine (Hpg), an alkyne-tagged analogue of methionine, to visualize newly synthesized proteomes. HeLa ceils grown in methmnine-deficient media supplemented with Hpg display an alkyne peak (e.g. FIG. 12a) about 20 times lower than that of 10 mM EdU solution (e.g. FIG. 10a). The corresponding alkyne-on image (e.g. FIG. 12f) shows the distribution of newly synthesized proteins with spatial enrichment in the nucleoli (arrow indicated), which experience rapid protein exchange. Similar to EdU in solution, the detection limit of alkynes in mammalian cells approaches 200 μΜ (with 100 ps pixel, dwell, time) based on an average signal-to-noise ratio of 2 as we obtained in HeLa cells. The Hpg signal is well retained in fixed cells (e.g. FIG. 1.4), indicating little contribution from freely diffusing Hpg. The alkyne-on image displays the Hpg distribution for the newly synthesized proteins. For the same set of cells, the off-resonant (alkyne-off) image shows vanishing signal, and the amide image shows total protein distribution. This result confirms that the detected signal is not from freely diffusive precursor Hpg itself (which is eliminated during the fixatio process). Scale bar, 10 μηχ Furthermore, adding methionine, which has a 500-fold -faster incorporation rate, to compete with Hpg causes the signal to disappear (e.g. FIG. 12f), Note that we verified the spatial patterns of EdU, EU and Hpg incorporation in live cells by performing click chemistry on fixed cells, with FIGS. 1.5A-C showing the fluorescence images of HeLa cells incorporated with a, EdU (for DMA); b, EU (for RNA); c, Hpg (for protein). Scale bars, 10 pm.
[0189] Lipid metabolism is critical for many functions in healthy and diseased tissues, but few non-per turbative tags are available to monitor lipids in the ceil. We thus moni tored the metabolic incorporation of alkyne-tagged choline and fatty acids. HippocampaS neurons grown on propargylcholine present a clear 21 2 cm- 1 Raman peak (e.g. FIG. 12a). Such a frequency shift from 2125 cm- 1 is due to the positive charge on the nitrogen near the alkyne (FIG. 10c). As revealed by enzymatic assays (e.g. FIGS. 16a- 1.6c), the alkyne-on signal (FIG. 12g) mainly originates from newly synthesized choline phospholipids at membranes. To label tatty acids, we incubated 17-octadecynoic acid (17-ODYA) wit THP-l macrophages, which actively scavenge cholesterol and .fatty acids. In FIG. 1 a, fixed M1H3T3 cells are seen after culturing with 0.5 mM propargylcholme for 48 hours. The alkyne-on image shows alkyne-tagged choline distribution, in FIG, 16b, treatment of fixed IH3T3 cells with phospholipase C, which removes Choline head groups of phospholipids only in the presence of calcium. The alkyne-on image shows the strong decrease of incorporated propargylcholine signal, supporting its main incorporation into membrane phospholipids. For FIG. 16c, treatment of fixed NIM3T3 cells with phospholipase C in the presence of EDTA (chelating calcium). Propargylcholine signal is retained in the alkyne-on image. For FIGS . 1.6a-c: in the same set of cells as in alkyne-on images, the alkyn.e-o.iT images show a clear background. The amide images display total protein distribtuion. Scale bars, 10 μηι. The alkyne-on image (FIG. Ϊ 2h) depicts the formation of numerous lipid droplets that indicates transtormation into foam cells, a hallmark of early atherosclerosis. Multicellular organisms are also capable of taking up 17-ODYA for lipid imaging. New fatty acids in C. elegans appeared mainly inside lipid droplets upon SRS imaging, known to exist largely in the form of triglycerides (e.g. FIG. 12t). Such a fat accumulation process could serve as a useful model for studying obesity and diabetes. We were also able to perform dual-color imaging of propargylcholine (2142 cni- 1) and EdU (2125 cra-1) incorporation due to the spectral sharpness a d separation of their two alkyne peaks (FIG. 12j).
[0190] Finally, we tracked alkyne-bearing drug delivery (FIGS. 17a-e and 18a-b) in animal tissues by taking advantage of the intrinsic 3D sectioning property of SRS.
(0191 } FIG. 17a depicts Rama spectra of a drug cream. Lamisil, containing 1% TH and mouse ear skin tissue. FIGS. 17b-e illustrate SRS imaging of tissue layers from stra tum corneum (z ~ 4 pm) to viable epidermis (z ~ 24 μηι), sebaceous gland (z ~ 48 μηι) and subcutaneous fat (z ~ 88 μηι). To facilitate tissue penetration, DM SO solution containing I ¾ TH was applied onto the ears of an anesthetized live mouse for 30 mm and the dissected ears are imaged afterwards. For all 4 layers shown in FIGS. 17b-e, alkyne-on images display TH penetration; alkyne-off images show off-resonant background (The bright spots in d are due to two-photon absorption of red blood cells).. The composite images show protein ( 1655 cm- 1) and lipid (2845 cm-!) distributions. Scale bars, 20 p.m. a.u,::::arbitrar units.
[0.192] FIGS, 1 Sa- show SRS imaging of the viable epidermis layer (z :::: 20 pm) and the sebaceous gland layer (sr. :::: 40 prn). For both a and b: the alkyne-on images display the TH penetration into mouse ear tissues through lipid phase. The composite images show both protein (1655 cm- 1) and lipid (2845 cm-i) distributions. Scale bars, 20 pm. [0193] Unlike bulky ftuorophores, alkvnes have little perturbation to pharmacokinetics and are common moieties in many pharmaceuticals. We chose terbkiafine hydrochloride (TH), a US Federal Drug Administration approved aUcyne-bearing antifungal skin drug, and imaged its drug delivery pathways inside mouse ear tissue to a depth of about 100 pan by targeting its internal alkyne at 2230 cm-1. TH images captured at various depths ail exhibit patterns that highly resemble lipid distributions but not protein distributions, suggesting that TH penetrates into tissues through the lipid phase, consistent with its lipophilic nature. Our technique should be applicable to tracking other drags after proper alkyne derivatization,
[01.94] in conclusion, we report a general strategy to image small and biologically vital molecules live ceils by coupling SRS microscopy with alkyne vibrational tags. The major advantages of SRS He in the superior sensitivity, specificity and compatibil ity with dynamics of live cells and animals. SRS imaging of alkynes may do for small hiomoleeules what fiuoresceoce imaging of fluorophores has done for larger species.
Example 3: Synthesis of bond-edited compounds
101951 A. Synthesis of Alkytie-D-G hi cose
Figure imgf000053_0001
(0196) Sodium hydride (138 mg, 5.8 mmol) was added to a solution of l ,:2:5 >-Di-0-isopropyIidene- -D-giucofuranose (Compound SI, 500 mg, 1.92 mmol, Aldrich D7600) in 10 niL dry D F at 0 °C. The solution was stirred at 0 °C for 30 min. before propargyS bromide (80% in toluene, 0.43 mL, 3.84 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 12 h before quenched with saturated ammonium chloride solution ( 10 mL). The mixture was extracted with ethyl acetate (2 x 25 niL), and the organic layer was combined- dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography on silica gel (0-50% Ethyl acetate in Hexanes) to give Compound S2 (518 mg, 90%) as a colorless oil. The }H NMR spectrum is in accordance with previously published values (A, Hansherr et a!., S nthesis, 2001, 1377).
10197] i l l NMR (400 MHz, CDC13) ό 5.88 (d, J - 3,6 Hz, 1 4.30 - 4,24 (m, 3 B), 4.14 (dd, J - 7.6, 2.8 Hz, 1 H), 4.11 - 4.06 (m, 2 E), 3.99 (dd, J - 8.8, 5.6 Ez, 1 H), 2.47 (t, J - 2.4 Hz, 1 H),
5.1 .50 (s, 3 B), 1.42 (s, 3 M), 1 .35 (s, 3 H), 1.31 (s, 3 H).
[0198] HRMS (FAB-i-) m/z Calcd. for CJSHBC¼ +B]": 299.1495. Found: 299.1496
Figure imgf000054_0001
[0199] Water (10 nil} and Dowex® 50WX.8 hydrogen form (600 rng, Sigma-Aldrich 21751 ) were added to Compound S2 (594.rag, i .99 ramol). The mixture was heated to 80 ®C for 20 h before filtered. The filtrate was concentrated in vacuo to give Compound S3 (416 mg, 1.91 ramol, 96%) as a white solid.
[0200] I B NMR (400 MHz, ¾0) 6 5.13 (d, J- 3.6 Hz, 1 H), 4.44 (d, 2.4 'Hz, 2 H), 3.79 -·■ 3.73 (m, 2 H), 3.70 - 3.61 (ra, 2 H), 3.51 (dd, J- 9.8, 3.8 Hz 1 H), 3.40 (t, J =:: 9.6 Hz, 1 H), 2.82 (s, 1 H). ,?C NMR (10 j MHz, D20) 6 92.1 , 80.8, 79.8, 75.9, 71.4, 71.2, 69.2, 60.4, 59.9.
102011 HRMS (FAB-i) m/z Calcd. for C¾FiwC¾Na [M+Naf: 241 .0688. Found: 241.0683 |0202] B. Synthesis of EdU-^C (Compound 2) and EdU-^CzfCompoimd 3)
Figure imgf000054_0002
Synthesis of Compound 4:
Figure imgf000055_0001
[0203] To a solution of 5-iodo-2'-deoxyuridine (Compound 1, 150 mg, 0.42 mmoi) in 1.5 ml of pyridrae was added 0.4 ml (0.42 mmoi) acetic anhydride at 0 °C. The resulting mixture was wanned up to room temperature and stirred for 4 h, then poured into 5 ml of cold i N NaHSC^ and extracted with ethyl acetate three times. The organic layer was washed with saturated NaHCO j and brine, dried over anbydrous NasSCu and concentrated. The crude product was purified by column chromatography on silica gel (0-70% Ethyl acetate in Hexanes) to give Compound. (157.3 mg, 0.36 mmoi, 85%) as a white solid.
[0204] Ή MR (400 MHz, CD(%) 5 ppm: 8.46 (s, 1 H), 7.97 (s, 1 H), 6.28 (dd, J - 8.2, 5.7 Hz, 1 H), 5.27 - 5.19 (m, 1 H), 4.41 (dd, J - 12.3, 3.2 Hz, 1 H), 4.34 (dd, J™ 12.3, 2.9 Hz, 1 H), 4.30 (q, J= 2.9 Hz, 1 H), 2,54 (ddd, J- 14.3, 5.7, 2.1 Hz, 1 H), 2,2 Ϊ (s, 3 H), 2.20 - 2.13 (m, 1 H), 2.12 (s, 3 11).
[0205] MS (APCI+) m z Calcd. for Cur½IN20? [ +Hf : 439.0. Found: 438.8
Syn thesis of Compound 5:
[0206]
Figure imgf000055_0002
[0207] To an oven-dried vial was added Compound 4 (72 mg, 164 pmol), Pd (OAc (3.6 mg, 16 μηιοΐ), PPh-3 (8.6 mg, 33 μτηοί), Cul (3.1 rag, 16 urnol), DM (2 ml), E¾N (50 mg, 69 μϊ, 492 pmol) and T S^C^CH. (25 mg, 250 pmol) under At. The yellow mixture was stirred at RT for 35 h before concentrated in vacuo. The residue was purified by column chromatography on silica gel (0-70% Ethyl acetate in Hexanes) to give Compound 5 (48.4 mg, 1 18 μτηοΐ, 72%) as a thin .film.
[0208] I E NMR (400 MHz, Methaaol-d4) δ 7.98 (d, I - 5.0 Hz, 1 H), 6.23 (dd, 1 = 7.8, 6.0 Hz, 1 H), 5.28 (dt, J - 6.7, 2.6 Hz, 1 H), 4.36 (t, J - 3.1 Hz, 2 H), 4.34 - 4.28 (tn, I H)5 2.50 (ddd, J = 14.5, 6.0, 2.5 Hz, 1 H), 2.39 (ddd, J - 14.5, 7.9, 6.6 Hz, 1 H), 2.16 (s, 3 H), 2.09 (s, 3 H), 0.20 (d, J - 2.5 Hz, 9 H). 13C NMR (101 MHz, MeOD) S 99.54 (d, J - 140.5 Hz), 96.95 (d, J - 140.5 Hz).
10209} MS (AFCI+) m/z Calcd. for 5(S¾2¾, 207Si [M÷Hf: 41 1.2. Found: 41 1.0 Synthesis of Compound 3:
Figure imgf000056_0001
[0210] To a solution of Compound 5 (3.5 mg, 8.5 μιηοϊ) in 0.9 ml MeOH and.O.'l ml 1¾0 was added KiCGs (6,0 mg, 43 μνηοΐ) at RT. The reaction was stirred overnight before concentrated in vacuo. The residue was purified by reverse phase HPLC to give compound 3 (1.6 rag, 6.4 μηιοί, 75%) as a thin film.
[0211] HPLC condition: 20 min gradient elution using H20:MeCN starting from 100:0 to 85: 15. Retention time: 15.4 min
10212] 1 H NMR (400 MHz, MeOD) ø: 8.39 (d, J - 5.6 Hz, I H); 6.24 (t, i = 6.4 Hz, 1 H); 4.40 (m, 1 H); 3.94 (dd, J ==== 6.4, 3.2 Hz, 1 H); 3.82 (dd, J - 12, 3.2 Hz, 1 H); 3.73 (dd, J - 12, 3.6 Hz, 1 H); 3.53 (dd, J ~ 250.4, 54.8 Hz, 1 H); 2.32 (ddd. J - 13.6, 6, .6 Hz, t H); 2.23 (m, 1 H). I3C MR (1 Q1 MHz, MeOD) δ 82.87 (d, J - 180.4 Hz), 75.85 (d, J - 180.3 Hz).
102131 MS (FAB+) m/z Caled, for C9I3C2H I3N20S [M+Hf : 255.09. Found: 255.11 Synthesis of Compound 9:
Figure imgf000056_0002
10214} To a solution Of ethynylnmgneskm bromide in THF (5.0 ml, 0.5 M solution, 2 mnioi) was added 15 ml THF under Ar, The solution was cooled to -78 °C and 2,4 ml «-BuLi hexane (1.6 M, 3.8 mmol) was added dropwisely. After 30 min, chloro(dimethyl)oetyisilane (1.21 ml, 1.06 g, 5.1 mmol) was added dropwisely. The reaction was then warmed to RT and stirred for another 3h before filtered through a short pad of silica. The solvent was removed trader reduced pressure and the residue was purified by column chromatography on silica ge! (pure Hexanes) to give Compound 9 (885 mg, 2,4 mmol, 96%) as a colorless liquid.
1 2151 \ B NMR (400 MHz, Cbloroform-d) δ 1.42 - 1.24 (as, 24 H). 0,88 (t, I - 6,6 Hz, 6 H), 0.60 (dd, J = 9.4, 6.2 Hz, 4 H), 0.13 (s, 12 H). 13C NMR ( 101 MHz. Chlorofora d) δ 1 13.94, 33.37, 32.12, 29.49, 29.43, 23,92, 22.85, 16.26, 14.27, -1.55.
j0216| HRMS (EH) m/z Caicd. for [Mf :366.3138. Found: 366.3134
Figure imgf000057_0001
[0217] In a glove box filled with Ar, catalyst 8 (36,5 μηιοΐ, 5 eq.) was prepared in 0.5 mL dry CCU in situ according to the procedure documented by Jyothish and Zhang (Ang w. Chem. fat Ed. Engl 50, 3435-8 (201.1 )). To the solution of catalyst 8 in CCL{ was added 9 (267 mg, 0.73 mmol) and a solution of Compound 5 (3.0 mg, 7.3 μηιοΐ) in 0.5 mL dry CCU, The mixture was heated to 70 °C for 8 h before concentrated in vacuo. The residue was purified by column chromatography on silica gel (0-70% Ethyl acetate in Hexanes) to recover Compound 5 (0.5 mg, 1.2 μηιοΐ) and to give Compound .10 (1 .0 rag, 2.0 raol, 27%, 33% B. .S.M.) as a thin .film.
102181 1H NMR (400 MHz, Methanol-d4) δ 7.97 (d, J - 5.6 Hz, 1 H), 6.23 (dd, J - 7.9, 5.9 Hz, 1 H), 5.28 (di J - 6,8, 2.4 Hz, I H), 4.36 (dd, J - 5.8, 3.4 Hz, 2 H), 4.31 (dd, J - 6.3, 3.2 Hz, 1 H), 2.50 (ddd, J - 14.5, 6.1 , 2.5 Hz, 1 H), 2.38 (ddd, J - 20.2, 7.7, 6.1 Hz, 1 H), 2.15 (s, 3 H), 2.0 (s, 3 H), 1.30 (s, 12 H), 0.90 (t, J - 6.9 Hz, 3 H), 0,70 - 0.62 (m, 2 H), 0.18 (s, 6 H). 13C NMR (101 MHz, MeOD) δ 97,56.
|02t9j MS (FAB+) m/z Caicd. for C24 15Cl½N2Na07Si p RNaf : 530.24. Found: 530.25 Synthesis of Compound 2:
Figure imgf000058_0001
[0221 J To a solution of compound 10 (0.4 nig, 0.8 μη ο!} in 0.5 ml eOO and 0.05 ml ¾0 was added K3CO3 (2.0 ng, 14 μηιοΐ) and TBAF (20 ΐ.,, 1 M m THF) at RT. The reaction was siined 7 h at RT before concentrated l« voczw. The residue was purified by reverse phase HPLC to give compound.2 (0.1 rag, 0,4 μιηοΐ, -50%) as a thin film,
[0222] HPLC condition: 20 mhi gradient elution using H20:MeCN starting from 100:0 to 85: 15. Retention time: 15.4 mm
[02231 The mass of the product is determined by UV-Vis ( abs == 288 n«i, ε == 12,000 cm- J ~l in methanol).
[0224] 1 H NMR (500 MHz, Methano!-d4) § 8.39 fd, J - 5.7 Hz, 1 H), 6.24 (t, J - 6.5 Hz, 1 H), 4.40 (*, J = 6.6, 3.6 Hz, 1 H), 3,94 (¾ j = 3.3 Hz, I H), 3.82 (dd, J = 12.0, 3. Ϊ Hz, 1 H), 3.73 (dd, J - 12.0, 3.4 Hz, 1 H), 3.53 (d, J - 51.3 Hz, 1 H), 2.32 (dikl i ^ i 3.6, 6.2, 3.7 Hz, I H), 2.27 - 2, 17 (m, 1 H). 13C NMR (101 MHz, MeOD) 76.00. MS (ESH) m/z Calcd. for C1013CB 13N2O5 [M÷Hj ;-: 254.09. Found: 254.70
Synthesis of Εϋ-ί3€2 (Compound 13)
Synthesis of SS:
Figure imgf000058_0002
S5 S6 0225] To an oven-dried vial was added compound S5 (15 mg, 50 μιηοϊ), Pd(OAc)a ( 1.1 rag, 5 μηιοΐ), PPh? (2.6 r g, .10 μηιοΙ) Cul (1.0 rag, 5 μηιοί), DMF (1 ml), Et3 ( 15 rag, 20.7 μ!, 150 umol) and T SK>0K>CH (7.5 rag, 10.8 μΙ, 75 μηιοί) under Ar. The mixture was stirred at T for 12 h before concentrated in vacuo. The residue was purified by column chromatography on silica get (0-50% methanol in dicWoromethane) to give compound S6 (9,0 rng, 26 μη οΐ, 52%) as a thin film.
[0226] m MR (400 MHz, Meraanol-d4) δ 8.4! (d, J - 4.9 Hz, 1 H), 5.91 -· 5.83 (m5 1 H), 4.21 ~ 4. ! 3 (ra, 2 H), 4.07 - 3.98 (ra, ! H>, 3.88 (dd, J - 12.2, 2.6 Hz, 1 H), 3.75 (dd, I = 12.2, 2.8 Hz, 1 H), 0.20 (d, J ' ==== 2.3 Hz, 9 B), BC NMR (10i MHz, MeOD) δ 9.24 (d, J === 141.0 Hz), 96.95 (d, J === 141.0 Hz).
[0227] MS (FAB+-) m/z Calcd. for Cf 2 BC2i l2iN2Of!Si [M+Hf: 343.12. Found: 343.17.
Synthesis of compound 13:
Figure imgf000059_0001
[0228] To a solution of compound S6 (3.0 mg, 8.8 μη οΐ) in 0.6 ml MeOH and 0,1 ml R2Q was added 2C03 (5,0 rag, 36 μηιοί at RX The reaction was stirred overnight before concentrated in vacuo. The residue was purified by reverse phase HPLC to give compound 13 (2.2 rag, 8.1 pmol, 92%) as a thin film.
10229] 1H NMR {400 MHz, Methanol-d4) δ 8.47 (d, J « 5.6 Hz, 1 H), 5.93 - 5.83 (m, 1 H), 4.21 - 4.13 (m, 2 H), 4.06 - 3.98 (m, 1 B), 3.88 (dd, J ==== 12.2, 2.6 Hz, 1 H), 3.75 (dd, I === 12.2, 2.8 Hz, IH), 3.54 (dd, 1 = 250.4, 54.6 Hz). 13C NMR (10i MHz, MeOD) δ 82.90 (d, J = 180.2 Hz), 75.74 (d, J ==== 180.2 Hz).
[•©230} MS (ESR) m/z Calcd for C9i3C2Hl3N206 [M+HJ- : 271.08. Found; 271.53
[0231] FIG. 38 shows a block diagram of an exemplary embodiment of a system according to the present disclosure. For example, exemplary procedures in accordance with the present disclosure described herein can be performed by a processing arrangement and/or a computing arrangement 3802. Such processing/computing arrangement 3802 can be, for example entirely or a part of, or include, but not limited to, a computer/processor 3804 that can include, for example one or more microprocessors, and use instructions stored on a computer-accessible medium (e.g., RAM, ROM, hard drive, or other storage device).
[0232] As shown in FIG 38, for example a computer-accessible medium 3806 (e.g., as described herein above, a storage device such as a hard disk, floppy disk, memory stick, CD-ROM, RAM, ROM, etc., or a collection, thereof) can be provided (e.g., in communication with the processing arrangement 3802). The computer-accessible medium 3806 can contain executable instructions 3808 thereon, in addition or alternatively, a storage arrangement 3810 can be provided separately from the computer-accessible medium 3806, which can provide the instructions to the processing arrangement 3802 so as to configure the processing arrangement to execute certain exemplary procedures, processes and methods, as described herein above, for example.
[0233) Further, the exemplary processing arrangement 3802 can be provided with or include an input/output arrangement 3814, which can include, for example a wired network, a wireless network, the internet, an intranet, a data collection probe, a sensor, etc. As shown in FIG 38, the exemplary processing arrangement 3802 can be in communication with an exemplary display arrangement 3812, which, according to certain exemplary embodiments of the present disclosure, can be a touch-screen configured for inputting information to the processing arrangement in addition to outputting information from the processing arrangement, for example. Further, the exemplary display 3812 and/or a storage arrangement 3810 can be used to display and/or store data in a user-accessible format and/or user-readable format.
[0234) The foregoing merely illustrates the principles of the disclosure. various modifications and alterations to the described exemplary embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated tha t those skil led in the art will be able to devise numerous systems, arrangements, and procedures which, although not expl icitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. Various different exemplary embodiments can be used together with one another, as well as interchangeably therexvith, as should be understood by those having ordinary skill in the art. in addition, certain terms used in the present disclosure, including the specification, drawings and cl ims thereof, can be used synonymously in certain instances, including, but not Limited to. e.g., data and information. It should be understood that, while thes words, and/or other words that can he synonymous to one another, can be used synonymously herein, that there can be instances when such words can be intended to not be used synonymously. Further, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly incorporated herein in its entirety. All piiblicatioiis referenced are incorporated herein by reference in their entireties.
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Claims

WHAT IS CLAIMED IS:
1. A method for imaging a living cell or a living organism, comprising:
introducing an effective amount of a bond-edited compound into a live cell or a living organism, wherein said bond-edited compound comprises a vibrational tag; and
detecting said vibrational tag hi said cell or said organism with stimulated Raman scattering (S. S) imaging.
2. The method of Claim I, wherein said bond-edited compound is a small molecule.
3. The method of Claim 1. wherein said bond-edited compound comprises one, two, three, four, five, six, seven, eight, nine, ten or more vibrational tags.
4. The method of Claim 3, wherein said vibrational tags are the same type of tags or a mixture of one or more different tags.
5. The method of Claim 1, wherein said vibrational tag is an. alkyne tag.
6. The method of Claim 1. wherein said vibrational tag is an. a ide tag.
7. The method of Claim I , wherein said vibrational tag is an isotope label.
8. The method of Claim 7, wherein said isotope label is a carbon-deuterium bond tag.
9. The method of Claim 1, wherein said vibrational tag is a combination of an alkyne tag and a carbon-deuterium bond tag.
10. The method of Claim 1, wherein said bond-edited compound comprises at least one vibrational tag selected from the group consisting of -C.≡C-> -O-N, ~N::: ~N, -OC-OC-,
-OC-ON, -C-D. and -O -D.
1 1. The method of Claim 1 , wherein said bond-edited compound comprises
at least one C atom or one deuterium atom,
12. The method of Claim I, wherein said bond-edited compound is an amino acid.
13. The method o Claim 12, wherein said ammo acid is a essential amino acid.
14. The method of Claim 13, wherein said essential amino acid is selected from the group consisting of histidine, isoleucine. leucine, lysing, methionine, phenylalanine, threonine, tryptophan and valine.
15. The method of Claim 1 , wherein said bond-edited compound is a nucleoside or a nucleotide,
16. The method of Claim 1, wherein said bond-edited compound is a fatty acid.
17. The method of Claim 1. wherein said bond-edited compound is a monosaccharide or a disaccharide.
18. The method of Claim 1 , wherein said bond-edited compound is gl ucose, a glucose derivative or propargyl glucose.
19. The method of Claim 1. wherein said bond-edited compound is selected from the group consisting of anti-cancer agents, anti -inflammatory agents, anii -bacterial agents, anti-fungal agents and anti -viral agents.
20. The method of Claim 1 , wherein said bond-edited compound is a cytokine or chemokine.
21. The method of Claim 1. wherein said vibrational tag is transferred from said bond-edited compound to a down-stream metabolite of said bond-edited compound, and is detected in said down-stream .metabolite.
22. A method for imaging a living cell or a living organism, comprising:
introducing into said live cell or organism a mixture of two or more bond-edited compounds wherein said two or more bond-edited compounds each comprises a different vibrational tag; and imaging with stimulated R aman scattering at two or more different -wavelengths t detect said vibrational tag on each of said two or more bond-edited compounds.
23. The method of Claim 22, wherein said two or more bond-edited compounds comprise EU-l3C2, EdU-l5C and 17-ODYA.
24. The method of Claim 22, wherein said two or more bond-edited compounds target different cellular components.
25. The method of C laim 22, wherein said two or more bond-edited compounds target the same cellular component but at different time period.
26. The method of Claim 22, wherein said two or more bond-edited eomoounds taraet different types of cells in said living organism.
27. The method o Claim 22, wherein said vibrational tag on each of said two or more bond-edited compounds is detected using a linear combination algorithm.
28. A method for making a alkyne-tagged compound, comprising:
adding propargyl bromide to a compound of formula S 1 in the presence of DMF and sodium hydride to produce a compound of formula S2;
Figure imgf000068_0001
and, adding water and an ion exchange resin to the compound of formula S2 to produce a compoirad
Figure imgf000069_0001
29. A method for making a i C-tagged compound, comprising:
reacting a compound of formula 5 with 2CO3, MeOH and _¾0 to produce the compound of formula 3:
Figure imgf000069_0002
30, A method for making a KX tagged compound, comprising:
reacting a compound of formula 10 with TBAF, 2CO.i, MeOH and ¾0 to produce the compound of formula 2:
Figure imgf000069_0003
31 , A method for .making a 1 'XT-tagged compoirad, comprising:
reacting a compound of formula S6 with K2C , MeOH and E2O to produce the compoirad of .formula 13:
Figure imgf000070_0001
A alkyne-tagged compound of formula S3,
Figure imgf000070_0002
33. A compound of formula 3:
Figure imgf000070_0003
34. A compound of tormisla 2:
Figure imgf000071_0001
35, A compoimd of formula 13:
Figure imgf000071_0002
13
36. A method for detecting a disease condition in a subject, comprising:
administering to said subject a composition comprising a bond-edited compound targeting a disease tissue or pathogen, wherein said bond-edited compound comprises a vibrational tag; and detecting said vibrational tag by stimulated Raman scattering imaging.
37. The method of Claim 36, wherein said disease condition is selected from the group consisting of cancer, metabolic syndrome, neurodegenerative diseases, inflammatory diseases and microbial infections.
38. The method of Claim.36, wherein said vibrational, tag is transferred from said bond-edited compound to a down-stream metabolite of said boad-edited compound, and is detected in said down-stream, metabolite.
39. A method for monitoring a treatment for a disease condition in a subject, comprising: administering to said subject a composition comprising a bond-edited compound and detecting said bond-edited compound by stimulated Raman scattering imaging at a first time point; further administering to said subject said composition comprising a bond-edited compound and detecting said bond-edited compound by stimulated Raman scattering imagmg at a second time point; and
comparing images obtained at the two time points,
40. The method of Claim 39, wherein said first, time point is a time point that is about or prior to the initiation of said treatment and the second time point is a time point thai is after the initiation of said treatment,
41. The method of Claim 39, wherein said first time point and said second time point are two time points during the course of said treatment
42. A method for screening a candidate agent, comprising:
administering said candidate agent and at least one bond-edited compound to a live cell or organism;
detecting said bond-edited compound in said li ve cell or organism using stimulated Raman scattering imaging; and
determining an effecti veness of said candidate agent based on one or more predetermined criteria selected from the group consisti ng of the uptake, accumulation, trafficking and degradation of said bond-edited compound in the said live cell or organism.
43. The method of Claim 42, wherein said candidate agent is an anti-cancer drug.
44. The method of Claim 42, wherein said candidate agent, is a skin regenerating agent.
45. A device for imaging bond-edited compounds by stimulated Raman scattering, comprising:
a first laser generator that produces a pulse laser beam of a first fixed wavelength;
a second laser generator that produces a pulse laser beam of a second fixed wavelength; a modulator that modulates the pulse laser beam of one of the first or second laser generator ; a photodetector that is adapted to detecting stimulated Raman scattering from a hiosaniple; and
a computer,
46. The device of Claim 45, wherein said first and second laser generators are configured to provide a pump radiation and a stokes radiation, each at a. fixed wavelength whose energy difference is between about 2000 and 2500 wavenumbers.
47. A non-transitory computer-accessible medium having stored thereon
computer-executable instructions for determining data associated with at least one tissue, wherein, when a computer hardware arrangement executes the instructions, the computer arrangement is configured to perform procedures comprising:
receiving first information related to at least one bond between at least two atoms attached to a metabolite; and
determining the data based on the at least one bond.
48. The non-transitory computer-accessible medium of Claim 47, wherein the bond is a carbon deuterium bond.
49. The non-transitory computer-accessible medium of Claim 48, wherei a deuterium to hydrogen ratio of the tissue is at least 1 to 5,000.
50. The non-transitory computer-accessible medium of Claim 48, wherein a deuterium to hydrogen ratio of the tissue is at least 1 to 1 ,000,
51. The non-transitory computer-accessible medium of Claim 48, wherei a deuterium to hydrogen ratio of the tissue is at least 1 to at most 100.
52. The non-transitory computer-accessible medium of Claim 48, wherein the bond is a triple carbon bond, a triple carbon nitrogen bond or an azide triple nitrogen bond.
53. The non-transitory computer-accessible medium of Claim 48, wherein the data includes a locatio of the at least one bond.
54. The non-transitory computer-accessible medium of Claim 48, wherein the computer hardware arrangement is further configured to determine the data based on an amplitude of a signal of the at least one bond.
55. The non-transitory computer-accessible medium of Claim 48, wherein the computer hardware arrangement is further configured to determine that data using a stimulated Raman microscopy arrangement.
56. The non-transitory computer-accessible medium of Claim 54, wherein the computer hardware arrangement is further configured to tune a laser of the stimulated Rama microscopy arrangement to a particular frequency based on the bond.
57. The non-transitory computer-accessible medium of Claim 48, wherein the computer hardware arrangement is further configured to determine the data using a least one of (i) a coherent anti-Stokes Raman scattering arrangement, (ii) an infrared absorption arrangement, (iii) a stimulated Raman excited photoihermal arrangement, or (iv) a stimulated Raman excited photoacousiie arrangement.
58. The non-transitory computer-accessible medium of Claim 48, wherein the at least one tissue includes at least one live animal ceil,
59. The non-transitory computer-accessible medium of Claim 48, wherein the metabolite includes at least one of (i) at least one deoxyribonitcleoside, (ii) at least one rihorsueleoside, (hi) at
7.1 least one amino acid, (iv) choline, (v) at least one fatty acid, (vi) at least one Adenosine tri hosphate, (mi) cholesterol, or (viti) at least one chemical drug.
60. A method for determining data associated wit at least one tissue, comprising;
receiving first information related to at least one bond between at least two atoms attached to a metabolite; and
using a comptiter hardware arrangement, determining the data based on the at least one bond.
61. A system for determining data associated with at least one tissue, comprising:
a computer processing arrangement configured to:
receive first information related to at least one bond between at least two atoms attached to a metabolite; and
determine the data based o the at least one bond,
62. A pre~mixed essential amino acid combination, comprising:
at least one nors -deuterated essential amino acid; and
at least 5 deuterated essential amino acids.
63. A pre-mixed essential amino acid combination, comprising:
at least one non-deuierated essential amino acid; and
at least 3 deuterated essentia! amino acids.
64. The cell culture medium of Claim 63, further comprising at least 4 deuterated essential amino acids.
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