WO2020243698A1 - Spectroscopie par corrélation croisée fluorescente interférométrique - Google Patents

Spectroscopie par corrélation croisée fluorescente interférométrique Download PDF

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WO2020243698A1
WO2020243698A1 PCT/US2020/035534 US2020035534W WO2020243698A1 WO 2020243698 A1 WO2020243698 A1 WO 2020243698A1 US 2020035534 W US2020035534 W US 2020035534W WO 2020243698 A1 WO2020243698 A1 WO 2020243698A1
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image detector
cross
phase
correlation function
fluorescence
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Saveez Saffarian
Ipsita SAHA
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University Of Utah Research Foundation
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    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0056—Optical details of the image generation based on optical coherence, e.g. phase-contrast arrangements, interference arrangements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/106—Beam splitting or combining systems for splitting or combining a plurality of identical beams or images, e.g. image replication
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/12—Beam splitting or combining systems operating by refraction only
    • G02B27/126—The splitting element being a prism or prismatic array, including systems based on total internal reflection
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/145—Beam splitting or combining systems operating by reflection only having sequential partially reflecting surfaces

Definitions

  • the present invention relates to spectroscopy. More generally, the invention relates to interferometric fluorescent cross-correlation spectroscopy. Therefore, the present invention relates generally to the fields of physics, optics, chemistry and biology.
  • FCS Fluorescence Correlation Spectroscopy
  • Single photon interference and fluorescence correlation spectroscopy can be used to simultaneously measure transport of fluorescent molecules within aqueous samples.
  • transport can be measured within seconds in thousands of homogenous voxels (100 nanometers (nm)) 3 while also eliminating photo-physical artifacts associated with blinking of fluorescent molecules.
  • This approach can be validated by measuring transport of quantum dots, associated with the G protein of vesicular stomatitis virus (VSV-G) receptor along cellular membranes and viscous gels.
  • VSV-G vesicular stomatitis virus
  • Interferometric fluorescence measurements were first introduced in interferometric photo-activated localization microscopy (PALM) microscopy in 2009.
  • PAM photo-activated localization microscopy
  • the photon wave-front interferes with itself with varying phase shifts and is imaged on multiple cameras.
  • the interference can increase the effective resolution of the optical microscope to (10nm) 3 for localizing single molecules within a sample.
  • a method for performing fluorescence cross correlation spectroscopy can include mounting a sample on a sample stage, the sample having a plurality of probe molecules.
  • the sample stage can be a micro-positioning stage although other stages may be useful.
  • the sample can be illuminated with laser (of a particular wavelength) to cause fluorescence from the probe molecules in the sample to pass simultaneously through the first objective and the second objective and which are recombined at a beam splitter.
  • the luminescence can then be phase shifted at the beam splitter to provide a first beam output with a first phase shift to a first image detector and a second beam output with a second phase shift to a second image detector.
  • a difference in signal strength can be measured between a first signal strength from the first image detector and a second signal strength from the second image detector. Interference is caused due to recombination of the beams from the first beam output and the second beam output. The difference in signal strength is due to the phase shift caused by combining the beam within the beam splitter which causes interference. At least one of diffusion and a flow vector of a probe molecule of the plurality of probe molecules can be detected using a cross-correlation function calculated from information received from the first image detector during an acquisition time and information received from the second image detector during the same acquisition time.
  • the illumination light can be a coherent light.
  • At least one of the first image detector and the second image detector can comprise one or more of: a complementary metal oxide semiconductor (cMOS) camera or a single-photon avalanche diode (SPAD) detector.
  • cMOS complementary metal oxide semiconductor
  • SPAD single-photon avalanche diode
  • a difference between the first phase shift at the first image detector and the second phase shift at the second image detector can be 120 degrees or 180 degrees.
  • the diffusion of the probe molecule can be detected with a voxel resolution, wherein each voxel is a symmetric voxel (i.e. cube).
  • the voxel resolution can be at least 150nm or in some cases 50-200 nm.
  • the detection can be applied to a plurality of homogenous voxels.
  • the detection can optionally include the diffusion of the probe molecule, using cMOS, frame rates of 100 - 1000 frames per second and, using a SPAD detector array, frame rates of 1000 to 1,000,000 frames per second.
  • the method can comprise detecting a flux of the probe molecule of the plurality of probe molecules when a symmetry of the cross-correlation function is asymmetric, wherein the symmetry of the cross-correlation function is metric when and asymmetric when
  • Such a cross-correlation function can be based on one or more of: a total number of frames, an integrated fluorescence minus background for a first region of interest (ROI), or an integrated fluorescence minus background for a second region of interest.
  • ROI integrated fluorescence minus background for a first region of interest
  • the cross-correlation function can use:
  • N is a total number of frames
  • F is the cross-correlation function
  • F is an integrated fluorescence minus background for a first region of interest (ROI) at a center position (x1, y1) with a phase f1
  • F’ is an integrated fluorescence minus background for a second ROI at a center position (x2, y2) with a phase of f2
  • t is a time duration.
  • the method can comprise calculating transport properties to map transport of the probe molecule of the plurality of probe molecules within a cytosol of a cell of the sample or along a membrane of the cell of the sample.
  • transport properties can include one or more of: a diffusion coefficient, a turbulent diffusion coefficient, correlative transport properties, or a directional flow.
  • the methods described herein may be implemented wholly or partially as computer readable program code executed by a processor and the computer readable code may be embodied on a non-transitory computer usable medium.
  • a system for fluorescence cross correlation spectroscopy can comprise: a light source configured to produce a coherent excitation light to cause fluorescence of a plurality of probe molecules in a sample; a sample stage adapted to receive the sample between a first objective and a second objective; a first objective configured to pass fluorescence from the plurality of probe molecules to a beam splitter; a second objective configured to pass fluorescence from the plurality of probe molecules to the beam splitter; the beam splitter configured to phase shift the fluorescence to pass a first beam with a first phase shift and a second beam with a second phase shift; a first image detector configured to receive the first beam with the first phase shift; a second image detector configured to receive the second beam with the second phase shift; and a controller configured to: measure a difference in signal strength between a first signal strength from the first image detector and a second signal strength from the second image detector, wherein the difference in signal strength is caused by interference arising from a difference in phase between the first beam and the second beam; and determine
  • FIG. la is a schematic diagram of an experimental setup using a three-way prism in accordance with one aspect.
  • FIG. lb is a schematic diagram of an experimental setup using a two-way prism in accordance with one aspect.
  • FIG. 2a is a graph of a calibration curve showing the interference effect and phase difference between the cameras. Calibration was performed by moving the sample in between the two objectives, in steps of 8 nm for 101 planes, along the axial direction.
  • FIG. 2b demonstrates that at each plane the three cameras collected fluorescence signal from a fiducial in accordance with one aspect.
  • FIG. 3 is a graph demonstrating the movement of the molecule between the cameras and their associated correlation functions in accordance with one aspect.
  • FIG. 4 illustrates the simulated system where the regions where the particles were subjected to flow are marked with arrows.
  • the flow regions along the axial plane have a volume of 2X25X0.3 micrometers (mm 3 ) and the ones along the optical axis have a volume of 2X21 mm 3 in accordance with one aspect.
  • FIGS. 5A-D illustrate transport measurements on particles in a box simulated by Monte Carlo dynamics.
  • C shows the correlation functions in a region where the particles are subjected to flow along the Z axis;
  • D shows the superimposed image from the simulated system for 20,000 frames with the regions marked which have been used for calculation in (A), (B) and (C).
  • FIG. 6A-F illustrate transport measurements in a sample of quantum dots in sucrose solution and vesicular stomatitis virus (VSV-G) diffusion on the membrane of HeLa cells.
  • a and C represent the correlation functions from two different regions of the sample.
  • B is the superimposed image from the experimental dataset for 20,000 frames with regions marked which have been used for calculation in (A) and (C). The correlation functions have been fitted to obtain diffusion coefficient of 2.68 +/- 0.28) x 10 -9 cm 2 /s and regions of flow of 0.21 - 0.55 mm /s.
  • D) and (F) represent the correlation functions from two different regions of the cell membrane.
  • E is the image of the cell in which the correlation functions has been measured. The correlation functions has been fitted and a diffusion coefficient of (1.28 +/- 1.00) x 10 -10 cm 2 /s was measured.
  • the correlation function calculation and representation is from Figure 5.
  • FIG. 7a is a flow diagram of a method for performing fluorescent cross correlation spectroscopy (FCCS) in accordance with an example of the present technology.
  • FIG. 7b is an apparatus for performing fluorescent cross correlation spectroscopy (FCCS) in accordance with an example of the present technology.
  • FIG. 7c is depicts a flowchart of a machine-readable storage medium having instructions embodied thereon for performing fluorescent cross correlation spectroscopy (FCCS) in accordance with an example of the present technology.
  • FCCS fluorescent cross correlation spectroscopy
  • FIG. 8 is a schematic diagram of a computing system for use in performing portions of the fluorescent cross correlation spectroscopy (FCCS) methods in accordance with an example of the present technology.
  • FCCS fluorescent cross correlation spectroscopy
  • the term“about” is used to provide flexibility and imprecision associated with a given term, metric or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art. However, unless otherwise enunciated, the term“about” generally connotes flexibility of less than 2%, and most often less than 1%, and in some cases less than 0.01%. Further, unless otherwise stated, the term“about” shall expressly include“exactly,” consistent with the discussion above regarding ranges and numerical data.
  • substantially when used in reference to a quantity or amount of a material, or a specific characteristic thereof, refers to an amount that is sufficient to provide an effect that the material or characteristic was intended to provide. Therefore, “substantially free” when used in reference to a quantity or amount of a material, or a specific characteristic thereof, refers to the absence of the material or characteristic, or to the presence of the material or characteristic in an amount that is insufficient to impart a measurable effect, normally imparted by such material or characteristic.
  • “adjacent” refers to the proximity of two structures or elements. Particularly, elements that are identified as being“adjacent” may be either abutting or connected. Such elements may also be near or close to each other without necessarily contacting each other. The exact degree of proximity may in some cases depend on the specific context.
  • the term“at least one of’ is intended to be synonymous with“one or more of.”
  • “at least one of A, B and C” explicitly includes only A, only B, only C, and combinations of each.
  • Numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a numerical range of“about 0.6 mm to about 0.3 mm” should be interpreted to include not only the explicitly recited values of about 0.6 mm and about 0.3 mm, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.4 mm and 0.5, and sub-ranges such as from 0.5 -0.4 mm, from 0.4 -0.35, etc. This same principle applies to ranges reciting only one numerical value. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
  • any steps recited in any method or process claims may be executed in any order and are not limited to the order presented in the claims.
  • Means- plus-function or step-plus-function limitations will only be employed where for a specific claim limitation all of the following conditions are present in that limitation: a)“means for” or“step for” is expressly recited; and b) a corresponding function is expressly recited.
  • the structure, material or acts that support the means-plus function are expressly recited in the description herein. Accordingly, the scope of the disclosure should be determined solely by the appended claims and their legal equivalents, rather than by the descriptions and examples given herein.
  • Fluorescence Correlation Spectroscopy can be used with interferometric single photon localizations to simultaneously measure transport properties in a cross section of a sample in 200 x 200 voxels with a voxel resolution of (lOOnm) 3 . Transport along the plasma membrane or within viscous gels can also be resolved. A transport map of the cytosol of living cells can be created with adequate detector speeds. Detector technologies are discussed to produce cytosolic measurements.
  • a system for fluorescence cross correlation spectroscopy can comprise a light source configured to produce a coherent excitation light to cause fluorescence of a plurality of probe molecules in a sample 104.
  • a sample stage 108 can be adapted to receive the sample 104 between a first objective 102a and a second objective 102b.
  • the two objectives 102a and 102b can be focused on the sample 104 from the top and the bottom.
  • the sample 104 can be positioned between a top coverslip 106a and a bottom coverslip 106b, and the sample 104 can be secured onto the sample stage 108 (e.g., a micro-positioning stage).
  • the first objective 102a can be configured to pass fluorescence from the plurality of probe molecules in a sample 104 to a beam splitter 110a.
  • the fluorescence passed from the first objective 102a can be directed through a first light path 114a and 114c to a beam splitter 110a, and to at least one image detector 120a, 120b, or 120c via 114e, 114f, or 114g, respectively.
  • the fluorescence can be directed along the first light path or the second light path by one or more prisms, mirrors , or the like 112a and 112b.
  • the second objective 102b can be configured to pass fluorescence from the plurality of probe molecules in the sample 104 to the beam splitter 110a.
  • the fluorescence passed from the second objective 102b can be directed through a second light path 114b and 114d to a beam splitter 110a, and to at least one image detector 120a, 120b, or 120c via 114e, 114f, or 114g, respectively.
  • the wave-front of the emitted photon within the sample 104 can travel through the first light path 114a and 114c and the second light path 114b and 114d.
  • the first light path and the second light path can be recombined in the beam splitter 110a (e.g., a two- way prism or a three-way prism). This recombined light can then be split by the beam splitter and produce phase shifted lights with a first phase shift, a second phase shift, or a third phase shift.
  • the three phase shifts can be about 0°, about 120°, and about 240°.
  • two phase shifts can include 0° and 180°.
  • the beam splitter 110a can be configured to phase shift the fluorescence to pass a first beam with a first phase shift and a second beam with a second phase shift.
  • the fluorescence with the first phase shift can be directed to and received by a first image detector 120a.
  • the first phase shift can be about 0°.
  • the fluorescence with the second phase shift can be directed to and received by a second image detector 120b.
  • the second phase shift can be about 120°.
  • the fluorescence with the third phase shift can be directed to and received by a third image detector 120c.
  • the third phase shift can be about 240°.
  • the system can further comprise a controller.
  • the controller can be configured to measure a difference in signal strength between a first signal strength from the first image detector 120a and a second signal strength from the second image detector 120b, wherein the difference in signal strength is caused by interference arising from a difference in phase between the first beam output 114e and the second beam output 114f.
  • the controller can be further configured to measure a difference in signal strength between a first signal strength from the first image detector 120a and a third signal strength from the third image detector 120c, wherein the difference in signal strength is caused by interference arising from a difference in path length between the first beam output 114e and the third beam output 114g.
  • the controller can be further configured to measure differences in signal strength between a second signal strength from the second image detector 120b and a third signal strength from the third image detector 120c, wherein the difference in signal strength is caused by interference arising from a difference in phase between the second beam output 114f and the third beam output 114g.
  • the controller can be further configured to determine at least one of a diffusion and a flow vector of a probe molecule of the plurality of probe molecules using a cross correlation function calculated from information from the first image detector 120a and information from the second image detector 120b.
  • the controller can be further configured to determine at least one of a diffusion and a flow vector of a probe molecule of the plurality of probe molecules using a cross correlation function calculated from information from the third image detector 120c. From the detector the intensity is taken into account which is directly proportional to the number of photons arriving at the detector.
  • At least one of the first image detector 120a, the second image detector 120b, and the third image detector 120c can be at least one of a complementary metal oxide semiconductor (cMOS) camera and a single-photon avalanche diode (SPAD) detector. At least one of the first image detector 120a, the second image detector 120b, and the third image detector 120c can be para-focal with the sample plane.
  • cMOS complementary metal oxide semiconductor
  • SPAD single-photon avalanche diode
  • the photon can be detected by one of the image detectors (e.g., cMOS cameras) based on the interferometric probability of its detection.
  • the image detectors e.g., cMOS cameras
  • a system for fluorescence cross correlation spectroscopy includes only two detectors, as illustrated in FIG. lb.
  • the system can comprise a light source configured to produce a coherent excitation light to cause fluorescence of a plurality of probe molecules in a sample 104.
  • a first objective 102a can be configured to pass fluorescence from the plurality of probe molecules in a sample 104 to a beam splitter 110b (e.g., a two-way prism).
  • the fluorescence passed from the first objective 102a can be directed through a first light path 114a and 114c to a beam splitter 110b, and to at least one image detector 130a or 130b via 114e or 114f, respectively.
  • a second objective 102b can be configured to pass fluorescence from the plurality of probe molecules in the sample 104 to the beam splitter 110b.
  • the fluorescence passed from the second objective 102b can be directed through a second light path 114b and 114d to a beam splitter 110b, and to at least one image detector 130a or 130b via 114e or 114f, respectively.
  • the wave-front of the emitted photon within the sample 104 can travel through the first light path 114a and 114c and the second light path 114b and 114d.
  • the first light path and the second light path can be recombined in the beam splitter 110a (e.g., a two- way prism) with a first phase shift and a second phase shift.
  • the two phase shifts can be about 0° and about 180°.
  • the beam splitter 110a can be configured to phase shift the fluorescence to pass a first beam with a first phase shift and a second beam with a second phase shift.
  • the fluorescence with the first phase shift can be directed to and received by a first image detector 130a.
  • the first phase shift can be about 0°.
  • the fluorescence with the second phase shift can be directed to and received by a second image detector 130b.
  • the second phase shift can be about 180°.
  • the system can further comprise a controller.
  • the controller can be configured to measure a difference in signal strength between a first signal strength from the first image detector 130a and a second signal strength from the second image detector 130b, wherein the difference in signal strength is caused by interference arising from a difference in phase between the first beam output 114e and the second beam output 114f.
  • the first image detector 130a and the second image detector 130b can be single-photon avalanche diode (SPAD) detectors. At least one of the first image detector 130a and the second image detector 130b can be para-focal with the sample plane.
  • the photon can be detected by one of the image detectors (e.g., SPAD detectors) based on the interferometric probability of its detection.
  • a plurality of lasers e.g., light sources
  • 405 nm, 488 nm, 561 nm, and 647 nm can be used as light sources.
  • Other wavelengths, numbers of light sources, and types of light sources can also be used.
  • specific light sources may be mentioned herein, other types of light sources can also be used to provide the functions as described herein.
  • the various optics, apertures, beam splitters, and so forth used in the system can be installed on a construction rail, micro-dovetail rail, or the like.
  • the system can be set up on a table or other surface, and may also include a computer having a memory and a processor configured to process data and operate the software.
  • the diffusion of the probe molecule can be measured with a voxel resolution of at least 150 nanometers (nm) with a range of about 50 nm to about 200 nm.
  • the voxel can be a symmetric voxel (e.g., a cube).
  • the transport properties of a plurality of homogenous voxels can be detected.
  • the diffusion of the probe molecule can be determined using a frame rate higher than 1000 frames per second.
  • the controller can be further configured to calculate transport properties to map the transport of a probe molecule within a cytosol of a cell of the sample. In one example, the controller can be further configured to calculate transport properties to map the transport of a probe molecule along a membrane of the cell of the sample.
  • the transport properties of the probe molecule of the plurality of probe molecules can include at least one of a diffusion coefficient, a turbulent diffusion coefficient, correlative transport properties, or a directional flow.
  • (x1, y1) is a first center position with a first phase f1
  • (x2, y2) is a second center position with a second phase f2
  • t is a time duration
  • the cross-correlation function can be calculated based on one or more of: a total number of frames, an integrated fluorescence minus background for a first region of interest (ROI), or an integrated fluorescence minus background for a second region of interest.
  • the cross-correlation function can be calculated using: is the cross
  • N is a total number of frames
  • F is an integrated fluorescence minus background for a first region of interest (ROI) at a center position (x1, y1) with a phase f1
  • F’ is an integrated fluorescence minus background for a second ROI at a center position (x2, y2) with a phase of f2
  • t is a time duration.
  • the point spread function (PSF) of the scope can therefore be a convolution of an optical microscope with an interferometric effect which approximates a sine wave as defined below and experimentally verified in Figure 2: which w can be the radial distance over which the intensity drops by 1/e 2 and aw can define the axial distance over which the intensity can drop by 1/e 2 and k p can be the phase factor that can be governed by the wavelength of excitation and numerical aperture of the objectives.
  • the f value can be the interferometric phase shift of each camera and in the cMOS system it can at least one of 0°, 120°, and 240°.
  • Fluorescence Correlation functions can be calculated based on probability density functions convoluted with the interferometric PSF function and the total internal reflection excitation intensity profile I excitation where d can be the total internal reflection fluorescence (TIRF) penetration depth.
  • the generalized correlation function can be calculated as:
  • f2— f1 and m can be the constant depending on the concentration of molecules in the observation volume.
  • the scope is further depicted in FIGS. 2a and 2b.
  • the fluorescence of the molecule can be detected either on a different camera or at a different pixel within the same camera.
  • 200 x 200 pixel images can be acquired on all three cameras.
  • a typical experiment can run for 20,000 frames with an exposure of 1 millisecond (ms).
  • fluorescence from a single molecule can be spread along an area of 3 x 3 pixels and therefore the fluorescence associated with each region of interest can be calculated by summing the fluorescence within a mask of 3 x 3 pixels with the center pixel at the center of the region of interest.
  • Experimental cross correlation functions can be calculated by multiplying the signal from two ROIs characterized by the center position (x, y) from each ROI and the phase f of the corresponding cMOS chip:
  • N can be the total number of
  • F can be the integrated fluorescence minus background from the 9 pixel ROI at (xl, y 1, f1 ) and F' can be the integrated fluorescence minus background from the 9 pixel ROI at (x2, y2, f2 ).
  • the background can be calculated based on the average fluorescence from the 200 x 200 pixels and subtracted from the total fluorescence intensity calculated in each region of interest.
  • region of interest and F j can be the fluorescence signal from the pixels in the entire image.
  • the fluorescence signal received by a first detector from this molecule can be:
  • v can be the flux vector.
  • the probability distribution function can be:
  • an anti-correlation can be predicted in various cross correlation situations.
  • the first and second detectors are 120° phase shifted and the molecule is in the plane of the first detector, interference can result in lowering of the registered signal of the first detector on the second and third detectors.
  • the signal strength in the second detector can increase while the signal in the first detector can decrease, which can result in a detectable anti -correlation.
  • the cross-correlation curves between the detectors which can be phase shifted by f > 0, can determine the diffusion and flow of the molecule along the optical axis with a very high resolution and also distinguish between fluctuations arising due to actual physical movement of the molecule and other photo-physical activity of a fluorescent proteins because when the molecule blinks or goes through any other photo-physical activity it can affect both the detectors simultaneously.
  • the characteristic anti-correlation between phase shifted cameras can be a signature of molecular motion along the z-axis and therefore anti -correlation can be detected during physical movement and not because of some other photo-physical effect.
  • the correlation function can also have an asymmetry when flux is present in the system.
  • the cross correlation functions can be symmetric and when there is flux the cross correlations can be
  • the method was validated using Monte Carlo simulations, as illustrated in FIG. 4.
  • the conditions for the simulated system approximated the real experiments.
  • 250 particles were taken in a cube including dimensions of 25 mpi along an x-axis 410, 1 mpi along a y-axis 430, and 25 mm along a z-axis 430 for a volume of 25 x 25 x 1 mm 3 .
  • the pixel size in the simulation was chosen to be 100 nm.
  • the particles underwent Brownian diffusion.
  • the initial position of the particles was generated from rand function of MATLAB.
  • the system had reflecting boundary condition.
  • a normalized random number generator (normrnd) determined the step sizes of each particle, the mean of which depended on the flux vector and the standard deviation were determined by the
  • Typical w was chosen to be 264.5 nm and a was 4.
  • the particles were excited by a 561 nm laser with a field depth of 300 nmand TIRF imaging conditions were maintained. Two detectors 120° phase shifted detected signals from these particles in the mentioned conditions for 20,000 frames with 1 ms of exposure. A Poisson random number determined the signals in the 200 x 200 pixel area with mean given by the PSF function as previously described.
  • the simulation code was written in MATLAB and run on the computer nodes with two Intel Xeon Gold 6130 CPUs, 32 CPU cores and 96 GB of RAM per node.
  • FIG. 5A-5D show the results of the simulation.
  • a superimposed image of the simulation is shown in FIG. 5D.
  • the areas undergoing flow shown in Figure 4 are also visible within this superposition.
  • FIG. 5A shows the calculated cross correlation functions for the ROI identified in FIG. 5D.
  • 8 cross correlation functions with the same phase can be calculated with ROIs separated by 2 pixels along the X and Y direction.
  • Two cross correlation functions can be calculated on the same ROI between two cameras with a 120° phase difference and one autocorrelation function.
  • the ROI in Figure 5A can be selected in an area where molecules were undergoing diffusion, hence the cross-correlation functions between cameras and adjacent ROIs can all be symmetric.
  • Figure 5B and 5C show similar calculations for the other ROIs shown in Figure 5D from areas undergoing flow along Y axis (5B) and Z axis (5C).
  • the correlation curves can be fitted to obtain a diffusion coefficient of (0.89 +/- 0.048) x 10 -9 cm 2 /s and flux of (0.36 +/- 0.028) mm /s.
  • a 25 mm 1.5 Hestzig coverslip and an 18 mm 1.5 coverslip were used to sandwich the sample.
  • the circular coverslips were thoroughly washed in 1 M sodium hydroxide solution followed by mQ water and then blow dried with nitrogen.
  • 70% sucrose solution was prepared by dissolving the sucrose in lOmg/ml casein solution in PBS.
  • the sucrose solution was prepared by heating it in a water bath at 80° C to dissolve crystal. Care was taken not to insert any air bubble.
  • 605 Quantum dots, by Thermo Fisher Scientific were added when the sucrose completely dissolved and produced a clear viscous solution. The solution was heated together to mix it. Then this sample was sandwiched between the coverslips and sealed with glue and allowed to cool down to room temperature before imaging.
  • the casein was used to prevent the quantum dots from sticking to the coverslips.
  • the instrument was schematically described in Fig. la.
  • the sample was placed between two Nikon 60X Apo TIRF objectives of NA 1.49 and was illuminated by a 315 mW LASER of wavelength 561 nm.
  • the 100 nm gold beads on the Hestzig slides were used to focus and calibrate the whole system.
  • the custom 3 -way beam splitter was adjusted so as to get the interference and 120° phase shift between the cameras (Hamamatsu Orca Flash 4.0 sCMOS) and was obtained, as seen in the calibration curves in Fig. 6a, 6c, 6d, and 6f.
  • Data was collected on a 200x200 pixel area with 1 ms of exposure for 20,000 frames.
  • the high viscosity of the sucrose created patches of inhomogeneity within the sample.
  • FIGS. 6a-f represent the correlation functions from two selected ROIs as shown in FIGS. 6a-f.
  • FIGS. 6a and 6c represent the correlation functions from two different regions of the sample.
  • FIG. 6b is the superimposed image from the experimental dataset for frames with regions marked which have been used for calculation in FIGS. 6a and 6c.
  • FIGS. 6d and 6f represent the correlation functions from two different regions of the cell membrane.
  • FIG. 6E is the image of the cell in which the correlation functions have been measured.
  • sucrose solution can be homogeneous
  • directional flow within the ROIs can be detected as characterized by the asymmetry in the measured cross correlation curves shown in Figure 6a, 6c, 6d, and 6f.
  • These flow vectors can be due to temperature gradients within the sample.
  • the 15-20 nm diameter quantum dots in 70% sucrose should theoretically yield a diffusion coefficient of approximately 2 x 10 -9 cm 2 /s at 33° Celsius (C) and the results obtained provided a diffusion coefficient of (2.68 +/- 0.28) x 10 -9 cm 2 /s.
  • HeLa cells were plated on 25 mm 1.5 Hestzig coverslip.
  • the cells were transfected with VSV-G GFP and then incubated at 37°C for 12 hours.
  • the coverslips were thoroughly washed in 1 M sodium hydroxide solution followed by mQ water and then blow dried with nitrogen and then plasma cleaned.
  • the coverslips were kept in UV for 2 hours before plating cells on them.
  • the cells were transfected with VSV-G GFP and then incubated at 37°C for 12 hours.
  • 2 pi of biotinylated VSV-G antibody, by abeam, and 2 m ⁇ of streptavidin conjugate 605 Quantum dots, by Thermo Fisher Scientific were mixed in 20 m ⁇ of CO2 independent media and incubated for 5 min.
  • this mixture was diluted in 180 m ⁇ of the media and added to the cells and incubated for 10 min.
  • the cells were then washed with the media and then sandwiched with 25 mm regular coverslips and vacuum grease so that the extra quantum dots are mostly removed from the solution.
  • the instrument was schematically described in Fig. la.
  • the sample was placed between two Nikon 60X Apo TIRF objectives of NA 1.49 and was illuminated by a 315 mW LASER of wavelength 561 nm.
  • the 100 nm gold beads on the Hestzig slides were used to focus and calibrate the whole system.
  • the custom 3 -way beam splitter was adjusted so as to get the interference and 120° phase shift between the cameras (Hamamatsu Orca Flash 4.0 sCMOS) and was obtained, as seen in the calibration curves in Fig. 6a, 6c, 6d, and 6f.
  • Data was collected on a 200x200 pixel area with 1 ms of exposure for 20,000 frames.
  • FIGS. 6a and 6c represent the correlation functions from two different regions of the sample.
  • FIG. 6b is the superimposed image from the experimental dataset for frames with regions marked which have been used for calculation in FIGS. 6a and 6c.
  • FIGS. 6d and 6f represent the correlation functions from two different regions of the cell membrane.
  • FIG. 6E is the image of the cell in which the correlation functions have been measured. The diffusion coefficient was .
  • the experimental validation can be limited by the speed of the cMOS cameras at 1 ms per frame which can limit the detection of diffusion to processes with diffusion coefficients below This limitation can be overcome with faster detectors.
  • FIG. 7a illustrates a flow diagram of a method according to the present technology.
  • the method is depicted and described as a series of acts.
  • acts in accordance with this disclosure can occur in various orders and/or concurrently, and with other acts not presented and described herein.
  • not all illustrated acts may be required to implement the methods in accordance with the disclosed subject matter.
  • a method for performing fluorescence cross correlation spectroscopy can include mounting a sample on a sample stage, the sample having a plurality of probe molecules, as shown in FIG. 7a, block 710a.
  • the method can further include illuminating the sample with coherent light to cause fluorescence from the probe molecules in the sample to pass through at least one of a first objective and a second objective to a beam splitter, as shown in block 720a.
  • the method can further include phase shifting the fluorescence at the beam splitter to provide a first beam output with a first phase shift to a first image detector and a second beam output with a second phase shift to a second image detectors, as shown in block 730a.
  • the method can further include measuring a difference in signal strength between a first signal strength from the first image detector and a second signal strength from the second image detector, wherein the difference in signal strength is caused by interference arising from a difference in phase between the first beam output and the second beam output, as shown in block 740a.
  • the method can further include detecting at least one of diffusion and a flow vector of a probe molecule of the plurality of probe molecules using a cross correlation function calculated from information received from the first image detector during an acquisition time and information received from the second image detector during the acquisition time, as shown in block 750a.
  • a system for fluorescence cross correlation spectroscopy can include a light source configured to produce a coherent excitation light to cause fluorescence of a plurality of probe molecules in a sample, as shown in FIG. 7b, block 710b.
  • the system can further include a sample stage adapted to receive the sample between a first objective and a second objective, as shown in block 720b.
  • the system can further include a first objective configured to pass fluorescence from the plurality of probe molecules to a beam splitter, as shown in block 730b.
  • the system can further include a second objective configured to pass fluorescence from the plurality of probe molecules to the beam splitter, as shown in block 740b.
  • the system can further include the beam splitter configured to phase shift the fluorescence to pass a first beam with a first phase shift and a second beam with a second phase shift, as shown in block 750b.
  • the system can further include a first image detector configured to receive the first beam with the first phase shift, as shown in block 760b.
  • the system can further include a second image detector configured to receive the second beam with the second phase shift, as shown in block 770b.
  • the system can further include a controller configured to: measure a difference in signal strength between a first signal strength from the first image detector and a second signal strength from the second image detector, wherein the difference in signal strength is caused by interference arising from a difference in phase between the first beam output and the second beam output; and determine at least one of a diffusion and a flow vector of a probe molecule of the plurality of probe molecules using a cross-correlation function calculated from information from the first image detector and information from the second image detector, as shown in block 780b.
  • a controller configured to: measure a difference in signal strength between a first signal strength from the first image detector and a second signal strength from the second image detector, wherein the difference in signal strength is caused by interference arising from a difference in phase between the first beam output and the second beam output; and determine at least one of a diffusion and a flow vector of a probe molecule of the plurality of probe molecules using a cross-correlation function calculated from information from the first image detector and information from the second image detector, as shown in
  • Another example provides at least one machine readable storage medium having instructions 700 embodied thereon for performing fluorescence cross correlation spectroscopy (FCCS), as shown in FIG. 7c.
  • the instructions can be executed on a machine, where the instructions are included on at least one computer readable medium or one non-transitory machine-readable storage medium.
  • the instructions when executed perform: measuring a difference in signal strength between a first signal strength from a first image detector and a second signal strength from a second image detector, wherein the difference in signal strength is caused by interference arising from a difference in phase between a first beam output and a second beam output, wherein the first beam output results from phase shifted fluorescence from a probe molecule of a plurality of probe molecules, the phase shifted fluorescence passed through a first objective and a beam splitter, while the second beam output results from phase shifted fluorescence passed through a second objective and the beam splitter, as in block 710c.
  • the instructions when executed perform: detecting a diffusion of the probe molecule of the plurality of probe molecules using a cross-correlation function calculated from information from the first image detector and information from the second image detector, as in block 720c.
  • the methods described herein may be implemented wholly or partially as computer readable program code executed by a processor and the computer readable code may be embodied on a non-transitory computer usable medium.
  • FIG. 8 illustrates a computing device 810 on which services or modules of this technology may execute.
  • a computing device 810 is illustrated on which a high level example of the technology may be executed.
  • the computing device 810 may include one or more processors 812 that are in communication with memory devices 820.
  • the computing device 810 may include a local communication interface 818 for the components in the computing device.
  • the local communication interface 818 may be a local data bus and/or any related address or control busses as may be desired.
  • the memory device 820 may contain modules 830 that are executable by the processor(s) and data for the modules.
  • a data store 822 may also be located in the memory device 820 for storing data related to the modules and other applications along with an operating system that is executable by the processor(s) 812.
  • the computing device 810 may further include or be in communication with a client device, which may include a display device.
  • the client device may be available for an administrator to use in interfacing with the computing device 810, such as to review operation of a virtual computing instance, make enhancements to machine learning models and so forth.
  • Various applications may be stored in the memory device 820 and may be executable by the processor(s) 812.
  • Components or modules discussed in this description that may be implemented in the form of software using high programming level languages that are compiled, interpreted or executed using a hybrid of the methods.
  • the computing device 810 may also have access to I/O (input/output) devices 814 that are usable by the computing devices.
  • I/O device 814 is a display screen that is available to display output from the computing devices.
  • Other known I/O device may be used with the computing device as desired.
  • Networking devices 816 and similar communication devices may be included in the computing device 810.
  • the networking devices 816 may be wired or wireless networking devices 816 that connect to the internet, a LAN, WAN, or other computing network.
  • the components or modules that are shown as being stored in the memory device 820 may be executed by the processor 812.
  • the term“executable” may mean a program file that is in a form that may be executed by a processor 812.
  • a program in a higher level language may be compiled into machine code in a format that may be loaded into a random access portion of the memory device 820 and executed by the processor 812, or source code may be loaded by another executable program and interpreted to generate instructions in a random access portion of the memory to be executed by a processor 812.
  • the executable program may be stored in any portion or component of the memory device 820.
  • the memory device 820 may be random access memory (RAM), read only memory (ROM), flash memory, a solid state drive, memory card, a hard drive, optical disk, floppy disk, magnetic tape, or any other memory components.
  • the processor 812 may represent multiple processors and the memory 820 may represent multiple memory units that operate in parallel to the processing circuits. This may provide parallel processing channels for the processes and data in the system.
  • the local interface may be used as a network to facilitate communication between any of the multiple processors and multiple memories. The local interface may use additional systems designed for coordinating communication such as load balancing, bulk data transfer, and similar systems.
  • modules may be implemented as a hardware circuit comprising custom VLSI circuits or gate arrays, off-the-shelf semiconductors such as logic chips, transistors, or other discrete components.
  • a module may also be implemented in programmable hardware devices such as field programmable gate arrays, programmable array logic, programmable logic devices or the like.
  • Modules may also be implemented in software for execution by various types of processors.
  • An identified module of executable code may, for instance, comprise one or more blocks of computer instructions, which may be organized as an object, procedure, or function. Nevertheless, the executables of an identified module need not be physically located together, but may comprise disparate instructions stored in different locations which comprise the module and achieve the stated purpose for the module when joined logically together.
  • a module of executable code may be a single instruction, or many instructions, and may even be distributed over several different code segments, among different programs, and across several memory devices.
  • operational data may be identified and illustrated herein within modules, and may be embodied in any suitable form and organized within any suitable type of data structure. The operational data may be collected as a single data set, or may be distributed over different locations including over different storage devices.
  • the modules may be passive or active, including agents operable to perform desired functions.
  • the technology described here may also be stored on a computer readable storage medium that includes volatile and non-volatile, removable and non-removable media implemented with any technology for the storage of information such as computer readable instructions, data structures, program modules, or other data.
  • Computer readable storage media include, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tapes, magnetic disk storage or other magnetic storage devices, or any other computer storage medium which may be used to store the desired information and described technology.
  • the computer readable storage medium may, for example, be in the form of a non-transitory computer readable storage medium.
  • the terms“medium” and“media” may be interchangeable with no intended distinction of singular or plural application unless otherwise explicitly stated. Thus, the terms “medium” and “media” may each connote singular and plural application.
  • the devices described herein may also contain communication connections or networking apparatus and networking connections that allow the devices to communicate with other devices.
  • Communication connections are an example of communication media.
  • Communication media typically embodies computer readable instructions, data structures, program modules and other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media.
  • a “modulated data signal” means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal.
  • communication media includes wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, radio frequency, infrared, and other wireless media.
  • the term computer readable media as used herein includes communication media.
  • a web service may be implemented by a software and/or hardware system designed to support interoperable machine-to-machine interaction over a network.
  • a web service may have an interface described in a machine-processable format, such as the Web Services Description Language (WSDL).
  • WSDL Web Services Description Language
  • Other systems may interact with the web service in a manner prescribed by the description of the web service's interface.
  • the web service may define various operations that other systems may invoke, and may define a particular application programming interface (API) to which other systems may be expected to conform when requesting the various operations.
  • API application programming interface
  • a web service may be requested or invoked through the use of a message that includes parameters and/or data associated with the web services request.
  • a message may be formatted according to a particular markup language such as Extensible Markup Language (XML), and/or may be encapsulated using a protocol such as Simple Object Access Protocol (SOAP).
  • SOAP Simple Object Access Protocol
  • a web services client may assemble a message including the request and convey the message to an addressable endpoint (e.g., a Uniform Resource Locator (URL)) corresponding to the web service, using an Internet-based application layer transfer protocol such as Hypertext Transfer Protocol (HTTP).
  • URL Uniform Resource Locator
  • HTTP Hypertext Transfer Protocol
  • web services may be implemented using Representational State Transfer (“RESTful”) techniques rather than message-based techniques.
  • RESTful Representational State Transfer
  • a web service implemented according to a RESTful technique may be invoked through parameters included within an HTTP method such as PUT, GET, or DELETE, rather than encapsulated within a SOAP message.

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Abstract

L'invention concerne un procédé (700a) permettant d'effectuer une spectroscopie par corrélation croisée de fluorescence (FCCS). Le procédé peut comprendre : la fluorescence de décalage de phase (730a) au niveau d'un diviseur de faisceau pour fournir une première sortie de faisceau avec un premier décalage de phase à un premier détecteur d'image et une seconde sortie de faisceau avec un second décalage de phase vers un second détecteur d'image ; la mesure d'une différence (740a) dans une intensité de signal entre une première intensité de signal provenant du premier détecteur d'image et une seconde intensité de signal provenant du second détecteur d'image, la différence d'intensité de signal étant provoquée par une interférence résultant d'une différence de phase entre la première sortie de faisceau et la seconde sortie de faisceau ; et la détection (750a) d'au moins l'un de la diffusion et d'un vecteur de flux d'une molécule sonde de la pluralité de molécules sondes à l'aide d'une fonction de corrélation croisée calculée à partir d'informations reçues en provenance du premier détecteur d'image et du second détecteur d'image pendant un temps d'acquisition.
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