WO2020016887A1 - Dispositifs optiques et procédés de mesure de l'efficacité du transfert d'énergie par résonance de type förster - Google Patents

Dispositifs optiques et procédés de mesure de l'efficacité du transfert d'énergie par résonance de type förster Download PDF

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WO2020016887A1
WO2020016887A1 PCT/IL2019/050794 IL2019050794W WO2020016887A1 WO 2020016887 A1 WO2020016887 A1 WO 2020016887A1 IL 2019050794 W IL2019050794 W IL 2019050794W WO 2020016887 A1 WO2020016887 A1 WO 2020016887A1
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donor
acceptor
fluorophore
excitation
fret
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PCT/IL2019/050794
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English (en)
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Eilon Sherman
Shai TSIPSHTEIN
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd
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Publication of WO2020016887A1 publication Critical patent/WO2020016887A1/fr
Priority to US17/149,586 priority Critical patent/US11674902B2/en

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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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

  • the present application relates in general to the field of detection of molecular interactions using fluorescence spectroscopy and microscopy.
  • the present application relates to optical devices and methods for measuring efficiency of the Forster resonance energy transfer (FRET) between a donor fluorophore and an acceptor fluorophore in a sample, for increasing resolution of a microscope suitable for single-molecule localisation (SML), and for imaging single molecular interactions by detecting single inter- or intramolecular interactions between a first molecular target labelled with the donor fluorophore and a second molecular target labelled with the acceptor fluorophore.
  • FRET Forster resonance energy transfer
  • Biochemical detection is likely the most primordial modality that emerged in the evolution of life. Without this ability to detect (bio)chemical compounds, life on earth would probably not exist. It is used for detecting nutrients, avoiding threats, finding mating partners and various forms of communication and social interaction between living forms.
  • biological pathogens including biological threat agents, are living organisms that reproduce and sustain a population, which amplify, grow and re-infect, thereby potentially resulting in an epidemic situation.
  • the biological pathogens represent an extremely diverse range of microorganisms, which have no seemingly common attributes other than infecting the human and animal populations. The problem is therefore to detect and identify them at the earliest stage of invasion and at the lowest concentration.
  • pathogen detection Majority of the modem (bio)molecular techniques used in pathogen detection are based upon sequence-based recognition of DNA, structural recognition of pathogens or pathogen biomarkers.
  • selection of the pathogen biomarkers introduces a serious challenge in the development of the sensors for detection of the biological pathogens. This is because most of the pathogen biomarkers have low selectivity and can distinguish between general classes of microorganisms but are not able to identify the specific species or strain of organism. For example, calcium dipicolinate is a unique component of endospores.
  • Dipicolinic acid can therefore be used to indicate the presence of endospores, but it cannot be able to distinguish between very dangerous Bacillus anthracis spores and other non-toxic Bacillus spores.
  • the presence of the DNA as an additional indicator will be able to determine that the unknown material is biological in nature but will not be able to identify its source (unless extensive sequence-based analysis is used).
  • cell metabolites are generally common to many different cell types and therefore extremely difficult to use for discrimination between specific microorganisms. In view of the above, there is a long-felt need for new methods and devices to detect and identify biological pathogens.
  • optical technologies intrinsically result in real-time (bio)chemical detection.
  • Optical devices based on these technologies have been available to military and civil defence for quite some time.
  • the common drawback of all the optical devices is low specificity. They mostly offer a generic detection capability at best, since the optical similarity of the target particles with benign, naturally occurring backgrounds makes them difficult to distinguish.
  • bio-agent detections strategies There are the some of the currently employed bio-agent detections strategies. Most represent a compromise between specificity, speed and cost.
  • Quantitative Polymerase Chain Reaction is capable of amplification and detection of a DNA sample from a single bio-agent cell within 30 minutes to hours. Knowing the pathogen nucleic acid sequence makes it possible to construct oligos for pathogen detection. These oligos are at the basis of many highly specific analytical tests now on the market.
  • Microarray-based detection can combine powerful nucleic acid amplification strategies with the massive screening capability of microarray technology, resulting in a high level of sensitivity, specificity, and throughput.
  • the cost and organizational complexity of performing a large number of PCR reactions for downstream microarray applications render this option feasible, but cumbersome, costly and thus, unattractive, particularly for the point-of-care diagnostics. This limitation has severely reduced the utility of this technique and impeded the continued development of downstream applications.
  • Proteins come together in order to facilitate various complex cell functions, including the DNA transcription and replication, enzymatic reactions, sensing and signal transduction. Since these protein interactions are stochastic in their nature, they can be heterogeneous and asynchronous. Thus, the identification and characterization of single protein interaction in cells are very important for understanding the complexity of the protein interactions. [0014] Various methods have been developed to measure protein interactions. The most popular method is immunoprecipitation. However, it operates in vitro and averages protein interactions over many interactions in millions of cells.
  • Advanced spectroscopic techniques such as Forster resonance energy transfer (FRET), proximity ligation (PLA), bimolecular fluorescence complementation (BiFC) and fluorescence cross-correlation spectroscopy (FCS), can report on protein interactions in single cells.
  • FRET Forster resonance energy transfer
  • PHA proximity ligation
  • BiFC bimolecular fluorescence complementation
  • FCS fluorescence cross-correlation spectroscopy
  • FRET is actually a non-radiative energy transfer between a pair of fluorescent molecules via dipole-dipole interaction.
  • Each such pair is made of a donor molecule and an acceptor molecule.
  • the typical distance over which the energy transfer occurs is in the range of 1 to 10 nm, which makes FRET a very useful tool for studying intra- and inter- molecular interactions in biological samples.
  • FRET FRET
  • overwhelming background as a result of residual direct excitation of the ensemble of acceptor molecules and 'bleed-through' of the ensemble of donor molecules.
  • the donor bleed-through and acceptor direct excitation are considered the significant obstacles in smFRET measurements. These contributions act on the bulk (or ensemble) of the emitters and compete with the faint signal of single emitters. These problems have actually limited the smFRET technique to in vitro measurements using a very low density of molecules.
  • the present invention relates to an optical device suitable for acquiring and measuring efficiency of the Forster resonance energy transfer (FRET) between a donor fluorophore and an acceptor fluorophore in a sample (10) to thereby resolving molecular interactions between said donor fluorophore and said acceptor fluorophore, said optical device comprising:
  • An excitation module comprising:
  • a) a first (11) and second (12) excitation source configured to emit a donor fluorophore excitation light (b) and an acceptor fluorophore excitation light (r), respectively, for exciting said donor fluorophore and said acceptor fluorophore in the sample (10);
  • a first excitation monochromator (14) configured to convert said donor fluorophore excitation light (b) into a donor fluorophore monochromatic excitation light beam (b'), and transmit said donor fluorophore monochromatic excitation light beam (b') to a beam combiner (13);
  • a second excitation monochromator configured to convert said acceptor fluorophore excitation light (r) into an acceptor fluorophore monochromatic excitation light beam (r'); and transmitting said acceptor fluorophore monochromatic excitation light beam (r') to a modulation unit (16);
  • the beam combiner (13) designed to combine said donor fluorophore monochromatic excitation light beam (b') and said modulated acceptor fluorophore monochromatic excitation light beam (r # ) into a single dichromatic excitation light beam (e);
  • B. A sample chamber containing a sample holder designed to hold said sample (10), to which said dichromatic excitation light beam (e) is directed;
  • An acquisition module comprising:
  • an emission monochromator configured to scan and transmit a predefined wave length range of a donor fluorophore emission (g), or donor and acceptor emission in a sequence;
  • a detector (19) configured to perform acquisition of the fluorescence emission (g) of said donor fluorophore, to measure intensity of the fluorescence emission (g) and to transfer the obtained fluorescence emission intensity data to a computing unit (20); and c) the computing unit (20) characterised in that :
  • ⁇ optionally display said fluorescence emission intensity data in a readable format
  • a FRET (first) algorithm characterised in that it is designed to acquire and measure the FRET efficiency between said donor fluorophore and said acceptor fluorophore in said sample (10), adapted for a lock-in detection and suitable for resolving weak and rare molecular interactions between the donor and acceptor in the sample (10);
  • FFT fast Fourier transform
  • the computing unit (20) is further designed to control the modulation unit (16) by providing a feedback to said modulation unit (16) for further modulating excitation intensity of the acceptor fluorophore monochromatic excitation light beam (r') and thereby modulating fluorescence emission intensity of said donor fluorophore in a predetermined frequency domain, resulting in reversible saturation of said acceptor fluorophore and consequently, frustration of the FRET process.
  • the optical device of the present invention is modular and may be configured to operate as a portable and highly sensitive fluorescence spectrophotometer (fluoro meter), lumino meter, fluorescence microscope or combinations thereof.
  • the excitation module, the sample chamber and the acquisition module of the optical device can be configured according to a desired application and adapted for the particular application.
  • the sample chamber (B) may be chosen as a fluorescence multiplate reader for laboratory high-throughput and rapid, multiplexing analysis of multiple samples for point-of-care diagnostics.
  • the detector (19) and the computing unit (20) are combined in a single unit designed to perform acquisition of the fluorescence emission (g), to measure its intensity, to process the fluorescent emission data and optionally display it in a readable format and/or output it to an external memory or user's interface.
  • the acquisition module (C) may be a part of a smartphone or any other mobile device or gadget suitable for performing the desired measurements.
  • the sample chamber (B) combined with the acquisition module (C) constitutes a fluorescence microscope, or said optical device is a combined fluorometer and a fluorescence microscope installed in a single case, or said optical device is incorporated inside a fluorescence microscope.
  • Said microscope is designed to generate raw data from single-molecule localisation as a video or as a series of static images and to further process said raw data generated by the microscope, to integrate said fluorescence emission (g) intensity data and said microscope raw data and to provide information on the molecular interactions and on the nanometre proximity of single molecules in a readable format or to output said information to an external memory or user's interface.
  • said sample chamber is a multiplexing spectrophotometric or imaging device, or part thereof, suitable for multiplexing multiple samples (10).
  • An example of such multiplexing device is a microplate reader.
  • the excitation sources (11) and (12) may be selected from a wide-spectrum halogen lamp, an arc-lamp or a mercury- vapour lamp, configured to emit said donor fluorophore excitation light (b) and said acceptor fluorophore excitation light (r) in a predetermined wavelength range or near peak wavelength of said donor fluorophore or said acceptor fluorophore, respectively.
  • the excitation monochromators (14, 15) in this case may be photomultiplier tubes (PMTs), and the emission monochromator (18) may be a diffraction grating.
  • first and second excitation filters (14, 15) are first and second excitation filters (14, 15), respectively, designed to select and transmit a narrow-wavelength beam of the excitation wavelength of light from the corresponding excitation source (11, 12), while said emission monochromator (18) is the emission filter (18) designed to transmit a narrow-wavelength beam of said donor fluorophore emission (g).
  • the optical device of the invention may further comprise a filter cube (17) installed between the excitation module (A), the sample chamber (B) and the acquisition module (C) in optical communication with said excitation module (A), said sample chamber (B) and said acquisition module (C), wherein said filter cube (17) comprises a two-channel dichroic mirror (41) configured to direct the modulated dichromatic excitation light beam (e) to the sample (10).
  • the acquisition module (C) may further comprise one or two mirrors (21, 2G), for example two-channel dichroic mirrors, configured to transmit the light emitted from the sample (10) to the emission filter (18).
  • the filter cube (17) may further comprise an excitation filter (42) and at least one optional emission filter (43) having two transmission windows, said emission filter (43) is configured to optionally filter out the light emitted from the sample (10) and to transfer it to the acquisition module (C).
  • the modulation unit (16) may be a modulating half-wave plate suitable for modulating polarisation of said acceptor fluorophore monochromatic excitation light beam (r'), or an acousto-optic modulator (AOM) suitable for modulating the frequency of said acceptor fluorophore monochromatic excitation light beam (r') using oscillating sound waves, or a vibrating mirror suitable for modulating the frequency of said acceptor fluorophore monochromatic excitation light beam (r') by mechanical diversion of the mirror.
  • AOM acousto-optic modulator
  • the sample chamber (B) of the optical device of the invention is further equipped with an objective configured to gather the fluorescence emission light (g) from the sample (10) to produce a fluorescence image, and optionally focus the excitation light beam (e) on the sample (10).
  • the detector (19) may be equipped with a magnification eyepiece (ocular) for viewing, imaging, focusing and increasing the overall magnification of a fluorescent image.
  • the detector (19) is an electron- multiplying charge-coupled device (EMCCD) imager, a charge-coupled device (CCD) imager, an avalanche photodiode (APD), a photomultiplier tube (PMT), scientific complementary metal-oxide- semiconductor (sCMOS) imager, or CMOS imager of a smartphone camera, a stand-alone camera, or a camera of any mobile device or gadget, said detector (19) optionally having a focusing apparatus and a computer link.
  • the detector (19) is a CMOS imager of a smartphone camera.
  • the computing unit (20) of said microscope further comprises: ⁇ a second algorithm for analysing said microscope raw data images obtained from single molecule localisation, said second algorithm is characterised in that it is designed to localise the donor fluorophore in the sample (10) and to transmit data on the localisation of said donor fluorophore molecules in said sample (10) to a third algorithm;
  • the third algorithm designed to receive and integrate the analytical data produced by, and received from the FRET (first) algorithm, the FFT algorithm and the second algorithm, and to output information on the molecular interaction and on nanometre proximity of the single donor and acceptor fluorophore molecules, in a readable format or to output said information to an external memory or user's interface.
  • the optical device of the present invention may further comprise a third excitation source suitable for photoactivation or photo switching of the donor fluorophore.
  • the optical device of the present invention may be used in various applications, due to its modular versatility. Thanks to its fluorometer's functionality, the optical device can be used in a method for resolving inter- or intramolecular interactions between a first molecular target labelled with a donor fluorophore and a second molecular target labelled with an acceptor fluorophore suitable for forming the FRET interactions with said donor fluorophore in the sample (10).
  • This method is carried out by placing said sample (10) in the sample chamber of the optical device of the present invention, and comprises the following steps:
  • the optical device of the present invention can be used in a method for increasing resolution of a fluorescence microscope suitable for single-molecule localisation microscopy (SMLM) and imaging single molecular interactions by detecting single inter- or intramolecular interactions between a first molecular target labelled with a donor fluorophore and a second molecular target labelled with an acceptor fluorophore capable of forming the FRET interactions with said donor fluorophore, or measuring the nanometre proximity between said first and second molecular targets, in the sample (10).
  • This method is carried out by placing said sample (10) on a microscope slide in the sample holder of the optical device, and comprises the following steps:
  • said donor fluorophore is either:
  • a photoactivatable fluorophore capable of switching from a non-emissive to an emissive state upon excitation with the third excitation source at an activating wavelength and then emitting fluorescence upon excitation at an excitation wavelength in a defined region of space at a given interval of time
  • ⁇ a photo switchable fluorophore capable of switching from one emissive state to another emissive state upon excitation with the third excitation source at an activating wavelength.
  • lock-in detection which comprises the following steps:
  • the first molecular target and the second molecular target can be fragments of the same molecule, thereby undergoing intramolecular interactions, or different molecules, thereby undergoing intermolecular interactions.
  • These first and second molecular targets are selected each independently selected from an antigen, antibody, antibody fragment, enzyme, substrate or inhibitor, receptor, protein or organic molecule, lectin, sugar, DNA, RNA and aptamer.
  • the first and the second molecular targets are hybridization, hydrolysis or similar (e.g. Scorpion ® or Molecular Beacon) probes suitable for binding closely to a common target DNA or RNA template, thereby facilitating the FRET between them and detecting the target.
  • the first and second molecular targets are a primary antibody and a secondary antibody, or a primary antibody and a fluorescent target, or antibody fragments (e.g. Fabs) acting as either primary and/or secondary antibody.
  • Fig. la schematically shows the sensitised emission FRET occurring in the donor- acceptor pair.
  • Fig. lb shows Jablonski diagram for the sensitised emission FRET process.
  • Fig. lc shows an exemplary excitation and emission spectrum of a donor and acceptor.
  • the overlapping areas of the two excitation spectra and two emission spectra create the problems of the acceptor direct excitation and donor bleed-through that are discussed in the present application.
  • Fig. 2a schematically shows the FRET frustration in the donor-acceptor pair, accompanied by increased emission intensity of the donor.
  • Fig. 2b shows a Jablonski diagram for the frustrated FRET process via acceptor saturation using intense acceptor excitation.
  • FIG. 3 schematically shows an optical device of the present invention.
  • FIG. 4 schematically shows a filter cube (17), which is an optional component in the optical device of the present invention.
  • Fig. 5a schematically shows the frames in the modulated excitation of the donor and of the acceptor.
  • Fig. 5b schematically demonstrates that for a FRET donor-acceptor pair, the frustrated FRET caused by the acceptor- modulated excitation, results in modulation of the donor fluorescence intensity.
  • FIG. 6 schematically shows a flowchart for the FRET calculation used with the optical device of the present invention operating in the frustrated FRET mode.
  • FIG. 7 schematically shows the optical device of the present invention having microscope functionalities.
  • Figs. 8a-8d schematically show four different configurations of the emission filter (18) and the detector (19).
  • Figs. 8a-8d show calculation of the figure of merit (FOM) for a time trajectory of a single molecule.
  • Fig. 9 schematically shows an algorithm for the FRET calculation used with the optical device of the present invention operating in a sensitised emission mode.
  • Fig. 10a is one exemplary frame out of a dSTORM imaging movie of a cell.
  • the PAGFP Actin was conjugated to Alexa Fluor ® 555 and Alexa Fluor ® 647.
  • Fig. 10b shows a time trajectory of the fluorescence intensity of the molecule.
  • Fig. 10c shows the absolute value of the Fourier transform of the time trajectory of the intensity.
  • the black circle with a dot marks the modulation frequency (the Nyquist frequency), while the dashed black line marks the median of the absolute values of the Fourier transform for all the frequencies.
  • Fig. lOd shows the accuracy of the experiment with the number of molecules as a function of s, the uncertainty in location of the single molecules (nm).
  • Fig. lOe shows a spectrogram that was built in MATFAB ® for the windowed Fourier transform of a single molecular trajectory.
  • Fig. lOf shows the comparative average FOM against all trajectories that were longer than a minimal length for the 'donor- acceptor' sample versus the 'donor-only' sample. Each dot marks the average FOM of all the molecules in a single cell. The average FOM is 2.05 ⁇ 0.06 for the donor-acceptor sample and 1.32 ⁇ 0.02 for the 'donor-only' sample. The p-value of the populations is 8xl0 10 . The error bars show the SEM (standard error of the mean).
  • Figs lla-lld show the dSTORM-FRET imaging in the present example.
  • Fig. 11a shows the dSTORM image of a cell (on the left) and magnification of a spot in a yellow frame (on the right), for the sample with protein-protein interactions, i.e. the cell has both donor and acceptor and hence, the FRET process between them. The FRET and the donor- acceptor distance are detected via the frustrated FRET process.
  • Fig. lib shows the dSTORM image of a cell (on the left) and magnification of a spot in a yellow frame (on the right), for the 'donor-only' sample with no protein-protein interactions (negative control).
  • Fig. 11c shows the energy transfer efficiency E distribution histograms of the molecular pairs from 18 'donor-acceptor' cells.
  • the average E value is 0.2 ⁇ 0.0l, as seen in the figure.
  • Fig. lid shows a distance distribution of the molecular pairs from 18 'donor-acceptor' cells.
  • the average distance between the donor and the acceptor is 6.5 ⁇ 1 nm, as seen in the figure.
  • Figs. 12a- 12b show two possible schemes for the SILM method using antibody detection of a target molecule.
  • Fig. 12a shows the first scheme, where the donor fluorescent protein (FP) is produced by the cell and is tagged to a specific target molecule to be measured.
  • the synthetic fluorophore which functions as the acceptor is conjugated to an antibody that is specific either to the target molecule or to the donor itself.
  • Fig. 12b shows the second scheme, where both the donor and acceptor are synthetic fluorophores.
  • the donor is conjugated to a primary antibody that is specific to some target molecule.
  • the acceptor is delivered to the donor by a secondary antibody to which it is conjugated.
  • Fig. 13 shows the PAGFP intensity in the absence of Alexa Fluor ® 555.
  • the lower series of dots mark the frames where excitation was done by 488-nm laser, and the upper series of dots mark the frames where excitation was performed by 488-and 56l-nm laser. Additional background that appeared due to 56l-nm excitation was subtracted from the respective frames.
  • Fig. 14 shows the saturation curve of Alexa Fluor ® 647 acceptor fluorophore, where Y axis is the fluorescence intensity mean values (arbitrary units).
  • Figs. 15a-15b show the fluorescence decay of Alexa Fluor ® 647 with the solid line fitting to double exponential decay and the residuals of this double exponential fit, respectively.
  • Figs. 15c-15d show the fluorescence decay of Alexa Fluor ® 555 with the solid line fitting to double exponential decay and the residuals of this double exponential fit, respectively.
  • FIGs. 16a-16c showing the images of the donor, acceptor and the sensitised emission, respectively, in a cell footprint.
  • Fig. 16a shows intensity of the donor fluorophore Alexa Fluor ® 555 in the donor channel.
  • Fig. 16b shows intensity of the acceptor fluorophore Alexa Fluor ® 647 in the acceptor channel.
  • Fig. 16c shows the cell image with the sensitised emission calculated for each pixel separately.
  • Figs. 17a-17b show the fluorescence decay of Alexa Fluor ® 555 in a 'donor-only' sample with the solid line fitting to double exponential decay and the residuals of this double exponential fit, respectively.
  • Figs. 17c-17d show the fluorescence decay of Alexa Fluor ® 555 in a 'donor-acceptor' sample with the solid line fitting to double exponential decay and the residuals of this double exponential fit, respectively.
  • Fig. 17e shows the modulation between intensity in even and odd frames of Alexa Fluor ® 555 in the 'donor-only' sample.
  • the modulation is caused by background that is generated by the 56l-nm excitation.
  • Fig. 17f shows the experimental differences between consecutive even and odd frames of Alexa Fluor ® 555 in the 'donor-acceptor' sample. The differences is the result of the frustrated FRET process, and it decays because the acceptor decays faster than the donor.
  • Fig. 19 shows a histogram of the length of intensity trajectories of molecules taken from 36 cells.
  • Fig. 20 shows the average of the frequencies' ratio for trajectories longer than a minimum length.
  • the upper dots are the average on samples with donor and acceptor, while the lower dots are the average of 'donor-only' samples.
  • the averages are taken on trajectories from 18 cells for each sample.
  • the error bars show the SEM (standard error of the mean).
  • Fig. 21a shows the average frequency ratio against the length of the trajectories for the sample with donor and acceptor taken from 18 cells.
  • Fig. 21b shows the average frequency ratio against the length of the trajectories for the sample with 'donor-only' taken from 18 cells.
  • Fig. 22 shows spectrograms built in MATLAB ® for the windowed Fourier transform of a single molecular trajectory with different time windows (8, 16, 24, 32, 48 and 64).
  • Fig. 23 shows the average FOM against all trajectories that were longer than a minimal length (same as Fig. 21 only that the FOM was calculated in this case with the windowed Fourier transform).
  • Fig. 24a shows the average FOM against the length of the trajectories for the sample with donor and acceptor taken from 18 cells.
  • Fig. 24b shows the average FOM against the length of the trajectories for the sample with 'donor-only' taken from 18 cells.
  • Fig. 25a-25c show an example of the FOM threshold determination.
  • Fig. 25a shows the total number of trajectories with the FOM larger than a specified value. Upper dots mark the donor-acceptor sample and the lower dots mark the 'donor-only' sample.
  • Fig. 25b shows the fraction of trajectories compared to the total number of trajectories.
  • Fig. 25c shows the minimal FOM ratio between the fractions in Fig. 25b.
  • Fig. 26a shows a FOM histogram for a cell containing both the donor and the acceptor.
  • the black dashed line in the histogram marks a threshold of FOM that is equal to 4.5. After thresholding, 47 molecules which were part of a FRET pair were identified in this 'donor- acceptor' cell (with 10% false positive).
  • Fig. 26b shows a FOM histogram for a cell containing only the donor.
  • the black dashed line in the histogram marks a threshold of FOM that is equal to 4.5. After thresholding, only two molecules which were part of a FRET pair were identified in this 'donor-only' cell.
  • Figs. 27a-27d show simulations and estimation of the distance between the donor and its nearest acceptor.
  • Fig. 27a shows a geometrical model of the interaction between the primary and secondary antibodies (considered as thin rigid rods).
  • Fig. 27b shows the histogram of all donor-acceptor in 1000 simulations, representing random geometries according to the model in Fig. 27a.
  • Fig. 27c shows the error in estimation of the effective energy transfer ( E mo dei - E) for the simulated data in Fig. 27b by Models 1 and 2 as a function of E.
  • Fig. 27d shows the error in estimation of the effective energy transfer ( E mo dei - E) for the simulated data in Fig. 27b by Models 1 and 2 as histograms.
  • Fig. 27e shows the relative error in the distance between the donor and the nearest acceptor, GPA""", as a function of the effective number of equidistant emitters (ranging from 1 in Model 1 to 2 in Model 2).
  • GPA the nearest acceptor
  • Figs. 28a-28b show the prototype device of the present invention having a smartphone as the acquisition module.
  • Fig. 29 shows the screenshot image of a smartphone generated with the CMOS camera for the emission of the short DNA molecules singly labelled with a fluorescent dye Cy3B in the sensitised emission experiment.
  • Figs. 30a-30c show the histograms of 'positive' and 'negative' FRET molecules that can be separated.
  • the Cy3B dye molecule which was used as a donor, was labelled at the 3-prime end of a short single DNA strand.
  • the Atto647N dye molecule which was used as an acceptor, was labelled at the 5-prime of the complementary single DNA strand.
  • the DNA molecules contained only the donor label, without the acceptor.
  • the term “about” is understood as within a range of normal tolerance in the art, for example within two standard deviations of the mean. In one embodiment, the term “about” means within 10% of the reported numerical value of the number with which it is being used, preferably within 5% of the reported numerical value. For example, the term “about” can be immediately understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In other embodiments, the term “about” can mean a higher tolerance of variation depending on for instance the experimental technique used.
  • adjacent another feature may have portions that overlap or underlie the adjacent feature.
  • the present invention essentially relates to an optical device suitable for measuring the Forster resonance energy transfer (FRET) in a sample.
  • the device can be operated in different FRET modes, which will be discussed below.
  • the device comprises a two-beam excitation FRET module and an image acquisition module; the modules may vary in their structure dependent on a specific application.
  • the applications of the optical device of the present invention are many and include, but not limited, to measuring single interactions in cells and detection of pathogens in a sample.
  • the method of measurements with the optical device of the present invention is essentially based on the combination of the FRET acquisition with lock- in detection. As an option, the FRET measurements may be combined with the single molecule localisation microscopy (SMLM).
  • SMLM single molecule localisation microscopy
  • the SILM of the present invention is based on the combination of the FRET measurement with the direct stochastic optical reconstruction microscopy (dSTORM) to detect and localise, for example, single molecular interactions in different cells, to measure the smFRET inside the cells, expressing a high density of molecules, and to detect pathogens in a sample using labelled DNA molecules.
  • dSTORM direct stochastic optical reconstruction microscopy
  • the FRET is a non-radiative energy transfer, where the term "non-radiative" is of particular importance.
  • the non-radiative energy transfer is essentially based on a dipole-dipole coupling mechanism between the donor and acceptor of the interacting molecular pair in their excited states. It is not a trivial emission of a photon, but a re-absorption by the donor and acceptor.
  • Sensitised emission is a direct two-channel imaging technique using an algorithm that corrects for excitation and emission crosstalk;
  • Acceptor photobleaching (sometimes called donor dequenching) is a technique capable of measuring increased donor emission when the acceptor is photobleached;
  • Fluorescence lifetime imaging microscopy FRET FLIM FRET is a technique capable of detecting fluorescence lifetime changes of donor
  • Fluorophore donor spectral imaging is a technique involving excitation at one or two wavelengths and measuring the spectral profiles of both donor and acceptor.
  • Figs la-lc schematically show the sensitised emission FRET process in a donor- acceptor pair of the molecules.
  • the donor such as green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • the donor is normally excited, for example with a blue light, it is very quickly relaxed from a high excited state by interconversion or preparative relaxation to the first electron excited state. From there it can go back to the ground state either through the non-radiative decay interconversion, or through the radiative pathway by emitting a photon.
  • the molecular orbitals of the donor can energetically couple with the orbitals of the acceptor, the dipole-dipole coupling occurs, thereby creating an extra channel for the non-radiative decay with much shorter excited state lifetimes.
  • the donor actually emitting less light, or in other words, quenching the donor when the sensitised emission FRET occurs.
  • the acceptor gets excited by this process as a result of the same dipole- dipole coupling and starts emitting fluorescence. So, by exciting the donor in this process, the emission is received also partially from the acceptor.
  • the sensitised emission is perhaps the simplest FRET method, because there is a single excitation source, from which the donor fluorophore is excited, and the signal is collected using emission filters chosen for both the donor fluorescence and the acceptor fluorescence.
  • the acceptor fluorescence increases in the presence of donor, whereas the donor fluorescence decreases in the presence of the acceptor.
  • the ratiometric change of fluorescence intensity can then be used to measure the FRET. This is the most straight-forward approach to measuring the process of the FRET. Figs, la-lb schematically show this process. It is inherently based on quenching of the donor molecules during the process, thereby increasing fluorescence intensity of the acceptor.
  • the images recorded in this approach are actually the donor emission upon excitation of the donor (DD) and the acceptor emission upon excitation of the donor (DA). The ratio between their intensities can indicate the FRET efficiency.
  • the direct measurement of the FRET is not practical because of the donor bleed-through and acceptor direct excitation, as will be explained below. This is essentially a crosstalk between the two fluorophores (donor and acceptor). Thus, it is very difficult to obtain quantitatively accurate FRET data with this approach. Additional control experiments are required in order to establish the presence or absence of the FRET in a sample.
  • the major parameter that is used to quantify the FRET is the FRET efficiency E, which is basically the number of excited donors that transfer the energy to the acceptor, divided by the number of photons absorbed by the donor. So, this is basically a fraction of donors that transfer their energy to the acceptor.
  • the FRET efficiency E can also be expressed as the following ratio:
  • R is the Forster radius (typically in the order of nanometres) that represents the distance between the donor and acceptor at which the FRET efficiency is 50% (when half of the excited donor molecules transfer their energy to the acceptor), and r is the distance between the donor and acceptor. Since it is r 6 , it makes the dependence very steep. So, measuring the FRET efficiency E allows to assess the distance r between the donor and acceptor.
  • the stoichiometry of the donor and acceptor can be kept constant in each pixel. This specifically works, for example, in sensors that can measure a change in a physiological parameter, such as calcium, or measure phosphorylation.
  • the sensitised emission measurements can be useful for detecting rapid dynamic changes and is especially useful when examining fluorescent proteins where the FRET dynamic range is large, and the stoichiometry of the donor and acceptor is fixed at a 1:1 ratio.
  • the sensitised emission FRET shown in Figs la-lc can be implemented in various types of microscopes, for example, in confocal microscopes or in wide-field imaging with proper filters, and it is very fast (suitable for live-cell imaging), because only two images need to be acquired in this approach.
  • this approach is not quantitative, because it allows to measure only differences in excited states, and the stoichiometry of donor and acceptor should be constant, which basically means that in many cases, they have to label different parts of the same molecule.
  • the sensitised emission is used as a simple FRET method for acquiring preliminary information about the presence or absence of the object of interest, such as a DNA molecule of a certain pathogen.
  • the answer in this case provides the initial indication for the presence of the molecule in the sample. Based on this initial indication, the system can be calibrated. Furthermore, the algorithm of the present invention will proceed to a further, quantitative sensitive measurement of the sample, which is based on another approach of the FRET measurement. This aspect will be described below.
  • the correction factors for the FRET measurements are determined as follows. First, for the sample containing only the donor, the intensity ratio Si between: (a) the donor emission at the acceptor emission maximum (the wavelength of the donor bleed-through into the acceptor detection channel), and (b) at the donor emission maximum, is determined. Second, for the sample containing only the acceptor, the intensity ratio S 2 between: (a) the acceptor excitation at the donor excitation maximum, and (b) the acceptor excitation maximum, is determined. Then, by measuring the sample containing both the donor and acceptor, by exciting only the acceptor in the presence of the donor, it is possible to determine how much acceptor is directly excited.
  • the bleed-through correction is initially determined.
  • the donor is excited in the sample in the absence of the acceptor, and therefore, the recorded are two images: the donor measured at its emission maximum (F D ) and at the emission wavelength of the acceptor (F F ).
  • the correction for the direct excitation is introduced.
  • the acceptor is measured in the sample in the absence of the donor.
  • the recorded are two images: the acceptor excited at the donor excitation maximum (F F ) and at the excitation wavelength of the acceptor (F A ).
  • the scaled image intensity F F is then divided by the scaled acceptor intensity F A to obtain the FRET apparent efficiency that is proportional to the real FRET efficiency and to the fraction of interacting molecules.
  • This scaling is easy to implement in most of the conventional microscopes, such as a confocal microscope or a wide-field microscope with appropriate filters.
  • the overall measurement is fast because one can switch the filters quickly on a filter wheel or on a confocal microscope.
  • this method is still semi-quantitative, because the resulted image obtained after scaling and correction is proportional to the relative concentration of interacting molecules, and it depends on the external calibration described above.
  • the signal-to- noise ratio of this method is also low because of the scaling that requires measuring three separate samples.
  • the spectra of the molecules should be invariant to the environment, for example it should be the same in a lipid environment and in a cytoplasmic environment. That is the case for many fluorescent proteins, notably (for more details on the calculation of the sensitised FRET and further discussion, see Example 11 below).
  • acceptor saturation is also referred here as "acceptor saturation"
  • fluorophores can undergo excitation through the absorbance of a photon and a following excitation of an electron from a low energy level (typically, the ground state) into a higher energy level (typically, the first excited state).
  • the electron may lose some energy via phonons, and then spontaneously return to the ground state while emitting a photon (a process known as fluorescence or spontaneous emission).
  • the lifetime for fluorescence emission is typically in nanoseconds. This process is schematically shown for the donor and acceptor fluorophores in Fig. la.
  • FD is the donor image recorded when the donor quenched by the active acceptor
  • F D 1 is the donor image recorded when the acceptor is photobleached. It will be more precisely defined in the Examples.
  • the FRET process can also be measured by the fluorescence lifetime imaging microscopy (FLIM) capable of measuring the excited state lifetime of a donor.
  • FLIM fluorescence lifetime imaging microscopy
  • the donor is excited, and instead of measuring its emission intensity, the excited state decay is measured as a function of time (fluorescence decay kinetics).
  • FRET fluorescence lifetime imaging microscopy
  • the donor and the acceptor are both excited, the energy transfer will be largely blocked (due to the mismatch of their spectral overlap) and thus, the donor fluorescence will be de-quenched and enhanced.
  • the acceptor fluorophore enters a short-lived and reversible dark state (e.g. a triplet state), it cannot undergo further excitation and the energy transfer will be largely blocked.
  • the frustrated FRET is inherently a nonlinear process requiring excitation of both donor and acceptor.
  • Fig. 3 schematically showing an optical device of the present invention, as described below.
  • the present application relates to an optical device suitable for acquiring and measuring efficiency of the Forster resonance energy transfer (FRET) between a donor fluorophore and an acceptor fluorophore in a sample (10) to thereby resolving molecular interactions between said donor fluorophore and said acceptor fluorophore, said optical device comprising:
  • An excitation module comprising:
  • a) a first (11) and second (12) excitation source configured to emit a donor fluorophore excitation light (b, for example blue) and an acceptor fluorophore excitation light (r, for example red), respectively, for exciting said donor fluorophore and said acceptor fluorophore in the sample (10);
  • a first excitation monochromator or filter (14) configured to convert said donor fluorophore excitation light (b) into a donor fluorophore monochromatic excitation light beam (b'), and transmit said donor fluorophore monochromatic excitation light beam (b') to a beam combiner (13);
  • a second excitation monochromator or filter configured to convert said acceptor fluorophore excitation light (r) into an acceptor fluorophore monochromatic excitation light beam (r'); and transmitting said acceptor fluorophore monochromatic excitation light beam (r') to a modulation unit (16);
  • said modulation unit is characterised in that it is designed to modulate excitation intensity of said acceptor fluorophore monochromatic excitation light beam (r') by tuning excitation intensity of said acceptor fluorophore monochromatic excitation light beam (r') from complete blocking to at least about 30% transmission, preferably at least about 50%, more preferably at least about 70%, such that said acceptor fluorophore becomes optically saturated in order to provide frustration (quenching) of said FRET process, and directing the modulated acceptor fluorophore monochromatic excitation light beam obtained (r # ) to said beam combiner (13); and
  • the beam combiner (13) designed to combine said donor fluorophore monochromatic excitation light beam (b') and said modulated acceptor fluorophore monochromatic excitation light beam (r # ) into a single dichromatic excitation light beam (e);
  • An acquisition module comprising:
  • an emission monochromator configured to scan and transmit a predefined wave length range of a donor fluorophore emission, or an emission filter (18) designed to transmit a narrow-wavelength beam of said donor fluorophore emission (g) or donor and acceptor emission in a sequence;
  • a detector (19) configured to measure intensity of the fluorescence emission (g) of said donor fluorophore and transfer the obtained fluorescence emission intensity data to a computing unit (20);
  • said computing unit (20) is characterised in that it is designed to be synchronised with said detector (19) and with said modulation unit (16), to analyse the fluorescent emission intensity data transferred from said detector (19), optionally display said fluorescence emission intensity data in a readable format and transfer said data to an external memory;
  • FFT fast Fourier transform
  • said computing unit (20) is further designed to control the modulation unit (16) by providing a feedback to said modulation unit (16) for further modulating excitation intensity of the acceptor fluorophore monochromatic excitation light beam (r') and thereby modulating fluorescence emission intensity of said donor fluorophore in a predetermined frequency domain, resulting in reversible saturation of said acceptor fluorophore and consequently, frustration of the FRET process.
  • donor fluorophore refers to a light-sensitive, fluorescence emitting molecule, which initially in its electronic excited state, may transfer energy to "acceptor fluorophore" through non-radiative dipole-dipole coupling.
  • the donor fluorophore must be bright (having high quantum yield and high absorption coefficient), stable (having long-living fluorescent excited state and low photo bleaching), and insensitive to the acceptor fluorophore excitation light.
  • acceptor fluorophore refers to a light-sensitive molecule, which initially in its ground-level electronic state may accept energy from "donor fluorophore" through non-radiative dipole-dipole coupling.
  • the acceptor fluorophore must have a large Forster distance Ro from the donor fluorophore (high spectral overlap of the absorption spectrum of an acceptor fluorophore with the fluorescence emission spectrum of a donor fluorophore), low photobleaching, must be insensitive to the donor fluorophore excitation light, having no cross-talk of its fluorescence emission spectrum with the fluorescence emission spectrum of the donor fluorophore, and must be capable of undergoing reversible saturation of its fluorescence emission under light excitation.
  • Synthetic fluorophores used in the present invention may include, but are not limited to generic or proprietary emitters listed in Table 1 below: Table 1. Generic or proprietary exemplary emitters suitable for use in the present invention
  • the first excitation source (12) can be a wide-spectrum halogen lamp, an arc-lamp or a mercury- vapour lamp, emitting said donor fluorophore excitation light (b) in a predetermined wavelength range or near peak wavelength of said donor fluorophore.
  • the optical device of the present embodiments then comprises the first excitation monochromator (14), which collimates said donor fluorophore excitation light (b) to obtain a donor fluorophore collimated excitation light beam, converts said donor fluorophore collimated excitation light beam into a donor fluorophore monochromatic excitation light beam (b'), and transmits said donor fluorophore monochromatic excitation light beam (b') to the beam combiner
  • the second excitation source (12) can also be a wide-spectrum halogen lamp, an arc- lamp or a mercury-vapour lamp, emitting said acceptor fluorophore excitation light (r) in a predetermined wavelength range or near peak wavelength of said acceptor fluorophore.
  • the optical device comprises the second excitation monochromator (15) that collimates said acceptor fluorophore excitation light (r) to obtain an acceptor fluorophore collimated excitation light beam, converts said acceptor fluorophore collimated excitation light beam into an acceptor fluorophore monochromatic excitation light beam (r'), and transmits said acceptor fluorophore monochromatic excitation light beam (r') to the modulation device (16).
  • the first and second excitation monochromators (14, 15) are optical devices that transmit a selectable narrow band of wavelengths of light chosen from a wider range of wavelengths available at the input.
  • the first excitation source (11) and the second excitation source (12) each is a laser, a light-emitting diode (LED) or a laser diode.
  • the first and second excitation monochromators (14, 15) are the first and second excitation filters (14, 15), respectively, and the emission monochromator (18) is the emission filter (18).
  • the optical device further comprises a first polariser (not shown in the figure) adjacent to said first excitation filter (14) for converting said donor fluorophore monochromatic excitation light beam (b') to a plane-polarised donor fluorophore monochromatic excitation light beam and directing said beam to said beam combiner (13); and a second polariser (not shown in the figure) adjacent to said second excitation filter (15) for converting said acceptor fluorophore monochromatic excitation light beam (r') to a plane- polarised acceptor fluorophore monochromatic excitation light beam and directing said beam to the modulation unit (16).
  • At least one of said first and second polarisers or each one of them comprises an adjustable Nicole prism for producing linearly-polarised excitation light beam from said plane-polarised excitation light beam.
  • at least one of said polarisers or each one of them further comprises a half-wave plate for shifting, and consequently adjusting, the polarisation direction of said linearly-polarised excitation light beam, wherein said Nicole prism is adjustable by rotating said half-wave plate to collimate said plane-polarised excitation light beam.
  • the modulation unit (16) of the optical device of the present invention is a modulating half-wave plate capable of modulating polarisation of said acceptor fluorophore monochromatic excitation light beam (r'), thereby modulating the excitation intensity of said acceptor fluorophore in said sample.
  • the modulation unit can be an acousto optic modulator (AOM) capable of modulating the frequency of said acceptor fluorophore monochromatic excitation light beam (r') using oscillating sound waves, thereby modulating the excitation intensity of said acceptor fluorophore in the sample (10).
  • AOM acousto optic modulator
  • it can be a vibrating mirror capable of modulating the frequency of said acceptor fluorophore monochromatic excitation light beam (r') by mechanical diversion of the mirror, thereby modulating the excitation intensity of said acceptor fluorophore in the sample (10).
  • the detector (19) of the optical device of the invention can be a photomultiplier tube (PMT), an avalanche photodiode (APD), a charge-coupled device (CCD) imager, an electron-multiplying charge-coupled device (EMCCD) imager, scientific complementary metal-oxide- semiconductor (sCMOS) imager or CMOS imager of a mobile phone camera, optionally with a focusing apparatus and a computer link.
  • the sample chamber of the optical device may further comprise other functionalities, for example a temperature control unit.
  • the sample holder of the optical device may be a coverslip, cuvette, slide, capillary tube or microfluidic chip.
  • the sample chamber may further comprise an objective for imaging and it can further assist in focusing.
  • the detector (19) and computing unit (20) are combined in a single unit to perform acquisition, data processing, display, user interface, uplink and communication.
  • the example of such combination is a mobile phone or any other mobile device capable of performing the aforementioned tasks.
  • the emission monochromator (18) of the optical device is a diffraction grating monochromator. It may further comprise dichroic mirrors and/or two polarisation filters for allowing anisotropy measurements. In certain embodiments, the optical device may further comprise a set of mirrors for directing the excitation light beams to the beam combiner (13) and/or to the sample holder.
  • the optical device of the present invention further optionally comprises a filter cube (17), schematically shown in Figs. 3-4. Since this is an optional component, it is shown in Fig. 3 with a dotted line.
  • Fig. 4 shows the magnified view of this filter cube (17) with the following optional components: a two-channel dichroic mirror (41), an excitation filter (42), and an emission filter(s) (43) having one or two transmission windows.
  • the excitation filter (42) and emission filter (43) are omitted, and the only component required in this configuration is the two-channel dichroic mirror (41) to direct the modulated dichromatic excitation light beam (e) to the sample chamber.
  • two similar emission filters (43) are needed with total optical density of up to 12 a.u.
  • the channels can then be switched or employed simultaneously.
  • the sample holder of the optical device of the present invention is a microscope.
  • the raw data obtained from the microscope is a video or a series of individual static images.
  • the optical device of the invention and the microscope are synchronised and controlled by said optical device, said microscope, or by a stand-alone controlling unit.
  • the present application provides a method for resolving inter- or intramolecular interactions between a first molecular target labelled with a donor fluorophore and a second molecular target labelled with an acceptor fluorophore capable of forming Forster resonance energy transfer (FRET) interactions with said donor fluorophore in a sample, said method being carried out by placing said sample in a sample chamber of the optical device of the present embodiments, and comprising the steps of:
  • FRET Forster resonance energy transfer
  • the acceptor and donor fluorophores are characterised by plotting a saturation curve for the acceptor fluorophore and determining the bleaching times of the donor and acceptor fluorophores. These pre determined values are considered in the above method when exciting the sample and modulating the excitation intensity of said acceptor fluorophore in a predetermined frequency domain, so not to destroy the fluorophores upon excitation with excessive light intensity. Further, the fluorescence emission intensity data of the donor fluorophore generated independently during the FRET process is analysed for predetermining the number of emitters of the donor fluorophore labelling the first molecular target and of the acceptor fluorophore labelling the second molecular target. This number of emitters is used in the interpretation of the distances between the molecular targets bearing the donor and the acceptor.
  • the FRET allows the measurements of intramolecular distances between donor and acceptor fluorophores of about 2-8 nm, with the precision of only a few Angstroms.
  • the FRET therefore often serves as a tool to measure intramolecular interactions between macromolecules, such as proteins, under physiological settings, for example in living cells and tissues.
  • the FRET measurements usually tend to be laborious and noisy due to the need to detect faint fluorescence signals in two spectral windows, and over a considerable background of non-interacting molecules and spectral interference between the detection channels.
  • One of the aspects of the present invention is the combination of lock-in detection and frustrated FRET for the detection of weak or sporadic interactions between molecular species.
  • This data acquisition method of the present invention is based on modulating the intensity of the acceptor in a predetermined frequency, thus causing frustration of the FRET process, which manifests itself subsequently in the modulation of the donor emission.
  • the FRET is then efficiently detected at the modulation frequency in the Fourier domain of the detected signal, while the non-modulated background is efficiently rejected.
  • the modulation frequency of the acceptor excitation depends on the system configuration, detector speed and on the fluorophore brightness. The frequency can range from about 1 Hz for imaging fixed cells or in-vitro samples to tens of kHz for non- imaging applications.
  • the method of the present invention allows for the detection of sparse FRET signals, down to the single molecule level, in samples densely labelled with fluorescence molecules.
  • FIG. 5a shows the modulated excitation of both the donor and the acceptor during the imaging when the first excitation source (11) for donor excitation operates continuously (in consecutive frames) and the second excitation source (12) for acceptor excitation operates in alternate frames.
  • Fig. 5b schematically shows that for a FRET donor-acceptor pair, the frustrated FRET caused by the acceptor-modulated excitation, results in modulation of the fluorescence emission intensity of the donor.
  • the intensity difference between the frames with the frustrated FRET and with the regular FRET is linearly related to the FRET efficiency (£), which is defined above.
  • the lock-in detection combined with the frustrated FRET is a very important and unique aspect of the present invention.
  • the present inventors surprisingly found that applying the lock-in detection to the frustrated FRET for the donor fluorophore attached to the first molecular target allows to differentiate between the donor molecules attached to said first molecular target and free donor fluorophore molecules in the sample.
  • the lock-in detection introduced in the above method comprises the steps of:
  • the FOM can be calculated by removal of a non-modulated part of the obtained FFT spectrum, followed by comparison of said spectral peak to its median value, wherein said non-modulated part of said FFT spectrum corresponds to a DC signal resulting from a non-specific background or from fluorescence emission of free donor fluorophore molecules.
  • the FOM in the above lock-in detection is essentially used for the detection of the interactions FRET (rejection of the false detections) as follows:
  • said negative control sample comprises either only said donor fluorophore, or a mixture of said donor fluorophore and an acceptor fluorophore that is incapable of forming FRET interactions with said donor fluorophore.
  • the sample holder of the optical device of the present invention is a microscope.
  • this microscope is capable of single-molecule localisation (SML) imaging, which allows measuring the frustrated FRET between a donor fluorophore and an acceptor fluorophore in the sample, thus resolving molecular interactions between said donor fluorophore and said acceptor fluorophore, and thereby increasing the resolution of said SML technique.
  • SML single-molecule localisation
  • the SML microscopy enables to localise single molecules to a precision of about 20 nm but cannot provide direct information on intermolecular interactions.
  • the FRET can be useful due to its higher distance sensitivity (2-8 nm).
  • the combination of these two techniques has never been realised so far due to the faint signal from the single molecule FRET (smFRET) and the overwhelming ensemble background, which is a result of the aforementioned problems of the residual direct excitation of the acceptor and bleed-through of the donor.
  • the method of the present invention allows to combine the direct stochastic optical reconstruction microscopy (dSTORM), which is an exemplary SMLM technique, and the FRET in order to detect and localise single intermolecular interactions in living cells and also to measure the smFRET inside densely labelled and intact cells or densely labelled in-vitro samples.
  • dSTORM direct stochastic optical reconstruction microscopy
  • FRET FRET
  • FIG. 6 showing the method of the present invention in a form of a scheme or algorithm.
  • the scheme actually summarises the method of the invention combing the two applications (frustrated FRET and SMLM).
  • the left branch in the scheme shows detection of low FRET signals in ensemble measurements, while the right branch shows the combination of both the frustrated FRET and SMLM in the same method.
  • the optical device of the present invention is limited to use of the excitation filters (14, 15) and emission filter (18) and is incorporated inside a microscope or constitutes a microscope.
  • Such optical device of the present invention comprises: A.
  • An excitation module comprising:
  • a) a first (11) and second (12) excitation source configured to emit a donor fluorophore excitation light (b, for example blue) and an acceptor fluorophore excitation light (r, for example red), respectively, for exciting said donor fluorophore and said acceptor fluorophore in the sample (10);
  • a first excitation filter (14) configured to convert said donor fluorophore excitation light (b) into a donor fluorophore monochromatic excitation light beam (b'), and transmit said donor fluorophore monochromatic excitation light beam (b') to a beam combiner (13);
  • a second excitation filter (15) configured to convert said acceptor fluorophore excitation light (r) into an acceptor fluorophore monochromatic excitation light beam (r'); and transmitting said acceptor fluorophore monochromatic excitation light beam (r') to a modulation unit (16);
  • the modulation unit (16) characterised in that it is designed to modulate excitation intensity of said acceptor fluorophore monochromatic excitation light beam (r') by tuning excitation intensity of said acceptor fluorophore monochromatic excitation light beam (r') from complete blocking to at least about 30% transmission, preferably at least about 50%, more preferably at least about 70%, such that said acceptor fluorophore becomes optically saturated to provide frustration (quenching) of said FRET process, and directing the modulated acceptor fluorophore monochromatic excitation light beam obtained (r # ) to said beam combiner (13); and
  • the beam combiner (13) designed to combine said donor fluorophore monochromatic excitation light beam (b') and said modulated acceptor fluorophore monochromatic excitation light beam (r # ) into a single dichromatic excitation light beam (e);
  • a sample holder designed to hold a microscope slide or a coverslip, or another compatible holder designed to carry said sample (10), to which said dichromatic excitation light beam (e) is directed;
  • An acquisition module comprising:
  • an emission filter configured to transmit a narrow-wavelength beam of said donor fluorophore emission (g) or donor and acceptor emission in a sequence;
  • a detector (19) configured to measure intensity of the fluorescence emission (g) of said donor fluorophore and transfer the obtained fluorescence emission intensity data to a computing unit (20);
  • said computing unit (20) is designed to be synchronised with said detector (19) and with said modulation unit (16), to analyse the fluorescent emission intensity data transferred from said detector (19), to control said modulation unit (16) by providing a feedback to said modulation unit (16) for further modulating excitation intensity of the acceptor fluorophore monochromatic excitation light beam (r') (which results in modulating excitation intensity of the acceptor fluorophore itself) and thus, modulating fluorescence emission intensity of said donor fluorophore in a predetermined frequency domain, thereby resulting in reversible saturation of said acceptor fluorophore and consequently, frustration of the FRET process, to analyse microscope raw data images obtained from single-molecule localisation, to integrating said fluorescence emission intensity data and said microscope raw data and to provide information on the molecular interactions and on the nanometre proximity of single molecules in a readable format; and
  • a first algorithm characterised in that it is designed to acquire and measure the frustrated FRET efficiency between the donor and acceptor fluorophores in the sample (10), adapted for a lock-in detection and suitable for resolving weak and rare molecular interactions between the donor and acceptor in the sample (10) and transmitting data on said molecular interactions to the third algorithm;
  • FFT fast Fourier transform
  • said second algorithm for analysing said microscopic raw data images obtained from single-molecule localisation, said second algorithm is characterised in that it is designed to localise the donor fluorophore in the sample (10) and to transmit data on the localisation of said donor fluorophore molecules in said sample (10) to a third algorithm;
  • the optical device of the present invention may further incorporate additional components or functionalities of a microscope.
  • Fig. 7 showing the optical device of the present invention having microscope functionalities, comprising:
  • An excitation module comprising:
  • a) a first (11) and second (12) excitation source configured to emit a donor fluorophore excitation light (b, for example blue) and an acceptor fluorophore excitation light (r, for example red), respectively, for exciting said donor fluorophore and said acceptor fluorophore in the sample (10);
  • a first excitation filter (14) configured to convert said donor fluorophore excitation light (b) into a donor fluorophore monochromatic excitation light beam (b'), and transmit said donor fluorophore monochromatic excitation light beam (b') to a beam combiner (13);
  • a second excitation filter (15) configured to convert said acceptor fluorophore excitation light (r) into an acceptor fluorophore monochromatic excitation light beam (r'); and transmitting said acceptor fluorophore monochromatic excitation light beam (r') to a modulation unit (16);
  • the modulation unit (16) characterised in that it is designed to modulate excitation intensity of said acceptor fluorophore monochromatic excitation light beam (r') by tuning excitation intensity of said acceptor fluorophore monochromatic excitation light beam (r') from complete blocking to at least about 30% transmission, preferably at least about 50%, more preferably at least about 70%, such that said acceptor fluorophore becomes optically saturated to provide frustration (quenching) of said FRET process, and directing the modulated acceptor fluorophore monochromatic excitation light beam obtained (r # ) to said beam combiner (13);
  • the beam combiner (13) designed to combine said donor fluorophore monochromatic excitation light beam (b') and said modulated acceptor fluorophore monochromatic excitation light beam (r # ) into a single dichromatic excitation light beam (e); and f) a filter cube (17) comprising a two-channel dichroic mirror (41), an excitation filter (42), and at least one emission filter (43) having two transmission windows, and configured to receive the single dichromatic excitation light beam (e), to transfer it to a sample holder for excitation of the sample (10), to filter out the emitted light from the sample (10) and to transfer it to an acquisition module;
  • a sample holder designed to hold a microscope slide, a coverslip, or another compatible holder designed to carry said sample (10), to which said dichromatic excitation light beam
  • sample chamber (B) is optionally equipped with an objective configured to gather the fluorescence emission light (g) from the sample (10) to produce a fluorescence image, and optionally focus the excitation light beam (e) on the sample (10);
  • An acquisition module comprising:
  • an emission filter configured to transmit a narrow-wavelength beam of the donor fluorophore emission (g) or donor and acceptor emission in a sequence;
  • a detector (19) configured to measure intensity of the fluorescence emission (g) of said donor fluorophore and transfer the obtained fluorescence emission intensity data to a computing unit (20), said detector (19) is optionally equipped with a magnification eyepiece (ocular) for viewing, imaging, focusing and increasing the overall magnification of a fluorescent image; and
  • said computing unit (20) is designed to be synchronised with said detector (19) and with said modulation unit (16), to analyse the fluorescent emission intensity data transferred from said detector (19), to control said modulation unit (16) by providing a feedback to said modulation unit (16) for further modulating excitation intensity of the acceptor fluorophore monochromatic excitation light beam (r') (which results in modulating excitation intensity of the acceptor fluorophore itself) and thus, modulating fluorescence emission intensity of said donor fluorophore in a predetermined frequency domain, thereby resulting in reversible saturation of said acceptor fluorophore and consequently, frustration of the FRET process, to analyse microscope raw data images obtained from single-molecule localisation, to integrating said fluorescence emission intensity data and said microscope raw data and to provide information on the molecular interactions and on the nanometre proximity of single molecules in a readable format; and
  • a first algorithm characterised in that it is designed to acquire and measure the frustrated FRET efficiency between the donor and acceptor fluorophores in the sample (10), adapted for a lock-in detection and suitable for resolving weak and rare molecular interactions between the donor and acceptor in the sample (10) and transmitting data on said molecular interactions to the third algorithm;
  • FFT fast Fourier transform
  • said second algorithm for analysing said microscopic raw data images obtained from single-molecule localisation, said second algorithm is characterised in that it is designed to localise the donor fluorophore in the sample (10) and to transmit data on the localisation of said donor fluorophore molecules in said sample (10) to a third algorithm;
  • the third algorithm designed to receive and integrate the analytical data produced by, and received from the first algorithm, the FFT algorithm and the second algorithm, and to output information on the molecular interaction and on nanometre proximity of the single donor and acceptor fluorophore molecules, in a readable format.
  • the acquisition module of the optical device of the above embodiment may further comprise a pair of two-channel dichroic mirrors (21, 2G) capable of transmitting the emitted dichromatic light beam from the filter cube (17) to the emission filter (18).
  • the beam combiner (13) and modulation unit (16) may further comprise additional excitation filters, if either the first excitation source (11) or the second (12) excitation source, or both, have a wide-spectrum excitation.
  • the sample chamber or holder further comprises an objective configured to directly gather emission light from the sample (10) being observed and to focus the emission light rays to produce a real image for observation by a user.
  • the objective used in the present invention can be a single lens or mirror, or combinations of several optical elements.
  • the numerical aperture for the lenses used in the present objective can range from 0.10 to 1.49, corresponding to focal lengths of about 40 mm to 2 mm, respectively.
  • the magnification achieved with this objective can range from x4 to xlOO.
  • the objective can be further equipped with a magnification eyepiece ranging from x2 to x20 to increase the overall magnification of the fluorescent image.
  • the filter cube (17) in the present configuration comprises a two-channel dichroic mirror (41) matching the donor and acceptor excitation and emission wavelength (reflecting two excitations and transmitting two emissions), an excitation filter (42) and emission filter (43) having two transmission windows.
  • the emission filter (18) is a rotating filter designed to transmit either donor emission or acceptor emission.
  • the emission filter (18) is a diffraction grating.
  • the emission filter (18) is a dichroic mirror. It may further comprise dichroic mirrors or polarisation filters for allowing anisotropy measurements.
  • the detector (19) can be an electron- multiplying charge-coupled device (EMCCD) imager, a charge-coupled device (CCD) imager, scientific complementary metal-oxide- semiconductor (sCMOS) imager or CMOS imager of a mobile phone camera, optionally with a focusing apparatus and a computer link.
  • the detector (19) can be optionally equipped with a magnification eyepiece (ocular) ranging from x2 to x20 to assist in focusing and increase the overall magnification of the fluorescent image.
  • Figs. 8a-8d schematically showing four different configurations combining the emission filter (18) and detector (19).
  • Communication link from the computing unit (20) may directly control excitations of the donor and acceptor from their corresponding excitation sources.
  • the detector (19) and computing unit (20) are components of a mobile phone or any personal gadget having the similar functionalities and computing power as a smartphone.
  • the processing unit (20) may transmit the results of the measurements to an external memory, which can be a mobile device (such as a smartphone), desktop computer, server, remote storage, internet storage, or diagnostics cloud.
  • the optical device of the present invention is a fluorometer. In another particular embodiment, the optical device of the present invention is a combined fluorometer and fluorescence microscope.
  • Using the optical device of the present embodiment which applies the single interaction localisation microscopy (SILM), allows to identify numerous molecules that constitute single FRET pairs and to localise them in densely labelled cells.
  • FRET pair is the donor-acceptor pair consisting of Alexa Fluor ® 555 as a donor fluorophore on a primary antibody and Alexa Fluor ® 647 as an acceptor fluorophore on a secondary antibody.
  • An upper limit in this case to the energy transfer efficiency E between the donor and acceptor undergoing the FRET process is as low as 1-3% with a resolution of about 0.01%.
  • Intramolecular donor-acceptor distances of 4-8 nm were measured with the resolution down to approximately 4-5 A and will be demonstrated in the Examples section below.
  • the present invention provides for the first time a super-resolved optical image of a cell in a single molecule detail and with distance measurements that continuously span from Angstroms to Microns.
  • the present invention also provides optimisation steps to improve the detection efficiency of the method of the invention, which will be described next. These optimisation steps include the optimisation of fluorophores for the process of the frustrated FRET, the optical configuration of the system, the modulation frequency and the decoding algorithm.
  • the present application provides a method for increasing the resolution of a microscope capable of single-molecule localisation and imaging single molecular interactions by detecting single inter- or intramolecular interactions between a first molecular target labelled with a donor fluorophore and a second molecular target labelled with an acceptor fluorophore capable of forming the FRET interactions with said donor fluorophore, or measuring the nanometre proximity between said first and second molecular targets, in a sample, said method being carried out by placing said sample on a microscope slide in a sample holder of said microscope with which the optical device of the invention is combined, and comprising the steps of:
  • a photoactivatable fluorophore capable of switching from a non-emissive to an emissive state upon excitation with the third excitation source at an activating wavelength and then emitting fluorescence upon excitation at an excitation wavelength in a defined region of space at a given interval of time
  • ⁇ a photo switchable fluorophore capable of switching from one emissive state to another emissive state upon excitation with the third excitation source at an activating wavelength.
  • the super-resolution microscopy technique used in the present invention is either photo activated localisation microscopy (PALM) or direct stochastic optical reconstruction microscopy (dSTORM).
  • PAM photo activated localisation microscopy
  • dSTORM direct stochastic optical reconstruction microscopy
  • the present invention is not limited to these techniques, but may use other super-resolution techniques, such as point accumulation for imaging in nanoscale topography (PAINT), binding activated localisation microscopy (BALM), reversible saturable optical fluorescence transitions (RESOLFT), spectral precision distance microscopy (SPDM), or super-resolution optical fluctuation imaging (SOFI).
  • PAINT point accumulation for imaging in nanoscale topography
  • BALM binding activated localisation microscopy
  • RESOLFT reversible saturable optical fluorescence transitions
  • SPDM spectral precision distance microscopy
  • SOFI super-resolution optical fluctuation imaging
  • the lock-in detection comprises the steps of:
  • C calculating the Figure of Merit (FOM) of individual donor molecules within said sample by optional removal of a non-modulated part of said FFT spectrum, followed by comparison of said spectral peak to its median value, wherein said non-modulated part of said FFT spectrum corresponds to a DC signal resulting from a non-specific background or from fluorescence emission of free donor fluorophore molecules.
  • FOM Figure of Merit
  • the FOM in the above lock-in detection is essentially used for the detection of the FRET interactions (rejection of the false detections) as follows:
  • said negative control sample comprises either only said donor fluorophore, or a mixture of said donor fluorophore on said target molecule and said acceptor fluorophore on a second target molecule that is incapable of forming molecular interaction with said target molecule, and thus does not demonstrate FRET with said donor fluorophore.
  • the first molecular target and the second molecular target are fragments of the same molecule, thereby undergoing the intramolecular interactions. In other embodiments, the first molecular target and the second molecular target are different molecules, thereby undergoing the intermolecular interactions. In a specific embodiment, the first and second molecular targets each independently is an antigen, antibody, antibody fragment, enzyme, substrate or inhibitor, receptor, protein or organic molecule, lectin, sugar, DNA, RNA, or aptamer.
  • the donor fluorophore or said acceptor fluorophore is a fluorescent protein, a synthetic dye, or a quantum dot.
  • the acceptor fluorophore is a fluorescence quencher.
  • the donor fluorophore is a photoluminescent emitter.
  • the FRET is measured in the present invention by observing the intensity of the donor in the frustrated FRET mode rather than the intensity of the acceptor. Observation of both the acceptor emission and the donor emission may contribute to the detection but is not required.
  • the method of the present invention is essentially based on modulation of the donor emission via modulation of the acceptor availability for the FRET process. Excitation of the acceptor consequently introduces frustration to the FRET, since the FRET can only occur as long as the acceptor is in the ground state and is available to receive energy. When the acceptor is excited, it is no longer available for energy transfer, which manifests in increased emission intensity of the donor.
  • optical devices and methods of the present invention are many and include, but not limited to, finding biomarker interactions, including weak or sporadic molecular interactions in cells and in tissues. Such interactions may report on the activity of cells, for example, via the report on enzyme- substrate interactions or the assembly of dimers or multi-molecular complexes of proteins or nucleic acids, the assembly of virus particles inside cells, the specific binding of antibodies to their target, the pathways of labelled drugs in the cell and more.
  • aberrant protein interactions may be involved in malignancies, and thus the optical devices and methods of the present invention can serve as a diagnostic tool for such malignancies.
  • the molecular targets used in the present invention are hybridisation, hydrolysis or similar (e.g . Scorpion ® or Molecular Beacon) probes that are suitable for binding closely to a common target DNA or RNA template, thereby facilitating the process of the FRET between them and detecting the target.
  • hybridisation hydrolysis or similar (e.g . Scorpion ® or Molecular Beacon) probes that are suitable for binding closely to a common target DNA or RNA template, thereby facilitating the process of the FRET between them and detecting the target.
  • the sample required for measurements with the optical devices and methods of the present invention is very small (as small as a single cell) and the detection can be performed either via a non-imaging system (for example, a miniature dedicated system, a flow cytometer, or a plate reader), or a microscope.
  • a non-imaging system for example, a miniature dedicated system, a flow cytometer, or a plate reader
  • microscope for example, a microscope
  • the first and second excitation sources (11) and (12) are alternated.
  • the emission filter (18) is rotated to capture either the donor or acceptor emission.
  • the optical device of the invention generates three images which are typically used:
  • the sensitised emission efficiency is calculated as detailed in Example 14 below.
  • the overall measurement is fast because the excitation sources (11) and (12) are alternated fast and the emission filter (18) rotates also fast on a filter wheel or on a confocal microscope.
  • An algorithm for the FRET calculation used with the optical device of the present invention operating in this sensitised emission mode is shown schematically in Fig. 9 and also detailed in the Example 14 below.
  • the donor excitation source (11) operates continuously (or in every frame), while the acceptor excitation source (12) is modulated. Only the donor emission is detected for further processing by the computing unit (20). In order to do this, the donor emission is isolated with the emission filter (18) as described above.
  • the optical device of the invention generates a movie of typically hundreds or thousands of frames. An algorithm for the calculation of the frustrated FRET used with the optical device of the present invention is shown in Fig. 6 and discussed above.
  • the device will primarily use the FRET mode to detect inter or intra-mo lecular FRET process between donor- and acceptor-labelled molecules.
  • This approach allows for highly sensitive measurements of FRET pairs in densely labelled samples, being either in vivo (i.e. in cells), or in vitro.
  • In-vitro measurements of prime interest include sensitive measurements for pathogen detection using either labelled DNA probes, antibodies or antibody fragments.
  • the method is especially valuable when most of the donor and acceptor molecules are free ⁇ i.e. they do not constitute FRET pairs), therefore giving rise to a large background in typical FRET measurements (such as sensitised emission) due to the problems of 'direct-excitation' and 'bleed-through'.
  • the FRET information is given for each FRET pair and is interpreted to report on the distance between them with very high spatial resolution (down to sub-nanometres).
  • the outcome data is presented and shared either in a detailed fashion (e.g. histograms) or as binary (e.g. the particular pathogen is detected or not).
  • this mode is an assisting mode, which is also supported by the device of the present invention, since the device is capable of detecting and comparing both the donor and acceptor emissions.
  • the sensitised emission can be taken relatively quickly (requiring essentially three frames) with minimal exposure of the sample to excitation light, particularly avoiding the repeated fluorophore excitations and acceptor saturation of the frustrated FRET mode. Still, it requires relatively high number of FRET pairs relative to a low number of free donor and acceptor fluorophores (i.e. low background). It also typically averages the FRET results across multiple FRET pairs. Therefore, the sensitised emission mode is employed before employing the frustrated FRET in the following exemplary cases:
  • the present inventors developed a method to image and measure single intermolecular (protein-protein) interactions in cells. This method, that the inventors named 'single interaction localisation microscopy' (SILM), is a FRET-SMLM combined technique. In order to overcome the FRET problems of acceptor direct-excitation and donor bleed-through, the process of the FRET was measured by observing intensity of the donor emission.
  • the donor emission was further modulated by reversible acceptor saturation that led to FRET frustration.
  • the lock-in detection of this modulation made it possible to detect smFRET and localise the measured molecules in densely labelled cells.
  • the combination of the FRET with dSTORM made it possible to work with a much higher density of donor molecules (about 100,000 molecules per 1,600 pm 2 , which is by a factor of five larger) than that was used in the previous smFRET studies described by Huppa et al in "TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity ", Nature 463 (7283), 963-7 (2010).
  • the donor fluorophore is any emitter of Types 1-5 or 7-10 (listed in the Table 1 above), although Types 8-10 are relatively dim.
  • the acceptor fluorophore is any molecule (emitter or absorber) of Types 1-7. It is chosen such that its emission is significantly red shifted from the donor emission, such that the detection window of the donor emission does not contain acceptor emission and that the acceptor direct excitation is minimal.
  • the detection of the FRET is conducted between labelled antibodies carrying the donor Alexa Fluor ® 555 and antibodies carrying the acceptor Alexa Fluor ® 647.
  • the antibodies stained actin in cells that were imaged using a fluorescence microscope (see the examples below).
  • the FRET levels down to 1-3% are detected using this method in individual cells.
  • the frustrated FRET process is actually combined with SMLM.
  • the donor fluorophore is for example, a primary or a secondary antibody, a fragment of an antibody, for example nanobody or Fab, an affibody, or a tag that can specifically bind a molecule of interest (for example, via click chemistry), stained with synthetic fluorophores that demonstrates blinking for SMLM. See the above Table 1 for Types 2 and 5 emitters.
  • the acceptor fluorophore in this case can be any one of the following:
  • a quencher whose absorption properties can be modulated reversibly and fast. Such a quencher may replace the fluorophore in a). See the Type 6 emitters.
  • a quantum dot whose absorption properties can be modulated reversibly and fast. Such a quantum dot may replace the fluorophore in a). See the Type 5 emitters.
  • the donor fluorophore is the one that can demonstrate transition from a dark state to a fluorescent state via molecular uncaging, for example by UV illumination. See Type 7 emitters in the Table 1 above to select.
  • the acceptor fluorophore may be any of a), b), c) or d), as specified above.
  • the donor fluorophore is the one that demonstrates transition from a dark state to a fluorescent state via encounter with an environment of different polarity. See, for instance, the Type 2 emitters in the Table 1 above to select.
  • the acceptor fluorophore may be any of a), b), c) or d), as specified above.
  • the donor fluorophore is the fluorescent protein that serves as a tag for a chimeric protein of interest and that undergoes photo-activation or photo-switching of its fluorescence emission.
  • the acceptor fluorophore may be any of a), b), c) or d), as specified above.
  • Example 2. Single interaction localisation microscopy ( SILM )
  • dSTORM buffer that made it blink. This is a combination of dSTORM and FRET.
  • the use of dSTORM buffer could make the acceptor enter a dark state as well as the donor.
  • acceptor excitation intensity just before optical saturation, thus leading to a fast return of the acceptor to the ground state after its excitation.
  • FIG. 10a is one representative frame out of a dSTORM imaging movie of a cell.
  • the PAGFP Actin was conjugated to Alexa Fluor ® 555 and Alexa Fluor ® 647.
  • the PAGFP was not excited and the emission of Alexa Fluor ® 647 was filtered out.
  • the crosses mark the position of single molecules that were localized in the analysis that was done after the imaging.
  • the white circle with a black cross in the center marks the position of the molecule that was analysed in Figs. lOb-lOc.
  • the photons that the molecule emits were collected from the pixel where the molecule was localised and the eight surrounding pixels.
  • Fig. 10b shows a time trajectory of the fluorescence intensity of the molecule. This molecule was detected in 132 consecutive frames.
  • Fig. 10c shows the absolute value of the Fourier transform of the time trajectory of the intensity. The dashed circle with a dot in the center indicates the modulation frequency (the Nyquist frequency), while the dashed line marks the median of the absolute values of the Fourier transform for all the frequencies.
  • Fig. lOd demonstrates the accuracy of the experiment with the number of molecules as a function of uncertainty in location of a single molecule.
  • Fig. lOe shows a spectrogram built in MATLAB ® for the windowed Fourier transform of a single molecular trajectory.
  • Time segments from 8 frames to 208 frames with jumps of 8 frames were used in the analysis.
  • the FOM for each time segment was then calculated.
  • the highest FOM was chosen to be the FOM of the detected molecule.
  • Fig. lOf shows the comparative average FOM against all trajectories that were longer than a minimal length for the 'donor- acceptor' sample versus the 'donor-only' sample.
  • the median was an estimation for the contribution of the background.
  • the calculation of the FOM was done by dividing the absolute value of the Fourier transform of the modulation frequency by the median of the absolute value. The same process was also done with windowed Fourier transform on different parts of the time trajectory. Calculation of the FOM with the Fourier transform yielded a value of 9.3. See further results on the FOM in FRET and control experiments in the examples below.
  • the imaging sequence in this experiment was as follows.
  • the first (donor) excitation source (11) was operating continuously while the second (acceptor) excitation source (12) was turned on and off in a predetermined frequency. Since the on-time of a fluorophore until it photobleaches ranges between milliseconds to seconds, we modulated the excitation of the acceptor as fast as possible. As a result, this frequency was actually set by the Nyquist frequency, which is half of the frame rate of the EMCCD camera that we used (130 fps). Every donor molecule that was a part of the FRET pair and was 'on' long enough had a distinct frequency component at the frequency of modulation. As seen in Fig.
  • this component showed as a high peak at the Nyquist frequency on the power spectrum, when compared to the median of the power of all the other frequencies.
  • Such identification of the emission modulation at the modulation frequency of the excitation source is known as "lock-in detection". This method can detect a faint signal with a known frequency in an overwhelmingly noisy environment.
  • the donor was excited by a 50-mW 56l-nm laser, and the acceptor was excited by a l25-mW 647-nm laser (Agilent MLC 400B).
  • a Chroma ET600/50m band filter For the imaging of the donor channel we used a Chroma ET600/50m band filter, and for the acceptor channel we used a Chroma ET705/72m band filter (bleed-through and direct excitation were measured on 10 different cells each).
  • the average ratio of the emission of the donor in the donor channel and in the acceptor channel (bleed-through) was determined in the absence of the acceptor to be 0.083 ⁇ 0.002 (for 10 cells).
  • the direct excitation of the acceptor was determined by measuring the intensity of the acceptor at the acceptor channel when it was directly excited by the 561 nm laser and when it was excited by the 647 nm laser and taking the ratio between these intensities. The direct excitation was 0.170 ⁇ 0.003 (for 10 cells).
  • the correction factor g was calculated and was found to be 3.18.
  • each pixel in our camera was 160 nm. Diffraction limit was about 200 nm and so is the order of the size of the PSF. Therefore, the photons that were emitted from a single molecule were also collected by the pixels of the camera that surround the pixel where the emitter was detected. As shown in Fig. 10a, the intensity of each emitter molecule in each frame was therefore determined by summing the intensity of the pixel where the molecule was localized and the intensities of the eight surrounding pixels (the grouping distance was set as four times the average uncertainty of all detected peaks, as mentioned in the Example 1 above).
  • the signal from peaks were grouped in consecutive frames into time trajectories of molecules, without allowing their disappearance (i.e . a gap time of 0).
  • the grouping distance was set as 105 nm, which was four times the average uncertainty of all detected peaks, as clearly seen in Fig. lOd.
  • the Thunder STORM algorithm was employed throughout the analyses to discriminate detections of single molecules via their single molecule characteristics, namely their spatial and temporal intensity profile (see Fig. 10b), and their localisation uncertainty (see Fig. lOd).
  • the time trajectories of intensities of all individual donor emitter molecules were determined, followed by determining a criterion to distinguish between the molecules that were influenced by the modulation of the acceptor in a FRET pair and those that were not. That was done by defining a figure of merit (FOM) that represented the extent of the FRET process for each donor molecule.
  • the FOM was set as the ratio between the absolute Fourier component at the Nyquist frequency and the median of the absolute Fourier components for all frequencies, except for the DC signal. Larger FOM values indicate higher strength of the modulation of the donor emission relative to the background, and thus, a higher confidence for the existence of a FRET pair. It should be noted that the inclusion of more extended surrounding pixels in the calculation of the intensity per the PSF resulted in much noisier background, and the effective reduction of the signal to background of the intensity measurement.
  • the acceptor should be fluorescently active.
  • the frustrated FRET process became negligible after about 3.6 sec from the initiation of the measurements, because of acceptor photobleaching (or entering prolonged dark-states).
  • the frustrated FRET would not be effective after that time. Since only the molecules that were emitting while the effect was still significant should be tracked, the focus was made only on the donor emitter molecules that appeared in the first 1.54 sec of each movie (which included about 210 frames). The signal was grouped from peaks in consecutive frames into time trajectories of molecules, without allowing their disappearance (gap time of 0).
  • Thunder STORM was employed to discriminate detections of single molecules via their single molecule characteristics (specifically, intensity and localisation uncertainty), and via their intensity drop to the background level when they disappeared. Indeed, the vast majority of detected molecules were outside of the saturated regions and most of the molecules showed in sparse areas in each frame.
  • FOM figure of merit
  • the FOM was the ratio between the absolute intensity at the Nyquist frequency and the median absolute intensity of all frequencies (except for the DC mode). The larger was the obtained FOM, the more likely the molecule was a part of the FRET pair. Since the acceptor also photobleaches and could have been in the "on” state for only some of the time period that the donor was "on”, we used the windowed Fourier transform in order to calculate the FOM value.
  • Fig. lOe showing a spectrogram built in MATLAB ® for the windowed Fourier transform of a single molecular trajectory.
  • Time segments from 8 frames to 208 frames with jumps of 8 frames i.e. segments with number of frames of 8, 16, 24, etc.
  • the FOM for each time segment was then calculated.
  • the highest FOM was chosen to be the FOM of the detected molecule.
  • the FRET values were assigned into a particular dSTROM image, and thus created an image that provided information both on the super-resolved locations and the interactions of the molecules. On average, 33 molecules per cell were detected that had the FRET with an acceptor molecule. A Fourier transform then was applied on the time trajectory of the intensity of each molecule separately, as explained in Example 1 above.
  • a dSTORM image is a super-resolved image which represents all the detected peaks in the form of a Gaussian.
  • the width of every Gaussian is the uncertainty in the location of the molecule.
  • the image In addition to the super-resolved location of each and every molecule, we wanted the image to contain information about the FRET between single emitters. This means the energy transfer efficiency from the FOM can be evaluated based on the modulation of the donor intensity, as follows:
  • I D and I sat D are the background- subtracted donor intensity values with and without the FRET, respectively, and a sat is the fraction of the acceptors that is undergoing saturation and become FRET incompetent.
  • a sat can also be interpreted as the fraction of time over which the acceptor is saturated (including its occupying time of short-lived and reversible dark states).
  • a sat was a-priori unknown.
  • the values I D and I sat D were obtained from the emission of single donor fluorophores in the FRET pairs. In order to do that, first, in each time trajectory of donor intensity, the segments, in which the donor was fluorescent and where it abruptly photobleached (or entered a prolonged dark state), were identified. The background was calculated from the segment after photobleaching. The donor intensity with and without acceptor saturation (I sat D and I D , respectively) was determined by averaging the intensity just before photobleaching, guided by the windowed Fourier analyses and by subtracting the background. This stage also assisted in the exclusion of erroneous data that passed the FOM test.
  • Figs lla-llb showing two STORM images, where the image in Fig. 11a is for 'donor-acceptor' and the image in Fig. lib is for 'donor-only'.
  • the E values were assigned to the molecules that were detected as part of a FRET pair using a colour code (on right). The rest of the molecules that were detected in the cell, but were not part of FRET pairs, were not assigned with an E value (shown in red). Note that the radii of the coloured discs representing individual FRET pairs were chosen to highlight the existence of multiple overlapping pairs in clusters (see the zoom in Figs lla-llb on the right panel), and do not represent the localisation errors of these pairs.
  • the red pixels in all images represent identified emitter molecules. These molecules were found in the cell using the super-resolution dSTORM analysis for which no FRET value was assigned.
  • the coloured Gaussians represent molecules for which the FOM was larger than the threshold of 4.5 that we chose. The Gaussians with the assigned FRET were overlaid on top of the other molecules.
  • the colour bars represent the energy transfer efficiency E calculated as described herein below.
  • Figs lla-llb the E values were assigned to the emitter molecules that were part of the FRET pair. The rest of the emitter molecules that were detected in cell were not assigned with any E value.
  • Figs llc-lld showing the histogram of E and the histogram of the distance distribution, respectively, as was calculated according to the first model for 18 cells having the donor and acceptor fluorophores. While the ensemble FRET measurements yield the average E and distance distribution, the smFRET provides detailed information about the distribution of these values. Indeed, the E values are not the same for all molecules and range from 0.08 to 0.7. The average E value of the 18 cells was 0.260+0.018.
  • Model 1 described in Example 4-2 considers the occurrence of energy transfer from a single donor at an 'on' state predominantly to the single nearest acceptor, when the FRET to the two other acceptors is neglected:
  • Model 2 considers the occurrence of energy transfer from a single donor at an 'on' state simultaneously to two equidistant acceptors, which are placed at the distance of the nearest acceptor, while the FRET to the third acceptor is neglected:
  • a third, intermediate model can also be considered that translates to 1.2 'equidistant acceptors'.
  • This model equally divides the errors in the simulated data and yields a nominal value for r DA mm between the upper and lower bounds.
  • Non- symmetric error for this value r DA mm around its nominal distance was -0.03 xRo and +0.08 xRo- Since in the present system, the Forster radius value Ro was 51 A (see Example 9 below), the obtained distance errors are -1.5A and +4.5A.
  • Another source for errors in estimating the donor-acceptor distance by FRET is the relative orientation of the donor and acceptor fluorophores.
  • Anisotropy error translates into an error in Ro of about +3.8A (see Example 9 below).
  • the RMS summation of this error with the error estimates due to the uncertainty in the system configuration (introduced above) results in total errors of -4 A and +5.6A.
  • a well-defined system having a single acceptor fluorophore in each FRET pair would eliminate the ambiguity in the physical arrangements of multiple acceptors.
  • the errors in estimating r DA are limited to +3.8A, i.e. the errors defined by our anisotropy measurements (see Example 9 below).
  • Fig. 11c shows the histogram of the intermolecular distance between the donor and its nearest acceptor, r DA mm .
  • This data was calculated according to a nominal model of the physical system for 18 cells labelled with donor and acceptor fluorophores.
  • the distance r DA mm ranged between 4 and 8nm and averaged at 6.05+0.04 nm, as seen in this figure.
  • the distances were calculated according to the first model with the total 527 emitter molecules in the cells. Heterogeneity in the E values and in the donor-acceptor distances is likely due to differences in the interaction geometry between the antibodies.
  • the developed method of SILM with the dSTORM-FRET of the present invention allows to detect single interactions between primary and secondary antibodies in densely labelled cells. An upper limit to the distance between the donor and the acceptor fluorophore-labelled antibodies is then provided.
  • the present invention made it possible to obtain a super-resolved optical image of a cell in single-molecule detail and then to measure intermolecular distances that continuously span from single Angstroms to Microns.
  • the Forster theory provides a calculation for the distance between the donor and the acceptor assuming the energy transfer efficiency E is given.
  • the primary antibody carried three donors.
  • the present single molecule imaging approach ensures that there is a single donor fluorophore in each localization event under study, as the probability of having two donors in a fluorescent state at the same PSF is kept very low. Since the average number of Alexa Fluor ® 647 fluorophores on the secondary antibody is 3 in the present assay, hence, the FRET between a single active donor and multiple acceptors may occur simultaneously.
  • the secondary antibody may bind the primary antibody at any point ⁇ 3 ⁇ 4 and at any angle Q which is proportional to [0, p ⁇ along the primary antibody length.
  • T O is the lifetime of the donor.
  • Co 8.8xl0 2S for Ro in nanometers
  • n the refractive index of the medium (usually 1.33 for water) and Qo is the quantum yield of the donor
  • k is a parameter that depends on the orientation between the fluorophores (i.e. interacting dipoles) and is usually 2/3 for the freely rotating dipoles
  • q( d, A) is the donor normalized emission spectrum
  • l is the wavelength
  • c(a, A) is the acceptor absorption spectrum multiplied by acceptor extinction coefficient.
  • E6.1 Jurkat line of T-cells As mentioned above, we used the E6.1 Jurkat line of T-cells to demonstrate the feasibility of our method. The origin of these cells is from patients who had T-cell leukaemia in the late l970s, as described by Schneider el al in " Characterization of EBV- genome negative 'null' and T' cell lines derived from children with acute lymphoblastic leukaemia and leukemic transformed non-Hodgkin lymphoma" , Int. J. Cancer 19(5), 621-626 (1977). These cells are immortal and continue splitting as long as they are fed and kept in a C0 2 incubator.
  • the experiments were done on stable cell lines of the Jurkat E6.1 T-cells that were introduced with a plasmid of EGFP-Actin and PAGFP-Actin.
  • the Jurkat T-cells were genetically encoded so that production of a protein named Actin will be accompanied with a production of the fluorescent proteins (FPs) EGFP (Enhanced Green Fluorescent Protein) or PAGFP (Photo Activated Green Fluorescent Protein) that are used as fluorescent markers for Actin.
  • FPs fluorescent proteins
  • EGFP Enhanced Green Fluorescent Protein
  • PAGFP Photo Activated Green Fluorescent Protein
  • the Jurkat E6.1 T-cells were kept growing in complete medium: RPMI (Roswell Park Memorial Institute Medium) (Gibco 21875-034), 1% Pen/Strep (Sigma p4333), 10% Foetal Bovine Serum (FBS) (Sigma F7524). The cells were kept at 37 °C in a humidified C0 2 incubator.
  • the cells immuno-staining procedure (in which the synthetic fluorophores are delivered to the cell) required the use of antibodies and was dependent on the target protein. This procedure was carried out before the sample preparation and cell fixation of the E6.1 cells, and after the sample preparation and cell fixation for the EGFP-Actin and the PAGFP-Actin cell lines.
  • the chambers were then covered with 0.01% poly-F-lysine (Sigma, P4707) for 15 minutes, then aspirated and dried in an oven for two hours. After the chambers dried, we covered them with 0.4 ml of 10 pg/ml CD3 antibody (Biotest, 16-0038-85) diluted in the PBS (Phosphate-Buffer Saline) for two hours in an oven at 37 °C. The chambers were washed three times with the PBS and were stored at 4 °C.
  • the cells were prepared before dropping them to the chamber as follows. We took 500,000 cells per chamber for a sample on the 4-wellchamber (or 250,000 for the 8-well chamber). The cells were then centrifuged for five minutes at 1200 rpm followed by resuspension with 0.15 ml imaging buffer for every 500,000 cells.
  • the cells were then resuspended in 1.5 ml solution of a secondary antibody conjugated with the synthetic fluorophore Alexa Fluor ® 647 (Invitrogen A21241, final concentration of 0.6 pg/ml) and incubated on ice for 30 minutes, followed by five-minutes centrifugation at 1200 rpm and resuspension in 1 ml FACS buffer. Then, the cells were washed with 1 ml PBS, and centrifuged for five minutes at 1200 rpm and again re-suspended in 0.15 ml imaging buffer. The cells were now ready to be dropped on the glass coverslips.
  • a secondary antibody conjugated with the synthetic fluorophore Alexa Fluor ® 647 Invitrogen A21241, final concentration of 0.6 pg/ml
  • Direct-STORM imaging requires the use of a special buffer in order to drive the synthetic fluorophores in a photoactivated mode.
  • the buffer is made of 89% B-buffer (50 mM Tris-HCl pH 8 and 10 mM NaCl prepared in double distilled water), 10% MEA diluted in 1 ml of 0.25 mM HC1, 1% GLOX (14 mg glucose oxidase with 50 pl catalase in 200 pl A-Buffer).
  • the A-Buffer is 10 mM Tris-HCl pH 8 and 50 mM NaCl prepared in double distilled water (see Schneider et al 1977, mentioned above).
  • the average number of fluorophores bound to the antibodies was determined by measuring the absorbance of the labelled antibodies using a Nano-drop ® spectrometer (Nano drop ® 2000 by ThermoFisher Scientific ® ).
  • the labelled antibody was diluted in the PBS, and absorbance of the labelled antibody was measured at 280 nm (A 2 so) and at the excitation wavelength of the relevant fluorophore attached to the antibody (A max ).
  • the protein concentration M was then calculated as follows:
  • Microscopy imaging was performed using a Nikon Ti-E inverted microscope and a lOOx oil- immersion objective with 1.49 NA (Nikon CFI Apo TIRF lOOx oil). The images were collected using an EMCCD camera (Andor iXon Ultra). Activation and excitation of the fluorophores was done using solid state lasers (Agilent MLC 400B). Activation was performed with 405 nm wavelength laser illumination, and excitation at either 488, 561 or 647 nm. The modulation of the lasers was done using an acousto-optic modulator with a rise-time of 1.5 microseconds and with a dynamic range of 1000:1.
  • the power of the lasers was 20 mW for the 405 nm laser line, 50 mW for the 488 and 561 nm lasers, and 125 mW for the 647 nm, measured at the tip of the fibre. Imaging frame rate was typically performed at 130 fps.
  • the total internal reflection fluorescence (TIRF) microscopy is a method used to excite only the molecules that are close to the coverslip, thereby minimizing the background from out of focus light.
  • TIRF total internal reflection fluorescence
  • the evanescent wave excites the molecules which are at the plasma membrane of spreading cells and are less than 100 nm away from the coverslip.
  • Fig. 12a illustrates the use of an FP donor and a synthetic fluorophore acceptor.
  • the main advantage of using the FP as a donor is that by inserting the cell with the appropriate DNA plasmid, the cell produces the FP, which acts then as a genetically encoded tag of a specific protein of interest.
  • the acceptor is a synthetic fluorophore
  • the synthetic fluorophore is conjugated to an antibody that is inserted to the fixed cell.
  • an antibody that is FP-specific In order to achieve a short distance for demonstrating the feasibility of this approach it is beneficial to use an antibody that is FP-specific. Note that the FP must be photoactivatable (in the present experiment, it is PAFP) for the donor to be localised.
  • both the donor and acceptor are synthetic fluorophores.
  • the donor is conjugated to a primary antibody that is specific to a measured target molecule.
  • the acceptor is delivered to the donor by a secondary antibody to which it is conjugated.
  • the secondary antibody is chosen such that it is complimentary to the primary antibody.
  • the target molecule does not play any role in the imaging, and its presence is only required so that the donor and acceptor will be present in the sample and in close proximity.
  • the SILM demonstrated in the present experiments is a combination of the PALM and the FRET.
  • the SMLM can be performed via PAFPs in the PALM mode or via synthetic fluorophores in the dSTORM mode.
  • the acceptor does not have to be photo activated in the imaging assay. However, the acceptor should have a long photobleaching life-time in comparison to the photobleaching lifetime of the donor.
  • the first pair that we employed for the frustrated FRET measurements was the pair of the PAGFP as a donor and the synthetic fluorophore Alexa Flour ® 594 as an acceptor. Calculation yielded Forster radius of 5.57 nm for this FRET pair.
  • Alexa Fluor ® 555 has the absorption maximum at 555 nm
  • Alexa Fluor ® 647 has the absorption maximum at 650 nm.
  • Table 2 summarizes the quantum yields, extinction coefficients and the excited state lifetimes for each fluorophore. Calculation of the Forster radius for the FRET pair Alexa Fluor ® 555 and Alexa Fluor ® 647 yields a value of 5.09 nm.
  • each antibody should be labelled with only one fluorophore. This ensures that the FRET will always occur between one donor and one acceptor. However, this is not the actual case for most of the labelled antibodies.
  • absorbance of the antibodies conjugated with Alexa Fluor ® 555 and Alexa Fluor ® 647 was measured and their concentration per antibody was determined. The results of these measurements were used to calculate the average number of fluorophores per antibody.
  • Table 3 shows the results and the correction factors for each antibody. In this table, the absorbance at 280 nm and at the maximum absorbance wavelength are displayed. Correction factors were taken from ThermoFisher Scientific ® (the supplier of the fluorophore molecules).
  • Alexa Fluor ® 555 and Alexa Fluor ® 647 average number of the fluorophores bound to antibodies.
  • the Forster theory provides calculation for the distance between the donor and the acceptor assuming the energy transfer efficiency E is given.
  • Our single molecule imaging approach ensures that there is a single donor fluorophore in each localisation event under study, as the likelihood of having two donors in a fluorescent state at the same PSF is kept very low.
  • the average number of Alexa Fluor ® 647 fluorophores bound to an antibody is 3.38, we used the two following models of the limiting cases to calculate the distance between the donor and the acceptor.
  • the saturation curve of Alexa Fluor ® 647 was generated. This was done by modulating the donor emission via the availability of the acceptor for the FRET process. This availability was reversibly blocked by saturation of the acceptor. Thus, on one hand, there is a need to bring the acceptor as close to saturation as possible so that most acceptors become unavailable for FRET at once. On the other hand, very strong excitation also drives the acceptor into metastable dark states, and ultimately causes its fast photobleaching. There is an optimal laser excitation intensity for the assay. Fig. 14 shows the saturation curve of Alexa Fluor ® 647 that was generated to identify this optimum and determine the desired excitation power of the acceptor in the present experiments. Excitation was done by 647-nm laser. Error bars were calculated by the SEM method (the standard error of the mean). The intensity is the average of measurements on ten different cells.
  • Alexa Fluor ® 647 begins saturating at about 80% of the 647-nm laser power. Working with this power should maximise the number of excited acceptor molecules such that the photobleaching rate will be as low as possible. Since the frustrated FRET depends on long lifetimes, the working laser power should not exceed 70% during the measurements.
  • the frustrated FRET imaging was done using band filters so that only the light emitted from the donor was collected.
  • two band filters two band filters: one filter (Chroma ET600/50m) was centred around 600-nm emission wavelength with the bandwidth of 50 nm, and the other (Chroma ET620/60m) was centred around 620 nm with the bandwidth of 60 nm.
  • Measuring the effectiveness of the band filters allowed to assess contribution of the acceptor fluorescence intensity to the donor channel. About 70% of the power of the 647 nm laser was used to excite Alexa Fluor ® 647.
  • C is the background and n and t 2 are characteristic decay times of the fluorophore.
  • This euqation assumes a double-exponential decay process, which is the result of two processes that govern the behaviour of photo activated fluorophore: photobleaching and entering a dark state.
  • photoblinking Such a decay has been observed for phoactivated fluorophores, for which the 'on' and 'off times fit to a double exponent decay times due to photoblinking (see the work by Sabanayagam et al, " Long time scale blinking kinetics of cyanine fluorophores conjugated to DNA and its effect on FRET', J. Chem. Phys. 123(22), p. 224708 (2005), for the reference).
  • Figs. 15a-15d showing fluorescence decay of the donor Alexa Fluor ® 555 and acceptor Alexa Fluor ® 647, respectively.
  • the measurements were performed on 10 cells. The intensity from each cell was divided by the maximum intensity of that cell, and then the data from all ten cells was averaged.
  • Fig. 15b shows the residuals of this double exponential fit.
  • Alexa Fluor ® 647 and Alexa Fluor ® 555 are very similar, the long-time scale of Alexa Fluor ® 555 is much longer than that of Alexa Fluor ® 647. This means that we should expect the FRET to decay in a typical time scale as that of the Alexa Fluor ® 647 acceptor.
  • the main obstacle when measuring FRET is the direct excitation of the acceptor due to overlap between the donor absorption spectrum and the acceptor absorption spectrum
  • the device of the present invention can operate in both modes: the sensitised emission and the frustrated FRET, dependent on the method used for excitation and acquisition.
  • excitation of the donor will result in light emission from the acceptor.
  • the acceptor intensity one has to consider the donor bleed-through and the direct excitation of the acceptor. Beside a sample that contains donor and acceptor, one should also prepare an 'acceptor only' and a 'donor-only' samples. Using an appropriate dichroic mirror and band pass filters, the light emitted from the sample is split into two different channels (one channel for the donor and another channel for the acceptor). The light emitted from the acceptor in the absence of the donor, while using an optimal donor excitation wavelength (direct excitation), and optimal acceptor excitation is measured in the acceptor channel. The light emitted from the donor in the absence of the acceptor, when using excitation that is optimal for optimal for the donor (donor bleed-through), is measured in each channel.
  • the donor- sensitised acceptor fluorescence was employed for the FRET imaging of the interaction between the primary and secondary antibodies.
  • three imaging channels can be defined:
  • the sensitised FRET process involves crosstalk between the imaged channels due to direct excitation of the acceptor at the donor excitation and bleed- through of the donor emission to the acceptor and FRET channels.
  • the background in all channels should be subtracted, yielding fluorescence signal for channels of the donor ( FD ), acceptor (FA) and FRET (F>).
  • FRET F>
  • the rate of relative detection sensitivity of the excited acceptor compared to the excited donor is described by another factor, a, as follows:
  • I x is the fluorescence intensity
  • L x is the labelling ratio
  • B x is the antigen ratio
  • e c is the extinction coefficient at maximal donor excitation
  • subscript x denoted either the donor (D) or the acceptor (A).
  • F A is the acceptor intensity in the acceptor channel after excitation by the donor excitation wavelength and in the presence of the donor
  • F D is the donor intensity in the donor channel after excitation by the donor excitation wavelength and in the presence of the acceptor.
  • Another relevant method is a donor recovery after acceptor photobleaching. Because of the energy transfer from the donor to the acceptor, the intensity of the donor in the presence of the acceptor is lower compared to when the acceptor is photobleached. This fact can be exploited to measure FRET by comparing the intensity of the donor in the same sample before and after the acceptor is photobleached.
  • the energy transfer in this case will be:
  • F F is the intensity of the donor in the presence of the acceptor and F D is the intensity of the donor after the acceptor is photobleached.
  • This method is also carried out with the device of the present invention and is relevant to the invention since it exploits the fact that the intensity of the donor goes up when the acceptor is unavailable for FRET. It is based on the fact that excitation can also make the acceptor unavailable for the FRET process. This excitation causes frustration to the FRET which is then used in the present invention to measure the energy transfer efficiency.
  • Figs. 16a-16c showing the images of the donor, acceptor and the FRET, respectively, in a cell footprint.
  • Fig. 16a shows intensity of the donor Alexa Fluor ® 555 in the donor channel
  • Fig. 16b shows intensity of the acceptor fluorophore Alexa Fluor ® 647 in the acceptor channel.
  • Fig. 16c shows the cell image with the sensitised emission calculated for each pixel separately. The average E value of this cell was found to be 0.068.
  • the device of the present invention After verifying that there is a FRET process in the donor-acceptor sample using the sensitised emission mode, the device of the present invention is switched to the next mode to proceed with the quantitative SILM measurements.
  • Fig. 17a showing the decay of Alexa Fluor ® 555 in a 'donor-only' sample
  • Fig. 17c showing the decay of Alexa Fluor ® 555 in a sample that contained both donor and acceptor.
  • the excitation of the samples was carried out with the two lasers simultaneously: continuous excitation with the first laser at 561 nm and modulated excitation with the second laser at 647 nm.
  • the data is generated for 18 cells.
  • the fluorescence intensity of each cell was normalized and averaged.
  • Figs. 17a and 17c the solid lines are the fit to a double exponential decay according to Equation 5 above. The error of the fit is smaller than 1%.
  • Fig. 17e shows the modulation between intensity in even and odd frames of Alexa Fluor ® 555 in the 'donor-only' sample. The modulation is caused by background that is generated by the 56l-nm excitation. The modulation is smaller than 1% and remains essentially constant throughout the imaging.
  • TAI and TA2 are the lifetimes of fluorescence decay the acceptor
  • TDA is the long decay time component of the donor in the presence of the acceptor
  • C is the background.
  • the sum of Ei and E 2 should scale as the energy transfer efficiency.
  • Fig. 17e shows the modulation between intensity in even and odd frames of Alexa Fluor ® 555 in the 'donor-only' sample.
  • the modulation is caused by background that is generated by the 56l-nm excitation.
  • Fig. 17f shows the experimental differences between consecutive even and odd frames of Alexa Fluor ® 555 in the 'donor-acceptor' sample. While the modulation of fluorescence intensity in the 'donor-only' sample is less than 1% of the intensity and remains constant for the entire duration of the imaging, the modulation of the donor-acceptor sample is more than three times larger and decays during the imaging. The decay in the modulation in the donor acceptor sample is explained due to the decay of the acceptor, which causes a decay in the FRET process.
  • FIG. 18a-18f showing examples of time trajectories of a measured molecule and the corresponding absolute value of the Fourier transforms with several different FOMs for each time trajectory.
  • Fig. 19 demonstrates the histogram of the length of intensity trajectories taken from 36 different cells. The vast majority of the trajectories (> 99%) of the molecule were shorter than 100 frames. This raises the question of how short a trajectory can be so that we can successfully apply our analysis to it. In order to answer this question, we calculated the average frequencies ratio for trajectories with a varying minimum length (average frequencies ratio for all trajectories that are longer than 8 frames, 16 frames and so on). Reference is now made to Fig. 20 showing the average of the frequency ratios for trajectories longer than a minimum length.
  • the series of the upper dots are the average on samples with donor and acceptor, while the lower dots are the average of 'donor-only' samples.
  • the averages are taken on trajectories from 18 cells for each sample.
  • the error bars show the SEM (standard error of the mean).
  • Fig. 21a-21b showing the distribution of the frequency ratio for different trajectory lengths for the sample with donor and acceptor and with 'donor- only', respectively, taken from 18 cells. Since the acceptor can photobleach or enter a dark state, it is possible that the energy transfer occurs only in a part of the time in which the donor is 'on'. Therefore, we also used windowed Fourier transform on segments of different sizes of the intensity trajectory and calculated the same ratio in every time segment. The shortest segment we used was of 8 frames from the same considerations of using trajectories of 8 frames, but not shorter.
  • Fig. lOe shows the spectrogram for the windowed Fourier transform of a single trajectory built with MATLAB ® .
  • the length of the trajectory was 132 frames and the size of the window was 64 frames. Between every two consecutive windows, there was an overlap of 62 frames. The two lowest frequencies are not drawn.
  • the Nyquist frequency was the dominant frequency in every window. This process was done for every window size between 8 and 200 frames. For each window, the FOM value was calculated, as in Fig.l7a-17f, and the maximal value was chosen. Colour bar in Fig. 22 indicates the absolute values of the Fourier transform (square root of the power), in arbitrary units.
  • Fig. 23 shows the average FOM against all trajectories that were longer than a minimal length (same as Fig. 20 only that the FOM was calculated in this case with the windowed Fourier transform).
  • Fig. 24a-24b show the distribution of the FOM on different trajectory lengths after the use of the windowed Fourier transform, from the samples with donor and acceptor (for 18 cells) and from the 'donor-only' sample (for 18 cells), respectively.
  • the increase in the average value of FOM is apparent.
  • Fig. lOf the average FOM of every cell was then calculated, followed by calculation of the p-value of the two populations.
  • the obtained p-value was 8xl0 10 , which indicates that there is a highly distinct difference between the donor-acceptor and donor-only populations.
  • the FOM threshold is determined to indicate whether a specific molecule was indeed a part of a FRET pair.
  • Fig. 25a-25c showing an example of how the FOM threshold is determined.
  • Fig. 25a shows the total number of trajectories with the FOM larger than a specified value.
  • Upper dots mark the donor-acceptor sample and the lower dots mark the 'donor-only' sample.
  • Fig. 25b shows the fraction of trajectories compared to the total number of trajectories. For example, for the minimal FOM that is equal to 3, the fraction was 0.201 because there was a total of 6708 trajectories. The fraction was higher in the donor-acceptor sample for all FOMs.
  • Fig. 25c shows the minimal FOM ratio between the fractions in Fig. 25b. For the minimal FOM value of 3, this ratio was 0.2632, which means that if the threshold is set at 3, the rate will have 26.32% false positive detections. From Fig. 25c, it is actually possible to obtain the percentage of false positive detections as a function of the FOM threshold. For 10% false positives, the threshold should be placed at 4.5. Every molecule with a higher FOM than this threshold was part of a FRET pair in 90% confidence. [0324] Reference is now made to Fig. 26a shows a FOM histogram for a cell containing both the donor and the acceptor.
  • the black dashed line in the histogram marks a threshold of FOM that is equal to 4.5.
  • the histogram of the donor- acceptor cell was shifted to the right, compared to that of the 'donor-only'. After thresholding, 47 molecules (out of 361 trajectories that were longer than eight frames), which were part of a FRET pair, were identified in this 'donor- acceptor' cell (with about 10% false positive).
  • Fig. 26b shows a FOM histogram for a cell containing only the donor.
  • the black dashed line in the histogram marks a threshold of FOM that is equal to 4.5. After thresholding, only two molecules (out of 363 long trajectories), which were part of a FRET pair, were identified in this 'donor-only' cell.
  • Example 13 Use of a smartphone for single molecule detection
  • Figs. 28a-28b show the prototype device of the present invention having a smartphone as the acquisition module.
  • the initial experiments performed with this set-up was to detect single molecules capable of FRET and to probe their FRET via sensitised emission.
  • the molecules were obtained from the short DNA molecules singly labelled with a fluorescent dye Cy3B using the CMOS camera of the smartphone (Xiaomi ® Note 3) as the detector.
  • the results of the experiment are shown in Fig. 29, which is the screenshot image of a smartphone generated with its CMOS camera for the fluorescence emission of the short DNA molecules singly labelled with a fluorescent dye Cy3B.
  • the sample prepared for this experiment contained short DNA molecules that were labelled with a FRET pair, as follows.
  • the Cy3B dye which was used as a donor, was labelled at the 3-prime end.
  • the Cy3B dye is a common donor for the FRET, when interacting with a red fluorophore as an acceptor (e.g. Atto647N) and is capable of undergoing photoactivation and blinking when used in a STORM buffer.
  • the Atto647N dye which was used as an acceptor, was labelled at the 5-prime.
  • the DNA molecules were diluted in a TE buffer to a final concentration of 10 nM and dropped into an ibidi ® chamber for imaging. In a negative control sample, the DNA molecules contained only the donor label, without the acceptor.
  • CMOS camera of a smartphone is suitable for detecting the emission of single molecules, and hence for measuring their FRET in the invented device.
  • the device of the present invention is designed to use the smartphone technology, such as the CMOS camera chip.
  • the field of view (FOV) of the camera chip is split into two spectral regions (e.g . as in 'dual-view' or using a diffraction grating) or switched mechanically between emission filters for the donor and the acceptor.
  • Autofocus of the camera is used for easily focusing onto a coverslip or microscope slide (positioned with relatively low mechanical tolerance) using the smartphone processor for decoding.
  • the CMOS imager of the smartphone can generate either three images (see the algorithm below) or short movies.
  • the smartphone can serve as the system control of the device, while the following algorithm is used to analyse the results.
  • the goal of this application is to analyse FRET images via the sensitized emission approach.
  • the algorithm gets three RGB images from the CMOS camera of the smartphone (under well-defined settings), converts them into HSV (hue, saturation, value), reduces background (assumed here as the median for sparse images), and calculates the FRET value per each pixel. Perfect registration of all images is assumed.
  • the three images are called ⁇ ', ⁇ ', and ⁇ ' according to their acquisition conditions.
  • the total energy in the detector for each image is defined as:
  • X is either D, A, or F
  • y is either R, G or B.
  • alphaD (alphax D $ );
  • Matlab ® syntax It may be necessary to reshape E (currently a matrix with the same dimensions of an image) into a vector first.
  • Matlab EE reshape(E ,size(E,2) 2); then do hist(EE(EE>0),50)
  • N T count(E > th E )
  • Designated rout could be email or any messenger application.
  • the present application relates to a new device and method employed by the device to image and measure single intermolecular (protein-protein) interactions in cells.
  • the method is the combination of the FRET and the SMLM technique.
  • the FRET was measured by detecting intensity of the donor emission. This emission was modulated by reversible acceptor saturation that led to the FRET frustration.
  • the lock-in detection of this modulation allows to detect smFRET occurring between individual FRET pairs, and to localise them in densely labelled cells.
  • the optical device and the methods of the present invention have made it possible for the first time to detect single interactions between primary and secondary antibodies in densely labelled cells and to further determine the intramolecular distance between the donor- and the acceptor-labelled antibodies with the Angstrom resolution.
  • a super-resolved optical image of a cell in single molecule detail and with distance measurements that continuously span Angstroms to microns was provided.
  • the present application has a strong proof of concept approach supported by Examples and is based on a fairly simple but ubiquitous case of protein-protein interactions between a primary and a secondary antibody.
  • the device of the present invention and the methods employing thereof are not limited to these examples.
  • Another interesting aspect of the present invention is the ability to use smartphone technologies, such as the CMOS camera chip, within the device of the invention for acquisition of the single molecule images and analysis of the inter-molecular interaction.
  • smartphone technologies such as the CMOS camera chip
  • This concept is revolutionary in all aspects as it allows significant miniaturisation and simplification of the devices and methods for point-of-care diagnostics, not mentioning the availability of further immediate technological advancements in this field.

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Abstract

La présente invention concerne un dispositif optique et des procédés utilisés par le dispositif pour imager et mesurer des interactions intermoléculaires uniques dans des cellules, en particulier dans des cellules marquées de manière dense, et pour déterminer en outre la distance intramoléculaire entre les molécules marquées donneuses et les molécules marquées acceptrices avec la résolution d'Angström. Le dispositif et les procédés sont basés sur la combinaison de la technique de transfert d'énergie par résonance de type Förster frustrée (f/FRET) et de microscopie unique de localisation de molécule (SMLM) avec la détection de verrouillage. Pour la première fois, une image optique super-résolue d'une cellule en un détail de molécule unique et avec des mesures de distance qui s'étendent de manière continue d'Angströms en microns est fournie par la présente invention. Le dispositif de la présente invention est configuré pour utiliser des technologies de téléphone intelligent, telles que la puce de caméra CMOS et le processeur de téléphone intelligent pour l'acquisition des images à molécule unique et l'analyse des interactions intermoléculaires, et peut être utilisé pour des diagnostics sur le lieu d'intervention.
PCT/IL2019/050794 2018-07-16 2019-07-15 Dispositifs optiques et procédés de mesure de l'efficacité du transfert d'énergie par résonance de type förster WO2020016887A1 (fr)

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CN113390844A (zh) * 2021-06-17 2021-09-14 中国药科大学 多尺度光纤荧光显微成像系统
US20230062169A1 (en) * 2021-03-22 2023-03-02 Kabushiki Kaisha Toshiba Optical apparatus, optical inspection method and non-transitory storage medium
EP4090951A4 (fr) * 2020-01-16 2023-09-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd Système et procédé pour une utilisation en microscopie à transfert d'énergie par résonance de type förster (fret)

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