Electrochemical fluorescence modulation for multicolour imaging techniques

Abstract

The present invention relates to a fluorescence microscope comprising a container for receiving a solution having a sample that comprises a plurality of biomolecules (such as cellular structures) to be imaged, and an electrode for contacting the solution. The solution also comprises an electrochemically active buffer composition for influencing the redox state of the fluorescent probes for modulating their brightness. The microscope further comprises a device configured to apply a varying electrical potential to the electrode, and a sensor for obtaining a resulting emission intensity profile during the application of the varying electrical potential, whereby when a plurality of fluorescent probe types that are capable of binding to a specific one of the plurality of biomolecules are disposed on or near the electrode, the varying electrical potential modulates the intensity of light emitted from the fluorescent probes. The resulting emission intensity profile may be subsequently colour un-mixed to obtain a plurality of colour un-mixed images that correlate to the fluorescence of each fluorescent probe type, thereby imaging each specific biomolecule / cellular structure which is bound to that specific fluorescent probe type. One or more of the coloured images may be merged to produce a composite image. The invention further relates to an attachment for a fluorescence microscope, a method of manufacturing and operating a fluorescence microscope, and a system for operating a fluorescence microscope.

Description

ELECTROCHEMICAL FLUORESCENCE MODULATION FOR MULTICOLOUR IMAGING TECHNIQUESRELATED APPLICATION

[0001] This application claims priority from Australian Provisional Patent Application No. 2024901257 filed 2 May 2024, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to the field of multicolour microscopy techniques. In particular, the invention relates to electrochemical fluorescence modulation for multicolour imaging. However, it will be appreciated that the invention is not limited to this particular field of use.BACKGROUND

[0003] The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0004] Fluorescence microscopy is widely employed for visualizing biological specimens with high specificity and sensitivity. Being able to visualize multiple molecular species within cells is important for understanding complex cellular processes, which makes multiplexing microscopy a valuable approach.

[0005] Life-science researchers can currently access a wide range of commercially available immuno-fluorescence probes and microscope systems marketed by a variety of companies. The most affordable of these systems are principally those using a single excitation laser and fluorophores with emissions in the visible and near IR. Increasing the number of fluorophores that can be used during a single experiment is technically feasible with current technologies, but relies on relatively expensive and / or complicated approaches, including: the development of novel fluorophores with excitation wavelengths and emission spectra outside of the conventional spectral window,selection of dyes has minimal spectral overlapping that compromise the overall number of dyes can be used,• using additional lasers to targeting novel excitation wavelengths,• the addition of hyperspectral cameras or light grating device to enable the recording of novel emission spectra, and / or• the implementation of lifetime unmixing, based on the decay of fluorescence.

[0006] Fluorescence imaging is a scheme for labelling a biomolecule within a sample with various fluorophores, exciting the fluorophores by using light, and indirectly monitoring the biomolecules within the sample by detecting light emitted from each of the fluorophores through an optical microscope. The fluorophores have different excitation spectra and emission spectra due to their unique chemical structures. In this case, the fluorophore emits light having a longer wavelength than the absorbed light. In general, the excitation and emission spectrum of the fluorophores have a wide width of about 100 nm within a visible ray range (400 to 700 nm). In order to simultaneously detect several biomolecules within one sample, it is necessary to label the several biomolecules with different fluorophores and then selectively obtain an image of each fluorophore. To this end, fluorophores whose excitation spectra and emission spectra do not overlap need to be used. If four or more fluorophores are simultaneously used, signals of fluorophores cannot be accurately distinguished because an emission spectrum overlap occurs between the fluorophores due to a wide width of an emission spectrum. Accordingly, in general, a maximum of four biomolecules are detected by exciting only one fluorophore per standard excitation wavelength (405, 488, 560, 633 nm).

[0007] Progress in optical setups has provided the opportunity for other multicolour imaging methods. With multichannel spectral detectors, fluorophores with substantial emission spectral overlap can be separated using a linear unmixing approach. Similar concepts in the art have been extended into an excitation spectral unmixing approach, where the excitation wavelength from a white light source was scanned through an acousto-optic tuneable filter (AOTF) while a single emission detection remains unchanged. In some cases, however, spectral unmixing alone is insufficient to resolve fluorophores that have almost identical spectroscopic characteristics, such as EGFP and Alexa fluor 488. Fluorescence lifetime is another option pursued in the prior art to discriminate fluorophores when they have nearly identical spectral features, but the fluorophores require sufficiently different lifetimes, and additional electronic devices are needed to measure the photon arrival time, which limits the applicability of this method. In addition to fluorescence spectral and lifetime, other photophysical properties have been explored for separation of mixed fluorophores. For instance, utilizing different fluorophores that exhibit varying levels ofphotostability, leading to the possibility of achieving separation based on their distinct characteristic photobleaching rates. Another prior art example is the unmixing of photoswitchable fluorophores based on their variation in modulation depth.

[0008] None of these methods provide a multicolour imaging strategy using dyes of similar colour on a standard widefield microscope, where a plurality of colours (e.g., four colours) can be resolved using a single excitation laser and filter setup. Additionally, the prior art is not able to simultaneously and efficiently image fluorophores with overlapping, or at least partially overlapping emission spectra.

[0009] It is a preferred object of at least one form of the present invention to provide novel methods for distinguishing fluorophores with similar or overlapping emission spectra.

[0010] It is an object of another preferred form of the present invention to provide novel methods for distinguishing fluorophores that are readily applicable for standard widefield microscopes, confocal, and stimulated emission depletion (STED) super-resolution microscopes, with little or no modification to the optical setups, and easy adaptation to different microscopes, thereby making multicolour imaging much simpler and more affordable.

[0011] It is an object of the present invention to overcome or ameliorate one or more the disadvantages of the prior art, or at least to provide a useful alternative.SUMMARY

[0012] The present disclosure provides methods for distinguishing fluorophores with similar or overlapping emission spectra by utilizing electrochemical signals to create a precise control of fluorophore modulation between relatively bright and dim states. The method of the disclosure additionally utilizes image processing techniques to simultaneously identify the contribution each fluorophore makes to the overall resulting emission intensity profile. Utilizing an electrochemical system as described herein provides a flexible and relatively low-cost solution compared to the prior art. In particular, the application of electrochemical stimulation during immunofluorescence imaging provides the ability to distinguish fluorophores, and which does not rely on an optical separation of their emission spectra. The application of an electrical potential simultaneously with stimulated fluorescence microscopy is a novel means of distinguishing the response of fluorescence dyes with the same, similar, or at least partially overlapping emission spectra. Previous attempts at distinguishing fluorophores (with similar or overlapping emission spectra) that have utilized the application of electric fields have modulated the intensity of fluorescence in single fluorophores (e.g., rhodamine derivatives), but to the inventor’s knowledge this is the first time that an electrical potential has been utilized to discriminate between multiple fluorophores.

[0013] The skilled person will appreciate that the present dislcosure may provide one or more significant advantages and improvements in the field and / or in view of the prior art. For example, these advantages may include the following.• The technique of the disclosure allows for the unmixing of fluorophores with overlapping spectra, allowing a greater density and variety of fluorophores to be deployed on current microscopes.• The technique of the disclosure advantageously provides for localisation and hyperresolution techniques to be applied on single-laser systems.

[0014] In preferred embodiments, the present disclosure involves the following elements: a) the integration of an electrochemical cell into a fluorescence optical microscope, preferably including an indium tin oxide (ITO) electrode surface, a counter electrode and reference electrode connected to a potentiostat, b) the application of a varying electrical potential to modulate the emissions of fluorescent probes during immunofluorescence imaging (i.e., varying their brightness), and c) the use of an image processing technique to analyse time-modulating images, and to correlate emissions to specific fluorophores, and thereby discriminate different biomolecules associated with those specific fluorophores, and thereby for example image different cellular structures within the same sample.

[0015] The present disclosure enables implementation of a relatively simple multicolour fluorescence imaging approach without hardware modification of the existing optical system (such as standard widefield microscopes, confocal, and stimulated emission depletion (STED) superresolution microscopes), thereby making multicolour imaging much simpler and more affordable. In preferred embodiments, the image processing technique utilized is linear unmixing. However, other image processing techniques will be known to the skilled person and will be discussed further below.

[0016] Disclosed herein are arrangements which seek to address the problems of the prior art by replacing photochemical processes with an electrochemical process. The electrochemical process according to the present disclosure uses an electrode disposed on or near a surface of a glass slide adjacent to the solution of the fluorescence microscope. A varying electrical potential is then applied to the electrode to thereby modulate the emissions of fluorescent probes during immunofluorescence imaging, followed by use of an image processing technique to analyse timemodulating images, and correlate emissions to specific fluorophores, thereby mapping the sampleunder study. How the brightness of specific fluorophores varies is a characteristic function of the electrical potential, enabling specific fluorophores to be differentiated. The present disclosure enables simultaneously studying multiple fluorophore labels in biological samples (biomolecules) at sub-cellular, cellular and tissue levels.

[0017] In one example, the inventors have been able to demonstrate the simultaneous acquisition of images with four distinct fluorescent labels with overlapping emission spectra, specifically Abberior STAR 580, mCherry H2B, Alexa Fluor 568 and Alexa Fluor 555. However, it will be appreciated that there will be many other combinations and permutations which will be possible, as will be discussed further below. Additionally, it is further demonstrated herein that the process of the disclosure is applicable across a range of fluorescence microscopy systems, including confocal and stimulated emission depletion (STED) microscopy. As such, the present disclosure has the potential to advance research across diverse fields, such as cell biology, disease study, and drug discovery.

[0018] According to a first aspect, the present invention provides a method for operating a fluorescence microscope, the fluorescence microscope comprising: a container for receiving a solution, the container having an electrode for contacting the solution, a device configured to apply a varying electrical potential to the electrode, a sensor for obtaining a resulting emission intensity profile, and the method comprising the steps of: providing the solution in the container, the solution having a sample comprising a plurality of different biomolecules, and an electrochemically active buffer composition for influencing the redox state of a fluorescent probe, providing a plurality of different fluorescent probes, wherein each different fluorescent probe binds to at least one of the different biomolecules, the fluorescent probes being disposed on or near the electrode, applying the varying electrical potential thereby modulating the fluorescence intensity of the bound fluorescent probes, obtaining the resulting emission intensity profile during the application of the varying electrical potential, andcolour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe.

[0019] In some embodiments, the emission spectrum of at least 2 of the fluorescent probe types is at least partially overlapping. In other embodiments, the emission spectrum of multiple fluorescent probe types is completely overlapping, or several may be partially / completely overlapping and others nonoverlapping. In further embodiments, the emission spectrum of multiple fluorescent probe types is non-overlapping.

[0020] According to a second aspect, the present invention provides a system for operating a fluorescence microscope, the system comprising: a container for receiving a solution having a sample comprising a plurality of different biomolecules (such as different cellular structures) and an electrochemically active buffer composition, the container having an electrode for contacting the solution, and a device configured to apply a varying electrical potential to the electrode, and whereby when a plurality of different fluorescent probes that are capable of binding to at least one of the plurality of different biomolecules are disposed on or near the electrode, the electrical potential is varied thereby modulating the fluorescence intensity of the fluorescent probes, and the resulting emission intensity profile is obtained during the application of the varying electrical potential, followed by colour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe.

[0021] Preferably the system for operating a fluorescence microscope includes a sensor for obtaining a resulting emission intensity profile during the application of the varying electrical potential.

[0022] Preferably the system for operating a fluorescence microscope includes a processor in communication with the sensor, wherein the processor is configured for receiving the resulting emission intensity profile from the sensor and for colour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each fluorescent probe type, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe type.

[0023] According to a third aspect, the present invention provides an attachment for a fluorescence microscope, the attachment comprising: a container for receiving a solution having a sample comprising a plurality of different biomolecules and an electrochemically active buffer composition for influencing the redox state of a fluorescent probe, the container having an electrode for contacting the solution, and a device configured to apply a varying electrical potential to the electrode.According to a fourth aspect, the present invention provides a method of manufacturing a fluorescence microscope, the method comprising: providing a container for receiving a solution having a sample comprising a plurality of different biomolecules and an electrochemically active buffer composition for influencing the redox state of the fluorescent probes for modulating their brightness, the container having an electrode for contacting the solution, and providing a device configured to apply a varying electrical potential to the electrode.

[0024] Preferably the method of manufacturing the fluorescence microscope includes the step of providing a sensor for obtaining a resulting emission intensity profile during the application of the varying electrical potential.

[0025] Fluorescence microscopes

[0026] As the skilled person would know, a fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances, and in particular uses fluorescence to generate an image. The present disclosure will be described in reference to a total internal reflection fluorescence microscope (TIRF) microscope. However, the invention described in the present disclosure can be used for other types of fluorescence microscopes, such as standard widefield microscopes, confocal, and stimulated emission depletion (STED) super-resolution microscopes.

[0027] The basic principle of the fluorescence microscope comprises illuminating a specimen with light of a specific wavelength (or wavelengths) which is absorbed by fluorophores, causing them to emit light of longer wavelengths (i.e., of a different colour than the absorbed light). Typically, the illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope are a lightsource (xenon arc lamp or mercury-vapor lamp are common; more advanced forms are high- power LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beamsplitter), and an emission filter.

[0028] In one embodiment, the fluorescence microscope is used for single molecule localization microscopy (SMLM). Preferably the SMLM comprises molecule counting.

[0029] In one embodiment, the fluorescence microscope is a total internal reflection fluorescence (TIRF) microscope.

[0030] In this disclosure, the illumination source radiation may include an illumination wave with a wavelength in the range of 300 nm to 1 ,300 nm. The illumination source radiation may include an illumination wave with a wavelength in the range of 300 nm to 700 nm. The illumination source radiation may include an illumination wave with a wavelength in the range of 690 nm to 1 ,300 nm. For example, the illumination source may be a one-photon excitation source that is capable of generating electromagnetic radiation in the range of 300 to 700 nm. For example, such one-photon excitation source may generate an electromagnetic radiation that may include a wave with a wavelength of about 405 nm, about 458 nm, about 488 nm, about 514 nm, about 554 nm, about 561 nm, about 592 nm, about 630 nm, or a combination thereof. In another example, the source may be a two-photon excitation source that is capable of generating electromagnetic radiation in the range of 690 nm to 1 ,300 nm. Such excitation source may be a tuneable laser. Yet in another example, the source may a one-photon excitation source and a two-photon excitation source that is capable of generating electromagnetic radiation in the range of 300 nm to 1 ,300 nm. For example, such one-photon excitation source may generate an electromagnetic radiation that may include a wave with a wavelength of about 405 nm, about 458 nm, about 488 nm, about 514 nm, about 554 nm, about 561 nm, about 592 nm, about 630 nm, or a combination thereof. For example, such two-photon excitation source may be capable of generating electromagnetic radiation in the range of 690 nm to 1 ,300 nm. Such two-photon excitation source may be a tuneable laser.

[0031] Imaging solutions

[0032] Examples of organic fluorophores (i.e., dyes) include, but are not limited to, Atto 488, Alexa Fluor 488, CF 488A, CF 568, Atto 527, Fluorescein, FITC, Cy2, Cy3B, Alexa Fluor 568, TAMRA, Cy3, Cy3.5, Atto 565, CF 633, Alexa Fluor 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, Atto 680, CF 680, Cy5.5, Dylight 750, CF 750, Cy7, Alexa Fluor 790, IRDye 800 CW, and the like. Other fluorophores will be known to the skilled person.

[0033] Examples of fluorescent proteins include, but are not limited to, PA-GFP, PA-mRFP, PAmCherry, PATagRFP, PS-CFP2, Kaede, EosFP, mlrisFP, asFP595, Dronpa, Padron, mlrisGFP, erCherry, rsCherryRev, mlrisFP, rsEGFP, and the like. Other fluorescent proteins will be known to the skilled person. The inventors contemplate that any fluorescent protein, such as BFP, CFP, GFP, YFP, mCherry, and TagRFP, which are sensitive to electric potentials would be suitable for the present invention.

[0034] The fluorescent probe types discussed above are capable of binding to a specific one of different biomolecules (which correspond to different cellular structures). In some embodiments the fluorescent probes are fluorescent proteins. In preferred embodiments, the fluorescent probes are genetically tagged to the biomolecules (or cellular structures).

[0035] In one embodiment of the invention, the solution comprises a species that reversibly binds / conjugates to the fluorescent probes, and that the species is an electrochemically active species, such as potassium ferricyanide. Examples of electrochemically active species that can reversibly bind / conjugate to a fluorescent probe include buffer solutions, buffer solutions with thiol, and buffer solutions with reducing agents. Examples of buffer solutions includes tris buffer, phosphate buffer, citric acid-disodium phosphate buffer, citric acid-sodium citrate buffer, sodium acetate-acetic acid buffer, imidazole (glyoxaline)-hydrogen chloride buffer, sodium carbonatesodium bicarbonate buffer, and the like. Examples of the buffer solutions with thiol include cysteamine, 2-mercaptoethanol, dithiothreitol, and the like. Examples of buffer solutions with reducing agents include tris(2-carboxyethyl)phosphine hydrochloride and ascorbic acid, and the like.

[0036] Without being bound by any particular theory, it is believed that the brightness of the fluorescent probes may be modulated by essentially oxidising and reducing the dye as the electrical potential is periodically varied. It will be appreciated that the composition of the buffer is preferably selected to cause a change in the redox state of the fluorescent probes in response to the varying electrical potential. In particular, the change in fluorescent intensity may be due to the electrochemically modulated redox reaction of the fluorophores. To enhance the amplitude of the electrochemical fluorescence modulation, it is preferable to include redox-active chemicals in solution. Redox active species (such as potassium ferrocyanide / ferricyanide, trolox, ascorbic acid) in the buffer solution are preferred for effective and fast electrochemical fluorescence modulation.

[0037] Electrodes

[0038] Figure 1 a shows a TIRF microscope 200 having a container for receiving a solution 210 where one side of the container is sealed by a slide 230. The slide 230 has an electrode 220(which is preferably ITO) attached to one surface of the slide 230. The electrode 220 is arranged to be in contact with the solution 210. The TIRF microscope 200 also includes immersion oil 240, and an objective 250.

[0039] The TIRF microscope 200 also includes a potentiostat 270. As known in the art, the potentiostat 270 includes a working electrode, a reference electrode 274, and a counter electrode 276. In the present disclosure, the electrode 220 is used as the working electrode and is connected to the potentiostat 270 via a working electrode connection 272 to the electrode 220. However, other devices having two electrodes (rather than 3) for applying the electrical potential to the electrode 220 may be used. Such a device has a working electrode and an auxiliary electrode, such that an electrical potential is maintained between the working electrode and the auxiliary electrode. When using such a device, the electrode 220 is the working electrode.

[0040] Preferred embodiments of the present invention utilize a reference electrode 274, which in preferred embodiments is a silver / silver chloride wire in 3 M KCL Other suitable materials for the reference electrode may also be used, such as hydrogen electrode, mercury-mercury oxide electrode, saturated calomel electrode, copper-copper(ll) sulfate electrode, silver-silver chloride electrode, palladium-hydrogen electrode, dynamic hydrogen electrode, mercury- mercurous sulfate electrode, and the like.

[0041] Preferred embodiments of the present invention utilize a counter electrode 276, which is preferably made of non-reactive materials. Examples of materials suitable for the counter electrode include platinum, carbon, iron, copper, palladium, silver, nickel, lead, magnesium, titanium, aluminium, tin, zinc; silicon, amorphous silicon, silicon carbide, gallium nitride, gallium arsenide, gallium antimonide, indium phosphide, indium arsenide, fluorine tin oxide, indium antimonide, cadmium selenide, cadmium sulfide, cadmium telluride, zinc oxide, zinc selenide, zinc sulfide, zinc telluride, lead selenide, lead (II) sulfide, zinc phosphide, titanium dioxide, tin oxide, gallium selenide, indium gallium nitride, haematite, magnesium, and the like.

[0042] As discussed above, the container includes an electrode 220 for contacting the solution. Examples of the materials that can be used for the electrode include indium tin oxide (ITO), gold, iron, copper, platinum, palladium, silver, nickel, lead, magnesium, titanium, aluminium, tin, zinc, carbon; silicon, amorphous silicon, silicon carbide, gallium nitride, gallium arsenide, gallium antimonide, indium phosphide, indium arsenide, fluorine tin oxide, indium antimonide, cadmium selenide, cadmium sulfide, cadmium telluride, zinc oxide, zinc selenide, zinc sulfide, zinc telluride, lead selenide, lead (II) sulfide, zinc phosphide, titanium dioxide, tin oxide, gallium selenide, indium gallium nitride, haematite, magnesium, and the like.

[0043] In one arrangement, the electrode 220 can be made to be, or is, optically transparent. For example, when used in a TIRF microscope, the electrode is optically transparent. However, when used on other types of fluorescence microscopes, the electrode may not need to be optically transparent.

[0044] The potentiostat 270 is configured to apply a varying electrical potential to the electrode 220 (via working electrode connection 272), thereby modulating the intensity of light emitted from the fluorescent probes. The varying electrical potential can constitute a variety of waveforms, including a sinusoidal pattern and other forms such as a step change on / off pattern. Whatever waveform is utilized, the intensity of light emitted from the fluorescent probes should preferably be modulated, rather than the fluorescent probes switched “on” and “off’.

[0045] Figure 1 b shows a TIRF microscope 200 in which the slide 230 is omitted. In this case, the slide 230 may also function as the electrode 220. The TIRF microscope 200 is configured to process a sample 100. The sample 100 is placed on the surface of the electrode 220 (i.e., on the surface within the container). Once the container is prepared, the immersion oil 240 is placed on the objective 250. The slide 230 is then disposed on the immersion oil 240. Fluorescent probes 100 are disposed on the electrode 220 by immersing the electrode 220 (and the glass slide 230) in a solution containing fluorophores. Examples of such a solution include organic dyes, fluorescent protein, and quantum dots. A laser 260 is used by the TIRF microscope 200 for imaging purposes. The inventors have used standard protocols of preparing cell-covered surfaces, which in particular is a ITO coated glass coverslip in the present invention. The biomolecules (cells) could be labelled by transfection with fluorescent proteins before seeding on ITO, fixed and permeabilized. For labelling with organic dyes, the cultured cells were plated in 6- well plates with ITO slides on the day before fixation. The next day, cells were fixed using 4% glutaraldehyde in PBS and permeabilized with 0.25% Triton X100. The cells were then immunostained with antibodies labelled with fluorophores.

[0046] The present inventors have surprisingly found that it is possible to adjust the frequency and / or amplitude of a varying electrical potential to enable differentiation of different fluorescent probes. In one embodiment, this can be achieved by increasing (or decreasing) the frequency (the number of oscillations / cycles that occur each period of time), and / or amplitude (the peak deviation of the function from zero) of the varying electrical potential so that different fluorescent probes that may have the same / similar spectral properties respond to that varying electrical potential in different ways, thereby enabling them to be differentiated. In preferred embodiments, the fluorescent probes are not completely switched on / off, but instead the intensity of their emissions are varied over time. The potential is cycled from a relatively negative to a relatively positive value. The relatively negative voltage may be selected from the group consisting of: -2, -1.9, -1.8, -1.7, -1.6, -1.5, -1.4, -1.3, -1.2, -1.1 , -1 , -0.9, -0.8, -0.7, -0.6, -0.5 ,-0.4 ,-0.3, -0.2, -0.1 , 0, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9V. The relatively positive voltage may be selected from the group consisting of: -1 .9, -1 .8, -1 .7, -1 .6, -1 .5, -1.4, -1.3, -1.2, -1.1 , -1 , -0.9, -0.8, -0.7, -0.6, -0.5 ,-0.4 ,-0.3, -0.2, -0.1 , 0, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, or 2V. In one example, the relatively negative voltage is -1 .8V and the relatively positive voltage is 0V. The frequency may be selected from the group consisting of 0.2, 0.4, 0.6, 0.8, 1 , 1 .2, 1 .4, 1 .6, 1 .8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 Hz. The waveform of the varying (periodic) electrical potential may be sinusoidal, square, triangular or sawtooth, and any combination thereof. In some embodiments the peak-to-peak voltage is kept the same each cycle. However, in other embodiments, the peak-to-peak voltage can be increased or decreased over time, or at each cycle. These embodiments will be suitable in situations where electric switching of 2 dyes are similar at small potentials, but only differ at larger electric potentials and where their modulation depths varies. These embodiments are additional ways to discriminate between different fluorescent probes.

[0047] Preferably the electrical potential applied to the electrode is maintained between the electrode and an auxiliary electrode.

[0048] Preferably the device comprises a potentiostat, wherein the electrode is a working electrode of the potentiostat and the auxiliary electrode is a reference electrode of the potentiostat.

[0049] Sensor

[0050] A sensor (or detector) is provided to obtain a resulting emission intensity profile during the application of the varying electrical potential.

[0051] In this disclosure, the sensor / detector is at least one optical detector and may have a configuration that detects electromagnetic radiation absorbed, transmitted, refracted, reflected, and / or emitted ("target radiation") by at least one physical point on the target. The target radiation may include at least one wave ("target wave"). The target radiation may include at least two target waves. Each target wave may have an intensity and a different wavelength. The at least one optical detector may have a configuration that detects the intensity and the wavelength of each target wave. The at least one optical detector may have a configuration that transmits the detected target radiation to a processor, with which the at least one optical detector is in communication. The at least one optical detector may have a configuration that transmits the detected intensity and wavelength of each target wave to the processor. The at least one optical detector may have any combination of these configurations.

[0052] The at least one optical detector may include a photodiode, a photomultiplier tube, a photomultiplier tube array, a digital camera, a hyperspectral camera, an electron multiplying charge coupled device, a Sci-CMOS, a digital camera, or a combination thereof. The digital camera may be any digital camera. The digital camera may be used together with an active filter for detection of the target radiation. The digital camera may also be used together with an active filter for detection of the target radiation, for example, comprising, luminescence, thermal radiation, or a combination thereof.

[0053] In this disclosure, the target radiation may include an electromagnetic radiation emitted by the target. The electromagnetic radiation emitted by the target may include luminescence, thermal radiation, or a combination thereof. The luminescence may include fluorescence, phosphorescence, or a combination thereof. For example, the electromagnetic radiation emitted by the target may include fluorescence, phosphorescence, thermal radiation, or a combination thereof. For example, the electromagnetic radiation emitted by the target may include fluorescence. In this disclosure, the at least one optical detector may detect the target radiation at a wavelength in the range of 300 nm to 800 nm. The at least one optical detector may detect the target radiation at a wavelength in the range of 300 nm to 1 ,300 nm.

[0054] The resulting emission intensity profile may be acquired at a rate of 1 , 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 Hz.

[0055] Colour un-mixinq image processing techniques

[0056] The present invention provides a processor and a computer-implemented method for un-mixing (also called demixing) a fluorescent-light signal, wherein the fluorescent-light signal comprises at least two fluorescence emission responses that at least partially overlap, wherein each of the at least two fluorescence emission responses being representative of an individual impulse response of a fluorophore to a fluorescence-triggering light pulse.

[0057] The present invention provides a computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out one embodiment of the inventive computer-implemented method, a computer-readable medium comprising instructions which, when executed by a computer, cause the computer to carry out one embodiment of the inventive computer-implemented method. The processor and computer- implemented method colour un-mixes the resulting emission intensity profile to obtain a plurality of colour un-mixed images, each of which correlate to the fluorescence of each fluorescent probe type, thereby imaging each specific cellular structure which is bound to that specific fluorescent probe type. The invention provides use of an image processing technique to analyse the time-modulating images and to correlate emissions to specific fluorophores. An unmixing algorithm is provided which is discussed further below.

[0058] A display is provided which can display false colour images or false colour video of the specimen under study.

[0059] Although some aspects have been described in the context of an apparatus, it is clear that these aspects also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step also represent a description of a corresponding block or item or feature of a corresponding apparatus.

[0060] Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some embodiments, some one or more of the most important method steps may be executed by such an apparatus.

[0061] Depending on certain implementation requirements, embodiments of the invention can be implemented in hardware or in software. The implementation can be performed using a non- transitory storage medium such as a digital storage medium, for example a floppy disc, a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory, having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable.

[0062] Some embodiments according to the invention comprise a data carrier having electronically readable control signals, which are capable of cooperating with a programmable computer system, such that one of the methods described herein is performed.

[0063] Generally, embodiments of the present invention can be implemented as a computer program product with a program code, the program code being operative for performing one of the methods when the computer program product runs on a computer. The program code may, for example, be stored on a machine-readable carrier.

[0064] Other embodiments comprise the computer program for performing one of the methods described herein, stored on a machine-readable carrier.

[0065] In other words, an embodiment of the present invention is, therefore, a computer program having a program code for performing one of the methods described herein, when the computer program runs on a computer.

[0066] A further embodiment of the present invention is, therefore, a storage medium (or a data carrier, or a computer readable medium) comprising, stored thereon, the computer program for performing one of the methods described herein when it is performed by a processor. The data carrier, the digital storage medium or the recorded medium are typically tangible and / or non- transitionary. A further embodiment of the present invention is an apparatus as described herein comprising a processor and the storage medium.

[0067] A further embodiment of the invention is, therefore, a data stream or a sequence of signals representing the computer program for performing one of the methods described herein. The data stream or the sequence of signals may, for example, be configured to be transferred via a data communication connection, for example, via the internet.

[0068] A further embodiment comprises a processing means, for example, a computer or a programmable logic device, configured to, or adapted to, perform one of the methods described herein.

[0069] A further embodiment comprises a computer having installed thereon the computer program for performing one of the methods described herein.

[0070] A further embodiment according to the invention comprises an apparatus or a system configured to transfer (for example, electronically or optically) a computer program for performing one of the methods described herein to a receiver. The receiver may, for example, be a computer, a mobile device, a memory device or the like. The apparatus or system may e.g. comprise a file server for transferring the computer program to the receiver.

[0071] In some embodiments, a programmable logic device (for example, a field programmable gate array) may be used to perform some or all of the functionalities of the methods described herein. In some embodiments, a field programmable gate array may cooperate with a microprocessor in order to perform one of the methods described herein. Generally, the methods are preferably performed by any hardware apparatus.DEFINITIONS

[0072] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[0074] Unless the context clearly requires otherwise, throughout the description and the claims, the terms “comprise”, “'comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.

[0075] The transitional phrase "consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0076] The transitional phrase "consisting essentially of" is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term "consisting essentially of" occupies a middle ground between "comprising" and "consisting of".

[0077] Where the applicant has defined an invention or a portion thereof with an open-ended term such as "comprising", it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms "consisting essentially of" or "consisting of." In other words, with respect to the terms “comprising”, “consisting of”, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of” or, alternatively, by “consisting essentially of”.

[0078] While reference may be made in this disclosure to the invention comprising a combination of a plurality of elements, it is also understood that this invention is regarded to comprise combinations which omit or exclude one or more of such elements, even if this omission or exclusion of an element or elements is not expressly stated herein, unless it is expressly stated herein that an element is essential to the applicant' s combination and cannot be omitted. It isfurther understood that the related prior art may include elements from which this invention may be distinguished by negative claim limitations, even without any express statement of such negative limitations herein. It is to be understood, between the positive statements of applicant's invention expressly stated herein, and the prior art and knowledge of the prior art by those of ordinary skill which is incorporated herein even if not expressly reproduced here for reasons of economy, that any and all such negative claim limitations supported by the prior art are also considered to be within the scope of this disclosure and its associated claims, even absent any express statement herein about any particular negative claim limitations.

[0079] As used herein, with reference to numbers in a range of numerals, the terms "about," "approximately" and "substantially" are understood to refer to the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to + 1 % of the referenced number, most preferably -0 .1 % to +0 .1 % of the referenced number. Moreover, with reference to numerical ranges, these terms should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth.

[0080] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0081] The complete disclosures of the patents, patent documents and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated.

[0082] Unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0083] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X," or "Y," or "X and Y." Similarly, "at least one of X or Y" should be interpreted as "X," or "Y," or "both X and Y."

[0084] The indefinite articles "a" and "an" preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, andthe singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0085] As used herein, wt.% refers to the weight of a particular component relative to total weight of the referenced composition.

[0086] It will be understood that use of the term “between” herein when referring to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, a temperature of between 80 °C and 150 °C is inclusive of a temperature of 80 °C and a temperature of 150 °C.

[0087] Various features of the embodiments of the invention disclosed herein are, for brevity, described in the context of a single embodiment, but may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the illustrative embodiments disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0088] In the foregoing paragraphs, where various ratios of components have been disclosed. It will be appreciated that these ratios of components can be combined in any disclosed combination. For example, the ratio of A:B (which may be between about 100:1 and 1 :100 or any range therein), may be combined with the ratio of C:D (which may be between about 50:1 and 1 :50 or any range therein), and may be combined with the ratio of E:F (which may be between about 10:1 and about 1 :10 or any range therein).

[0089] As used herein, the term "cellular structures" is understood to refer to the different structures typically found in cells. The present disclosure may image a wide range of biomolecules, including proteins labelled with several antibodies or mRNA labelled with DNA. The present disclosure is not limited to the above listed kinds of samples and kinds of biomolecules, and it may be used for imaging all biomolecules which may be labelled with fluorophores in all kinds of samples in which fluorophores may be used.

[0090] In molecular biology and biotechnology, and as used herein, a “fluorescent tag”, also known as a “fluorescent label” or “fluorescent probe” or “dye” or “fluorescent species”, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labelling, uses a reactive derivative of a fluorescent molecule known as a “fluorophore”. The fluorophore selectively binds to a specificregion or functional group on the target molecule and can be attached chemically or biologically. “Fluorescent probe types” simply refers to different types of fluorophores, including fluorescent proteins and quantum dots.

[0091] Fluorophores are photoreactive chemical compounds that absorb light energy of a certain wavelength and emit that light at a longer wavelength. This capacity makes them useful as detection reagents in the study of cells and tissues. Because of their novel electronic configurations, fluorophores have unique and characteristic spectra for absorption (usually similar to excitation) and emission. These absorption and emission spectra show relative intensity of fluorescence, with the relative intensity classically plotted on the vertical axis versus wavelength on the horizontal axis. As used herein, the term "emission intensity profile" is understood to refer to the relative intensity of fluorescence measured at various wavelengths, which are produced as a result of exciting a fluorophore.

[0092] As used herein, the term “electrochemical modulation” is understood to mean to any process whereby changing the potential of an electrode alters the emission intensity of fluorescent species.BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The aspects described above, as well as other apparent aspects, advantages, and objectives of the present invention are apparent from the detailed description below in combination with the drawing, in which:

[0094] Figure 1 shows a TIRF microscope.

[0095] Fig. 2 shows linear unmixing of two fluorophores using simulation. Fig. 2a is a schematic illustration of a microscope integrated with the electrochemistry set-up of the present invention. A 3-electrode system is utilized, which includes a transparent ITO coated glass slide as the working electrode (WE), a silver / silver chloride wire in 3 M KCI as the reference electrode (RE), and a platinum mesh counter electrode. Each electrode is connected to a potentiostat, which applies a defined potential difference between the WE and RE. Fig. 2b shows simulated fluorescence signals from a non-switching (black line), switching (red line) fluorescent labels and 1 :1 mixture of the two fluorophores (blue line), in response to the oscillated electrochemical potential. The least square fitting with a constraint of non-negative value was applied to extract the contribution of each fluorophore from the mixture. The fitting residual is shown in Fig 2c. Simulation of the mixed non-switching and switching fluorophore strips. Gaussian noise was added along the vertical axis, ranging from 0 to 50% of the signal intensity. To aid in interpretationof the result, the non-switching (red) and switching (green) fluorophores were colour coated after the unmixing procedure.

[0096] Fig. 3 shows colour unmixing of six fluorophores in three spectral channels based on electrochemical fluorescence modulation. Fig. 3a shows a raw image of a multi-target labelled HeLa cell prior to colour unmixing. Fig. 3b shows the electrochemical fluorescence response spectra of three fluorophore pairs. Time-lapse image sequences were recorded during a triangular potential scan from -0.8 V to +0.15 V and back to -0.8 V at a scan rate of 500 mV s-1, with each sequence comprising 19 frames acquired at 200 ms per frame. Fig. 3c shows the unmixed fluorescence images of the HeLa cell, where actin filaments appear in yellow, paxillin in red, microtubules in green, transferrin in magenta, vimentin filaments in blue, and the nucleus in white. Fig. 3d shows the merged image after unmixing, demonstrating spatially resolved labelling of six different cellular targets. Fig. 3e shows a quantitative analysis of spectral crosstalk based on the Pearson correlation coefficient, calculated from six independent unmixing experiments across three spectral channels. Scale bar = 10 pm in Fig. 3a, 3c, and 3d.

[0097] Fig. 4 shows normalized fluorescence response of fluorophores in (Fig. 4a) 561 nm and (Fig. 4b) 642 nm channels to the sinusoidal electrochemical potential applied. The electrochemical potential was oscillated between -0.8 V and 0.1 V at the frequency of 2 Hz. Images were acquired at 10 Hz rate. Fig. 4c shows a comparison of the electrochemical fluorescence responses for Alexa 647 and ATTO 655 dyes. The electrochemical potential was scanned between -0.7 V and 0.5 V with a scan rate of 500 mV s-1.

[0098] Fig. 5 shows colour unmixing of four fluorescent labels within a single spectral detection channel based on electrochemical fluorescence modulation. Fig. 5a shows a raw fluorescence image of a HeLa cell labelled with four distinct targets prior to colour unmixing. The mitochondria, actin filaments, cell nucleus, and transferrin receptor were labelled with Alexa 594, Alexa 555, mCherry, and Alexa 568, respectively. Fig. 5b shows representative electrochemical fluorescence response spectra for Alexa 568, mCherry, Alexa 594, and Alexa 555. The potential was scanned linearly between -0.8 V and +0.15 V at a rate of 500 mV s-1, and time-lapse fluorescence sequences were recorded at 200 ms per frame over 19 frames. Fig. 5c shows the unmixed images corresponding to different subcellular targets. Fig. 5d shows a merged image after unmixing, where actin filaments are rendered in yellow, mitochondria in green, transferrin in magenta, and the nucleus in blue. Fig. 5e shows box plots representing the crosstalk factor calculated from six independent unmixing experiments (n = 6), quantified by the Pearson correlation coefficient. Scale bar = 10 pm in Fig. 5a, 5c, and 5d.

[0099] Fig. 6 shows two colour STED imaging based on electrochemical fluorescence switching. Fig 6a shows HeLa cells co-immunostained with tubulin STAR-Red and phalloidin ATTO 655. The image was generated by summing a total of four collected frames. Scale bar = 25 gm. Fig 6b shows normalized fluorescence response of phalloidin ATTO 655 (red dots) and tubulin STAR-Red (green dots). Across frames 1 to 4, the electrochemical potential was sequentially set to +0.4 V, -0.7 V, +0.4 V, and -0.7 V. Colour unmixed images of phalloidin tagged with ATTO 655 and microtubules labelled with STAR-Red the merged image was colour coded, green for microtubule and red for phalloidin.[000100] Fig. 7 shows confocal microscopy images for phalloidin tagged with ATTO 655 and microtubules labelled with STAR-Red after colour unmixing. The merged image in Fig. 7c was colour coded - green for microtubule and red for phalloidin. A zoomed in colour unmixed confocal (Fig. 7d) and STED (Fig. 7e) image of the microtubules that highlighted by yellow dotted line in (Fig. 7b). Comparison of the intensity line profile across two microtubule fibres highlighted in (Fig 7d) that acquired in confocal and STED mode.[000101] Fig. 8. shows four colour STED imaging based on electrochemical fluorescence modulation, a, A U2OS cell was co-immunostained with STAR Red-tubulin, ATTO 655-phalloidin, SiR-DNA, and STAR 635-paxillin. The raw image was generated by summing a total of six image frames, b, EC spectra of four dyes were measured at the respective labelled potential values, the STED images were captured with a frame rate of approximately 27 s. c, Electrochemical unmixed images of U2OS cells showing the structures for actin filaments, nucleus, paxillin, and microtubules, d, The merged unmixed image was color-coded, with yellow representing microtubules, magenta representing paxillin, red representing the nucleus, and blue representing actin. Scale bar = 10 pm in c and d, Scale bar = 2 pm in f.[000102] Fig. 9. Shows a list of potential-fluorescence curves for various fluorophores. The image panels are highlighted in green, orange, and red, corresponding to the green, red and far- red spectral channels for the respective fluorophores. The fluorescence signal was collected from the immunostained cells with the given dye or the fluorescent protein transfected cell and imaged over a widefield epi fluorescence microscope. During the acquisition of the EC spectra, the electrochemical potential was scanned from -0.8 V to 0.4 V and back to -0.8 V at a scan rate of 500 mV s’1.[000103] Fig. 10. Shows electrochemical colour unmixing demonstration across various z-focal planes, a, Raw fluorescence images of U2OS cells with ATTO 655-labeled actin and STAR Red- labelled mitochondria at different z-distances from the ITO surface, with the indicated distances of 0, 0.5, 1 , 2, 3, and 4 pm, respectively, b, EC-spectra for the two dyes, green for ATTO 655,magenta for STAR red, while the electrochemical potential was scanned from -0.8 V to 0.4 V and then back to -0.8 V with a scan rate of 0.5 V / s, the frame rate is 250 ms. c, Effective separation achieved at various z-focal planes, ATTO 655-actin was labelled in green colour, STAR red- mitochondria was labelled in magenta. Scale bar = 10 pm.[000104] Fig. 11. Shows an analysis of crosstalk as function of number of frames used for electrochemical colour unmixing, a, Raw images showing ATTO 655-labeled actin and STAR Red-labelled mitochondria for a U2OS cell, b, EC-spectra for STAR red (green line) and ATTO 655 (orange line), while the electrochemical potential was scanned from -0.8 V to 0.4 V and then back to -0.8 V with a scan rate of 0.5 V / s, the frame rate is 250 ms. c, Unmixed images obtained using 2, 4, 6, and 8 frames, demonstrating progressive improvement in separation, d, Crosstalk factor as a function of the number of frames used, showing decrease as the frame number increases from 2 to 8 frames. Scale bar = 10 pm.DETAILED DESCRIPTION AND EXAMPLES[000105] The skilled addressee will understand that the invention comprises the embodiments and features disclosed herein as well as all combinations and / or permutations of the disclosed embodiments and features. The present invention will now be described with reference to the following examples which should be considered in all respects as illustrative and non-restrictive.[000106] Linear unmixing of simulated signals with different responses to external electrochemical potential[000107] The working principle of the present invention is described in Fig. 1. An indium tin oxide (ITO) coated glass coverslip, which is both conducting and transparent, serves as both the imaging surface and electrode. By connecting the ITO coated glass coverslip to a potentiostat, the potential at the ITO surface can be precisely controlled. Consequently, the fluorescence intensity of the fluorophores attached to the surface becomes brighter under positive potential and dimmer under negative potential. In this work, the potential at the ITO was modulated as a function of time in a sinusoidal wave pattern, and hence, the fluorescence intensity of the fluorophores show their characteristic switching patterns.[000108] To validate whether fluorescent labels could be separated under a modulated potential, the inventors first created simulated datasets. In Fig. 2b, a pair of fluorophores that exhibited differing response to an oscillated electrochemical potential was simulated (red and black lines), considering the presence of photobleaching. A third sample was generated by combining the signals from two dyes in a 1 :1 ratio (blue line). A 20% Gaussian noise was introduced to the third sample to incorporate the camera noise and fluorescence background. Toascertain the precise contribution of each fluorophore within the mixed sample, the proportions of the switching and non-switching fluorophores were adjusted until the combined signal aligned perfectly with the profile from the provided mixed sample. In this case, the inventors utilized a least square fitting approach while enforcing the constraint of non-negative variable to determine the optimal solution for linear equations. Prior to fitting, the raw fluorescence signals from both the reference dye and sample were normalized by their means. This eliminated the potential interference from the heterogeneity of dye brightness and spatial distribution, and only the extent of relative deviation from the means in response to the oscillating electrochemical potential was taken into consideration. The fitting procedure gave a fractional contribution of 0.46 and 0.54 for the electrochemical potential responsive and non-response fluorophore, respectively. The residuals obtained from the fitting were symmetrically distributed around 0, indicating a satisfactory fit (see Fig. 2b).[000109] To further evaluate the unmixing algorithm, two fluorophores were un-mixed in a 100x100-pixel area, and their distribution patterns are shown in Fig. 2c. The Gaussian noise was increased from 0 to 50% of the signal along the vertical axis as illustrated in Fig. 2c. The unmixing process was performed after applying 8 cycles of oscillating electrochemical potentials. The results surprisingly showed the successful separation of the two fluorophores. Surprisingly, the increased noise showed no impact on the unmixing algorithm. The results indicate the capability of an unmixing algorithm for separating the signals with low signal to noise ratios. The simulation described herein verifies that the linear unmixing approach can precisely extract the contribution of each fluorophore within a mixed signal. The unmixing algorithm was employed for separating fluorophores in the experiments that follow.[000110] Six colour imaging in three spectral channels[000111] The following provides an experimental demonstration of multicolour imaging by unmixing fluorescent labels from their distinctive responses to the electrochemical potential, in which three pairs of fluorophores were imaged on a confocal microscope. The spectra of each pair of fluorophores have significant overlapping, and lie in green, red, and far-red spectral channels, respectively. In the green spectral channel, microtubules were genetically tagged with EGFP, while the intermediate filament vimentin was immunolabelled using an anti-vimentin antibody conjugated with Alexa Fluor 488. In the red channel, the nuclear protein Histone H2B was genetically fused with mCherry, and the transferrin receptor was immunostained using an anti-transferrin antibody conjugated with Alexa Fluor 568. In the far-red channel, the actin cytoskeleton was visualized using phalloidin conjugated with ATTO 655 dye, and the focal adhesion protein paxillin was labelled with an anti-paxillin antibody tagged with STAR 635. The 488 nm, 561 nm, and 638 nm laser lines were used for excitation. Sequential line scanning wasperformed to minimize cross-excitation and crosstalk in the emission. The fluorescence was separated using the tunable Acousto-Optical Beam Splitter (AOBS) system, with the bandwidth for green, red, and far-red spectral detection set to 495-550 nm, 570-630 nm, and 650-720 nm, respectively. Three time-lapse image series were acquired across three spectral channels in sequential line-scanning mode, while the electrochemical potential was scanned from -0.8 V to +0.15 V and back in a single cycle. In the raw images (Fig. 3a), distinguishing individual stained cellular structures was challenging due to signal overlap. However, the electrochemical fluorescence spectra (Fig. 3b) revealed distinct responses for each fluorophore pair. For example, Alexa Fluor 488 exhibited a more pronounced intensity modulation than EGFP in the green channel. In the red channel, Alexa Fluor 568 showed a symmetric bell-shaped response, in contrast to the asymmetrical profile of mCherry. In the far-red channel, ATTO 655 displayed a sharper and more symmetric spectrum compared to the broader profile of STAR 635. These differences enabled effective separation of fluorophores via linear unmixing, as illustrated in the unmixed (Fig. 3c) and merged (Fig. 3d) images. To evaluate unmixing performance, crosstalk between unmixed image pairs was quantified using the Pearson correlation coefficient. As shown in Fig. 3e, the crosstalk factor remained below 0.03 for GFP-microtubule vs. Alexa 488-vimentin and mCherry-H2B vs. Alexa 568-transferrin, confirming effective spectral unmixing. A slightly elevated coefficient of 0.05 was observed for STAR 635— paxillin and ATTO 655-actin, reflecting true biological overlap at focal adhesions where these proteins colocalize functionally.[000112] Four colour imaging in a single channel[000113] After demonstrating the feasibility of separating two fluorescent labels in different spectral channels, the potential of resolving more dyes within the same channel was explored. Specifically, four red-emitting fluorophores have been trialed, (i.e., Alexa Fluor 568, mCherry, Alexa Fluor 555, and STAR-580), to label distinct cellular components: the transferrin receptor for cell tagging, the H2B protein for nucleus labelling, phalloidin for cytoskeletal staining, and the TOM20 for mitochondria identification, respectively.[000114] The samples were imaged on confocal microscope, with the 561 nm laser for excitation, and the detection bandwidth was set to 570-645 nm. Frame averaging of 6 was employed to increase the signal-to-noise ratio, resulting in a frame rate of approximately 200 ms.. A time-lapse series of images was acquired as the electrochemical potential was scanned from - 0.8 V to 0.15 V and back. Fig. 5a shows the raw image, averaged from the series, where cellular structures are challenging to distinguish. From electrochemical fluorescence spectra shown in Fig. 5b, the four fluorophores exhibited varying fluorescence modulation depths between -0.8 V and 0.15 V. Alexa 568 showed the most pronounced change, followed by mCherry, Alexa 594, and Alexa 555. More importantly, there are clear differences at the inflection points, shoulderpeaks, and troughs on each electrochemical fluorescence spectra, making it possible to identify and separate each fluorophore through the unmixing algorithm. After performing linear unmixing, the individual unmixed images and the colour-coded overlay image (Fig. 5c-d) showed all four labeled structures as distinctly discernible. This unmixing process also effectively separated different fluorescent labels within the same pixel, resolving structures such as mitochondria and actin located beneath the cell nucleus. The Pearson correlation coefficient was applied to analyse the unmixed image pairs, as shown in Fig. 5e. The results demonstrated minimal crosstalk, confirming the capability of our unmixing approach to separate four distinct fluorophores within a single imaging channel.[000115] Expanding the colour unmixing approach to confocal and STED microscopes[000116] The inventors have shown above surprisingly successful colour unmixing using wide field and confocal microscope demonstrated through electrochemical switching. In the following, the feasibility of electrochemical switching-based colour unmixing for STED microscopy is shown. Early implementations of multicolour stimulated emission depletion (STED) microscopy often involved complex optical setups, as each fluorophore utilized a pair of excitation and depletion lasers. Achieving the precise alignment of the laser pairs poses an optical complexity, furthermore due to the first blue-shifted depletion laser frequently results in direct excitation and subsequent photobleaching of the second, red-shifted dye, sequential imaging is required. A prior art technique involving two excitations lasers for red and far-red dyes, utilizing a shared single depletion laser, has been adopted for two-colour STED imaging, and the same concept has been previously extended up to 3 and 4 fluorophores. To reduce spectral crosstalk, interleaved excitation is often utilized to individually excite each dye over time. However, due to the limited spectral overlap between the red dye and the depletion laser, the resolution of the red dye is often compromised. In more recent developments, lifetime information has been integrated with spectral imaging for three-colour STED imaging.[000117] The present invention achieves multicolour STED by using a single pair of excitation and depletion lasers, but differentiating fluorophores by electrochemical switching them during the imaging process. This simplifies the optical setup while enabling the simultaneous imaging of fluorophores that shows distinct response to the electrochemical potential. Two widely used STED dyes, abberior STAR-Red and ATTO 655, were utilized to label microtubules and actin fibers within COS-7 cells attached on ITO. Opting for dyes with similar spectra, and simultaneously collecting their signals, offers the advantage of reducing drift related artifacts. For colour unmixing, four frames of STED images were acquired with electrochemical potentials settings at 0.4 V, -0.7 V, 0.4 V, and -0.7 V, respectively. Due to the substantial fluorescence intensity changes with the potential switch for both dyes (see Fig. 6b), the microtubules and actin filaments became distinctlyseparable through linear unmixing (see Fig. 6c). This separation was challenging prior to unmixing (see Fig. 6a), particularly in the central area of the cell. For the same cell, colour unmixing in confocal mode was conducted (see Figure 7). Surprisingly, the spatial resolution of the STED image remained uncompromised after colour unmixing. The zoomed-in region of the unmixed tubulin structure (Figure 7d-e) clearly demonstrated that the STED image exhibited higher spatial resolution. A line profile taken across two closely spaced microtubules was visibly resolved in the STED image, whereas it was not discernible in the confocal image (Figure 7f).[000118] To extend the applicability of electrochemical modulation in multicolour STED imaging, four widely used STED-compatible dyes — STAR Red, STAR 635, SiR (a silicon-rhodamine-based fluorophore), and ATTO 655 — were employed to label microtubules, paxillin, DNA, and actin in U2OS cells (Fig. 8a). To reduce photobleaching during repeated imaging, only six STED images were acquired at discrete electrochemical potentials (-0.7 V, -0.4 V, -0.35 V, -0.3 V, -0.15 V, and 0.15 V) to construct the EC spectrum of each dye (Fig. 8b). As shown in Fig. 8c, successful unmixing of the four dyes was achieved using a linear unmixing algorithm. Each unmixed image revealed clear and specific cellular structures, with minimal signal bleed-through across channels (Fig. 8d-e). The zoomed-in region of the unmixed microtubule image (Fig. 8f) further demonstrated the super-resolution performance of STED imaging.[000119] The present inventors have introduced a multicolour imaging method by electrochemical modulation of fluorophores and unmixing the fluorophores based their characteristic fluorescence response to varying the electrochemical potential. This approach enables visualization of up to four distinct cellular structures with a single excitation laser and filter setup, which has significantly expanded the number of imaging channels without requiring any modifications to the microscope itself. The present invention enables multicolour confocal and STED imaging by applying electrochemical fluorescence modulation and unmixing. The versatility of the techniques described herein, including their simplicity in setup, and the wide range of available fluorophores, make the invention a powerful and easily adaptable method for multicolour imaging on virtually any microscope system.[000120] Methods - Chemicals and materials[000121] Commercially manufactured ITO coated glass coverslip (06489-AB, SRI supplies, PA, USA) contains 750 nm thick layer of ITO deposited on 170 pm thick glass coverslip. It produces 8-12 Q electric resistance. Prior to placing cells on top, the ITO coverslip was plasma cleaned for 3 min and washed by 70% ethanol.[000122] The electrochemical imaging composition was prepared in the following way: (1 ) stock buffer A containing 50 mM Tris, 10 mM NaCI (adjust to pH 8), (2) 10% glucose was added tobuffer A, followed by adding 0.5 mg / mL glucose oxidase and 40 pg / mL catalase. Cysteamine and potassium ferricyanide was added to the oxygen scavenging tris buffer at final concentration of 50 mM and 1 mM, respectively.[000123] Immunofluorescence staining. The 6-dye electrochemical dye unmixing was performed using the EGFP-alpha-tubulin and H2B-mCherry stable transfected HeLa cell line (Cell Lines Service, Germany). The cells were cultured in Dulbecco's Modified Eagle's Medium fortified with 10% FBS, penicillin and streptomycin, and incubated at 37°C with 5% CO2. Cells were plated in 6-well plates with ITO slides in at -10,000-20,000 cells per well on the day before fixation.[000124] The immunostaining procedure was performed as following: Prior to fixation, cells were incubated in Transferrin conjugated with Alexa Fluor 568 (ThermoFisher, T23365) at -5 pg mL-1for 20 mins, and washed with PBS. Then cells were fixed for 7 minutes at 37°C using 4% paraformaldehyde (Sigma) in PBS, followed by washing with PBS. Permeabilization was carried out for 5 minutes using 0.2% Triton X-100 and 3% BSA in PBS. Subsequently, cells were incubated for 1.5 hours with a mixture of primary antibodies, which included mouse anti-Paxillin antibody (ThermoFisher, AHO0492), mouse anti-TOM20 F-10 antibody (Santa Cruz, sc-17764 AF488), and Alexa Fluor 488-tagged rabbit anti-Vimentin antibody (Abeam, ab185030), all at a concentration of 2 pg mL-1in blocking buffer (3% BSA in PBS). Cells were then washed with PBS and incubated to secondary antibodies for 30 mins, this includes Alexa 647 labelled goat antimouse IgG secondary antibody (ThermoFisher A-21235), and Abberior STAR RED conjugated goat anti rabbit secondary antibody (STRED-1002-500UG, Abberior, Gottingen, Germany), and Abberior STAR-580 conjugated goat anti mouse secondary antibody (ST580-1001 -500UG, Abberior, Gottingen, Germany) at a concentration of -2.5 pg mL"1in PBS; washed in PBS, secondary fixation for 5 min with 4% paraformaldehyde in PBS and finally washing with PBS.[000125] Electrochemistry. The sigmoidal oscillating potential were applied using SP-200 potentiostat (Bio-Logic, France). All the electrochemistry was carried out in a custom chamber (Chamlide EC 22, Live Cell Instrument Co., Ltd., Republic of Korea) containing an Ag | AgCI | 3M KCI reference electrode and a Pt-wire counter-electrode, where the working electrode was the indium tin oxide (ITO) coated coverslips (8-12 Q, 22x22 cm, SPI Supplies, USA).[000126] Image acquisition and analysis. The widefield images shown in Figs. 2 and 4 to 9 were collected on Zeiss Elyra 7 Super-resolution microscope (Zeiss, Germany) equipped with two metal oxide semiconductor (sCMOS) camera. For excitation, 488, 561 and 642 nm laser was reflected from a 405 / 488 / 561 / 642 nm quad band dichroic mirror (Chroma, TRF89901 v2,) and focused at the back focal plane of the 100 X 1 .46 NA Oil objective. The focus is laterally shifted alone the back focal plane to transit from EPI (0°) to HiLo (55.2°) or TIRF (66.7°) illumination. The fluorescence was collected by the same objective and guided to one of the two PCO Edge 4.2sCMOS camera through either a 560 nm long pass filter or a dual band pass filter (BP 490-560 nm / LP 640 nm) depending on the sample. The denoise option on the sCMOS camera was used to reduce noise patterns of the sCMOS camera. Laser intensity used were between 1 -4 kW cm-2. For the electrochemical dye unmixing, typically 100 frames were collected with exposure time of 100 ms.[000127] Dataset shown in Fig. 3, Fig. 5, and Fig. 10-1 1 were collected on a Leica Stellaris 8 Falcon FLIM microscope equipped with a resonance scanner, a white light laser, and HyD detectors. For the 6-colour imaging shown in Fig. 3, the 488 nm, 561 nm, and 638 nm laser lines from the white light laser were used for excitation. A 488 / 561 / 638 nm notch filter was placed in front of the white light laser to block any leaked laser light from other spectral regions. Sequential line scanning was performed using the resonance scanner in a bidirectional scan mode at 8k Hz, with sequential detection used to minimize cross-excitation and crosstalk in the emission. The fluorescence was separated using the tunable Acousto-Optical Beam Splitter (AOBS) system, with the bandwidth for green, red, and far-red spectral detection set to 495-550 nm, 570-630 nm, and 650-720 nm, respectively. For the 4-colour imaging shown in Fig. 5, the 561 nm laser line was used for excitation, and the detection bandwidth was set to 570-645 nm. The analog mode of the HyD detector was used, with the gain set to approximately 30 to prevent pixel saturation. Frame averaging of 6 was employed to increase the signal-to-noise ratio, resulting in a frame rate of approximately 200 ms.[000128] The laser scanning STED super-resolution imaging was performed on the PicoQuant microtime 200 STED microscope (PicoQuant, GmbH) equipped with a FLIM bee galvanometer scanner (PicoQuant GmbH, Berlin, Germany). A 640 nm picosecond pulsed laser (LDH series, PicoQuant GmbH, Berlin, Germany) and a 765 nm pulsed STED laser (VISIR-STED, PicoQuant GmbH, Berlin, Germany) was set to pulse at 40 MHz to excite and deplete the dyes, respectively. The STED pulse was electronically delayed by -200 ps relative to 640 nm laser to provide the maximal depletion efficiency for the two dyes selected. The STED pulse was widened to -300 ps to avoid two-photon excitation. The two lasers were reflected to the objective by a 640 / 760 nm dichroic mirror (Chroma, Bellows Falls, VT). Samples were imaged through a 100X Oil immersion objective (NA1 .40, UPlanSApo, Olympus, Tokyo, Japan). The fluorescence emitted was collected by the same objective and spatially filtered by a 100 pm pinhole at the conjugated focal plane. The pinhole purified fluorescence was guided and focused onto the Single Photon Avalanche Diodes (SPAD) (SPCM AQRH-14 TR, Excelitas Technologies, Waltham, MA). A 690 / 70 band pass filter (Chroma, Bellows Falls, VT) was inserted in front of a SPAD detector to clean up the reflected excitation laser, the STED laser and the stimulate emissions.[000129] Unmixinq algorithm. The linear unmixing algorithm used here is reminiscent to the colour unmixing of spectral imaging, where each pixel in the dye mixed sample is assumed to contain a mixture of reference dyes. Its electrochemical pattern (IA) is a total summation of electrochemical pattern of individual reference dyes (R iA) that is weighted by their concentrationrepresents the index of the fluorophore. The best solution of Ci is determined by least square fitting with constrains to be non-negative values. In which case, the sum of squared residuals between the experimental fluorescence intensity at each point of the electrochemical pattern in the form lAi, i = 1 , 2, 3,... n, and the parameter model intensity y= f(Ai, C1,C2,C3...Cm) is minimized, which is defined as S = Sf (^Ai—) . c is the parameter vector specifying the concentration of each dye, with m in total. To solve the equations, ds f the partial deferential equation in relation tois set to zero: '= 0 with a constrain thatC > 0. The set of Ci produces the smallest S from the experimental IA is regarded as the best fit. The linear unmixing algorithm requires the electrochemical switching pattern of individual reference dyes. This data can be collected separately using a reference dye-only sample or acquired by selecting the region containing only the reference dye within the sample containing mixed dyes. The challenge with the former approach lies in the requirement for fully synchronizing the electrochemical potential with the camera. In the current study, we obtained all reference dye spectra using the latter approach. To enhance the contrast between different dyes, a pseudo colorRGB image was generated, in which the sum R=, standard deviation G = maxfe) / the ratio of max to min B= / minV i) at each pixel across the domain of electrochemical pattern lAi.[000130] The person skilled in the art will understand that the unmixing algorithm described above is a convenient and powerful methodology to unmix a spectral image. However, other unmixing algorithms could be used instead of the approach described above. For example, another approach can be spectral phasor. In this case, the intensity modulation of the dyes under electric potentials would be treated as a ‘spectrum’ of the dye. In spectral phasor, the spectrum of the dyes are Fourier transformed, and the real and imaginary part of the transformed as plotted as 2D histogram, the peak position and width of the spectrum determines the 2D position of the dye. When multiple dyes are present in the sample their contribution influence the phasor position in an additive way. For instance, when the dyes are mixed, the phasor position lies on the linebetween the reference phasor position of the two reference dyes, and the position shift depend on the ratio of the mixture, which can be used to infer the contribution of each dye in the mixture.[000131] Another method is supervised or unsupervised machine learning, where a classification algorism can be employed to separate the dyes with certain confidence. For supervised learning, a ground truth data would need to be provided to train the algorithm, while for unsupervised learning, a clustering algorithm may be used to separate the data, k-mean clustering or hierarchical tree clustering could also be implemented.[000132] In some embodiments, the fluorescence microscope described herein is a total internal reflection fluorescence microscope (TIRF), and may be used for single molecule localization microscopy (SMLM), and wherein the SMLM comprises molecule counting.[000133] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, and in particular features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.[000134] Other embodiments of the invention as described herein are defined in the following paragraphs:1. A method for operating a fluorescence microscope, the fluorescence microscope comprising: a container for receiving a solution, the container having an electrode for contacting the solution, a device configured to apply a varying electrical potential to the electrode, a sensor for obtaining a resulting emission intensity profile, and the method comprising the steps of: providing the solution in the container, the solution having a sample comprising a plurality of different biomolecules, and an electrochemically active buffer composition for influencing the redox state of a fluorescent probe,providing a plurality of different fluorescent probes, wherein each different fluorescent probe binds to at least one of the different biomolecules, the fluorescent probes being disposed on or near the electrode, applying the varying electrical potential thereby modulating the fluorescence intensity of the bound fluorescent probes, obtaining the resulting emission intensity profile during the application of the varying electrical potential, and colour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe. ystem for operating a fluorescence microscope, the system comprising: a container for receiving a solution having a sample comprising a plurality of different biomolecules (such as different cellular structures) and an electrochemically active buffer composition, the container having an electrode for contacting the solution, and a device configured to apply a varying electrical potential to the electrode, and whereby when a plurality of different fluorescent probes that are capable of binding to at least one of the plurality of different biomolecules are disposed on or near the electrode, the electrical potential is varied thereby modulating the fluorescence intensity of the fluorescent probes, and the resulting emission intensity profile is obtained during the application of the varying electrical potential, followed by colour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe. attachment for a fluorescence microscope, the attachment comprising: a container for receiving a solution having a sample comprising a plurality of different biomolecules and an electrochemically active buffer composition for influencing the redox state of a fluorescent probe, the container having an electrode for contacting the solution, and a device configured to apply a varying electrical potential to the electrode.3. A method of manufacturing a fluorescence microscope, the method comprising: providing a container for receiving a solution having a sample comprising a plurality of different biomolecules and an electrochemically active buffer composition for influencing the redox state of the fluorescent probes for modulating their brightness, the container having an electrode for contacting the solution, and providing a device configured to apply a varying electrical potential to the electrode.4. The method, system or attachment according to any one of the preceding paragraphs wherein the fluorescent probes are organic fluorophores selected from: Atto 488, Alexa Fluor 488, CF 488A, CF 568, Atto 527, Fluorescein, FITC, Cy2, Cy3B, Alexa Fluor 568, TAMRA, Cy3, Cy3.5, Atto 565, CF 633, Alexa Fluor 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, Atto 680, CF 680, Cy5.5, Dylight 750, CF 750, Cy7, Alexa Fluor 790, and IRDye 800 CW.5. The method, system or attachment according to any one of the preceding paragraphs wherein the fluorescent probes are fluorescent proteins selected from: PA-GFP, PA-mRFP, PAmCherry, PATagRFP, PS-CFP2, Kaede, EosFP, mlrisFP, asFP595, Dronpa, Padron, mlrisGFP, erCherry, rsCherryRev, mlrisFP, rsEGFP, BFP, CFP, GFP, YFP, mCherry, and TagRFP.6. The method, system or attachment according to any one of the preceding paragraphs wherein the electrochemically active buffer composition comprises redox active species selected from the group consisting of potassium ferrocyanide / ferricyanide, trolox, and ascorbic acid.7. The method, system or attachment according to any one of the preceding paragraphs wherein the electrode is optically transparent, and is preferably indium tin oxide (ITO).8. The method, system or attachment according to any one of the preceding paragraphs wherein the varying electrical potential applied to the electrode is maintained between the electrode and an auxiliary electrode.9. The method, system or attachment according to any one of the preceding paragraphs wherein the device comprises a potentiostat.10. The method, system or attachment according to any one of the preceding paragraphs wherein the electrode is a working electrode of the potentiostat and the auxiliary electrode is a reference electrode of the potentiostat.1 1. The method, system or attachment according to any one of the preceding paragraphs wherein the potentiostat is configured to apply a varying electrical potential to the electrode, thereby modulating the intensity of light emitted from the fluorescent probes.12. The method, system or attachment according to any one of the preceding paragraphs wherein the varying electrical potential comprises waveforms selected from sinusoidal, square, triangular, sawtooth, and any combination thereof.13. The method, system or attachment according to any one of the preceding paragraphs wherein the varying electrical potential has a frequency selected from the group consisting of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 Hz.14. The method, system or attachment according to any one of the preceding paragraphs wherein the varying electrical potential is cycled from a relatively negative voltage to a relatively positive voltage, wherein the relatively negative voltage is selected from the group consisting of: -2, -1.5, -1 , -0.5, or 0V, and the relatively positive voltage is selected from the group consisting of: -1 , -0.5, 0, 0.5, 1.0, or 1.5V.15. The method, system or attachment according to any one of the preceding paragraphs further comprising a processor in communication with the sensor, wherein the processor is configured for receiving the resulting emission intensity profile from the sensor and for colour unmixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe.16. The method, system or attachment according to any one of the preceding paragraphs whereby imaging each specific biomolecule that is bound to a specific fluorescent probe images different cellular structures within the sample.17. The method, system or attachment according to any one of the preceding paragraphs wherein each colour un-mixed image is coloured with a pre-determined or an arbitrary colour to produce a false-coloured image of the specific cellular structures (which is / are bound to that specific fluorescent probe).18. The method, system or attachment according to any one of the preceding paragraphs wherein one or more of the false-coloured images are merged to produce a composite image.19. The method, system or attachment according to any one of the preceding paragraphs wherein the emission spectrum of at least 2 of the fluorescent probes is at least partially overlapping, or the emission spectrum of multiple fluorescent probes is completely overlapping.20. The method, system or attachment according to any one of the preceding paragraphs wherein when a plurality of different fluorescent probes that are capable of binding to a specific one of the plurality of different biomolecules that are disposed on or near the electrode, the varying electrical potential modulates the fluorescence intensity of the fluorescent probes bound to the biomolecules.21. The method, system or attachment according to any one of the preceding paragraphs whereby when a plurality of different fluorescent probes that are capable of binding to a specific one of the plurality of different biomolecules are disposed on or near the electrode, the varying electrical potential modulates the fluorescence intensity of the fluorescent probes.

Claims

CLAIMS1. A method for operating a fluorescence microscope, the fluorescence microscope comprising: a container for receiving a solution, the container having an electrode for contacting the solution, a device configured to apply a varying electrical potential to the electrode, a sensor for obtaining a resulting emission intensity profile, and the method comprising the steps of: providing the solution in the container, the solution having a sample comprising a plurality of different biomolecules, and an electrochemically active buffer composition for influencing the redox state of a fluorescent probe, providing a plurality of different fluorescent probes, wherein each different fluorescent probe binds to at least one of the different biomolecules, the fluorescent probes being disposed on or near the electrode, applying the varying electrical potential thereby modulating the fluorescence intensity of the bound fluorescent probes, obtaining the resulting emission intensity profile during the application of the varying electrical potential, and colour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe.

2. The method according to claim 1 wherein the fluorescent probes are organic fluorophores selected from: Atto 488, Alexa Fluor 488, CF 488A, CF 568, Atto 527, Fluorescein, FITC, Cy2, Cy3B, Alexa Fluor 568, TAMRA, Cy3, Cy3.5, Atto 565, CF 633, Alexa Fluor 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, Atto 680, CF 680, Cy5.5, Dylight 750, CF 750, Cy7, Alexa Fluor 790, and IRDye 800 CW.

3. The method according to claim 1 or 2 wherein the fluorescent probes are fluorescent proteins selected from: PA-GFP, PA-mRFP, PAmCherry, PATagRFP, PS-CFP2, Kaede, EosFP,mlrisFP, asFP595, Dronpa, Padron, mlrisGFP, erCherry, rsCherryRev, mlrisFP, rsEGFP, BFP, CFP, GFP, YFP, mCherry, and TagRFP.

4. The method according to any one of the preceding claims wherein the electrochemically active buffer composition comprises redox active species selected from the group consisting of potassium ferrocyanide / ferricyanide, trolox, and ascorbic acid.

5. The method according to any one of the preceding claims wherein the electrode is optically transparent, and is preferably indium tin oxide (ITO).

6. The method according to any one of the preceding claims wherein the varying electrical potential applied to the electrode is maintained between the electrode and an auxiliary electrode.

7. The method according to any one of the preceding claims wherein the device comprises a potentiostat.

8. The method according to claim 7 wherein the electrode is a working electrode of the potentiostat and the auxiliary electrode is a reference electrode of the potentiostat.

9. The method according to claims 7 or 8 wherein the potentiostat is configured to apply a varying electrical potential to the electrode, thereby modulating the intensity of light emitted from the fluorescent probes.

10. The method according to any one of the preceding claims wherein the varying electrical potential comprises waveforms selected from sinusoidal, square, triangular, sawtooth, and any combination thereof.1 1 . The method according to any one of the preceding claims wherein the varying electrical potential has a frequency selected from the group consisting of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 Hz.

12. The method according to any one of the preceding claims wherein the varying electrical potential is cycled from a relatively negative voltage to a relatively positive voltage, wherein the relatively negative voltage is selected from the group consisting of: -2, -1.5, -1 , -0.5, or 0V, and the relatively positive voltage is selected from the group consisting of: -1 , -0.5, 0, 0.5, 1 .0, or 1 ,5V.

13. The method according to any one of the preceding claims further comprising a processor in communication with the sensor, wherein the processor is configured for receiving the resulting emission intensity profile from the sensor and for colour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence ofeach different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe.

14. The method according to claim 13 whereby imaging each specific biomolecule that is bound to a specific fluorescent probe images different cellular structures within the sample.

15. The method according to claim 14 wherein each colour un-mixed image is coloured with a pre-determined or an arbitrary colour to produce a false-coloured image of the specific cellular structures (which is / are bound to that specific fluorescent probe).

16. The method according to claim 15 wherein one or more of the false-coloured images are merged to produce a composite image.

17. The method according to any one of the preceding claims wherein the emission spectrum of at least 2 of the fluorescent probes is at least partially overlapping, or the emission spectrum of multiple fluorescent probes is completely overlapping.

18. A system for operating a fluorescence microscope, the system comprising: a container for receiving a solution having a sample comprising a plurality of different biomolecules (such as different cellular structures) and an electrochemically active buffer composition, the container having an electrode for contacting the solution, and a device configured to apply a varying electrical potential to the electrode, and whereby when a plurality of different fluorescent probes that are capable of binding to at least one of the plurality of different biomolecules are disposed on or near the electrode, the electrical potential is varied thereby modulating the fluorescence intensity of the fluorescent probes, and the resulting emission intensity profile is obtained during the application of the varying electrical potential, followed by colour un-mixing the resulting emission intensity profile to obtain a plurality of colour un-mixed images that each correlate to the fluorescence of each different fluorescent probe, thereby imaging each specific biomolecule which is bound to that specific fluorescent probe.

19. An attachment for a fluorescence microscope, the attachment comprising: a container for receiving a solution having a sample comprising a plurality of different biomolecules and an electrochemically active buffer composition for influencing the redox state of a fluorescent probe, the container having an electrode for contacting the solution, anda device configured to apply a varying electrical potential to the electrode.

20. An attachment according to claim 19 wherein when a plurality of different fluorescent probes that are capable of binding to a specific one of the plurality of different biomolecules that are disposed on or near the electrode, the varying electrical potential modulates the fluorescence intensity of the fluorescent probes bound to the biomolecules.21 . A method of manufacturing a fluorescence microscope, the method comprising: providing a container for receiving a solution having a sample comprising a plurality of different biomolecules and an electrochemically active buffer composition for influencing the redox state of the fluorescent probes for modulating their brightness, the container having an electrode for contacting the solution, and providing a device configured to apply a varying electrical potential to the electrode.

22. A method of manufacturing a fluorescence microscope according to claim 21 whereby when a plurality of different fluorescent probes that are capable of binding to a specific one of the plurality of different biomolecules are disposed on or near the electrode, the varying electrical potential modulates the fluorescence intensity of the fluorescent probes.

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