IMAGING A TARGET FLUOROPHORE IN A BIOLOGICAL MATERIAL IN THE PRESENCE OF AUTOFLUORESCENCE

[0001] This application is a non-provisional application that claims the benefits of priority of U.S. Provisional Application No. 62/056,830, filed on Sep. 29, 2014, pending, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates generally to fluorescence imaging, and in particular to imaging a target fluorophore in a biological material in the presence of autofluorescence.

[0003] In the life sciences, fluorescence is typically used as a non-invasive method of identifying and analyzing biological materials. Specific targets in the biological material such as for example, proteins, nucleic acids, lipids, cells and cell components, stem cells or small molecules can be labeled with an extrinsic or exogenous fluorophore, and thus subsequently imaged. Biological materials also naturally fluoresce, which is known as intrinsic fluorescence or “autofluorescence” because it occurs in the absence of exogenously administered fluorophores. Autofluorescence is believed to originate from various endogenous fluorophores in biological materials, including for example nicotinamide adenine dinucleodde (NADH), elastin, collagen, flavins, amino acids and porphyrins.

[0004] Autofluorescence and fluorescence emission can be generated and recorded as images when light with the appropriate excitation wavelengths illuminates the biological material. However, autofluorescence, which is the result of a combination of fluorophores and is characterized by broad emission spectra extending over several hundred nanometers, can interfere with the ability to detect the emission of a specific fluorophore, when the emission spectra of the fluorophore and the autofluorescence overlap. In such instances, in addition to reducing signal detection sensitivity by masking the fluorescence of the fluorophore of interest, autofluorescence may also decrease the specificity of detection by providing false positive results.

[0005] One approach to addressing this problem is to utilize means to reduce or minimize the detected emission signal that is contributed by autofluorescence of the biological material. The prior art describes methods to reduce autofluorescence by employing various pre-treatments of the biological material prior to image acquisition. However, such techniques may also degrade the quality of the biological material itself, and are typically not suitable for in vivo applications. Alternatively, if the autofluorescence emission itself cannot be mitigated, it is possible to minimize the contribution of signal from autofluorescence to image data by means of digital manipulation of any acquired fluorescence images. For example, in images containing the combined signal from both the fluorophore of interest and autofluorescence, some of these methods rely on acquiring estimates of the “pure” autofluorescence signal and using such estimates to remove autofluorescence by a weighted subtraction. Other methods use statistical correlation techniques to correct for the additive autofluorescence signal. These image data manipulation techniques are described in prior art references and are generally limited by poor accuracy, by the need for small (i.e., low resolution) data sets, or by the need for significant post-processing. It is consequently desirable to establish a high resolution image processing technique to quickly and accurately distinguish the fluorescence emitted by a fluorophore of interest in a biological material from the autofluorescence emission in that same biological material.

[0006] In accordance with one aspect of the invention, there is provided a method for extracting an image of a target fluorophore in a biological material wherein a waveband for the target fluorophore emission overlaps a waveband for autofluorescence emission in the biological material. The method includes illuminating the biological material with a first excitation light to induce a first fluorescence emission arising from both autofluorescence of the biological material and fluorescence of the target fluorophore and with a second excitation light to induce a second fluorescence emission arising from the autofluorescence of the biological material, acquiring a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission, and processing the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore, wherein relative intensities of the first and second excitation lights are modulated prior to acquiring the first and second fluorescence images. The processing may for example involve subtracting the second fluorescence image from the first fluorescence image.

[0007] According to an embodiment, the modulation of the relative intensities includes identifying a wavelength region in the first and second fluorescence emissions, wherein the wavelength region is a region where emission arising from the fluorophore is present in the first fluorescence emission and absent in the second fluorescence emission, selecting a waveband outside the wavelength region, calculating at the selected waveband a ratio of relative intensities of the first and second fluorescence emissions, and adjusting the relative intensities of the first and second excitation lights to adjust the corresponding first fluorescence emission, second fluorescence emission or both until a suitable calculated ratio is achieved. According to an embodiment, the ratio of relative intensities of the first and second fluorescence emissions may be calculated by dividing an area-under-the curve value corresponding to the first fluorescence emission by an area-under-the curve value corresponding to the second fluorescence emission.

[0008] In accordance with another aspect of the invention, there is provided a system for extracting an image of a target fluorophore in a biological material wherein a waveband for the target fluorophore emission overlaps a waveband for autofluorescence emission in the biological material. The system includes a light source configured to illuminate the biological material with a first excitation light to induce a first fluorescence emission arising from both autofluorescence of the biological material and fluorescence of the target fluorophore and with a second excitation light to induce a second fluorescence emission arising from the autofluorescence of the biological material, an image acquisition assembly configured to acquire a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission, a modulator configured to modulate relative intensities of the first and second excitation lights prior to acquisition of the first and second fluorescence images, and a processor assembly configured to process the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore. According to an embodiment, the light source configured to illuminate the biological material includes an illumination module, the image acquisition assembly includes a fluorescence emission acquisition module, and the processor assembly includes a processor module.

[0009] In the embodiments where the target fluorophore is porphyrin, for example, the first excitation light has a wavelength of about 405 nm, the second excitation light has a wavelength of about 450 nm, the selected waveband is about 600 nm, and the calculated ratio is about 1.

[0021] Reference will now be made in detail to implementations and embodiments of various aspects and variations of the invention, examples of which are illustrated in the accompanying drawings.

[0022] According to one aspect of the invention, there is provided a method for extracting an image of a target fluorophore in a biological material wherein a waveband for the target fluorophore emission overlaps a waveband for autofluorescence emission in the biological material. FIG. 1 schematically illustrates the method of the present invention according to an embodiment. Referring to FIG. 1, the method comprises illuminating the biological material with a first excitation light to induce a first fluorescence emission arising from both autofluorescence of the biological material and fluorescence of the target fluorophore and with a second excitation light to induce a second fluorescence emission arising from the autofluorescence of the biological material, acquiring a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission, and processing the first and second fluorescence images to obtain a third fluorescence image representing the target fluorophore, wherein relative intensities of the first and second excitation lights are modulated prior to acquiring the first and second fluorescence images.

[0023] In various embodiments, the biological material includes a material derived from, obtained from, or located in a biological subject (e.g., a mammal), and further includes a material in vitro, in situ or in vivo. Examples of the biological material include a biological tissue or fluid or a fraction thereof, an organ, a cell, a cell line, a cell constituent derived from or located in mammals including humans. The biological material includes a collection of cells obtained from, derived from or in a tissue of the subject such as, for example, epithelium, connective tissue, blood vessels, muscle, nerve tissue, bone from any time in development of the subject. In various embodiments, the biological material includes healthy, diseased, or malignant tissue (e.g., cancerous or tumour tissue) comprising the target fluorophore (e.g., porphyrin). An example of the biological material further includes bacteria, including bacteria present in the subject (human, animal). Examples of the biological material which is a fluid include urine, serum, blood plasma, or blood. In various embodiments, the biological material may be a tissue section used in histochemistry, immunohistochemistry, cytochemistry, immunofluorescence, immunoblotting or other fluorescence-related imaging applications.

[0024] In various embodiments, the target fluorophore in the biological material is a fluorophore which when excited by a particular wavelength of light emits a light at a different, typically longer, wavelength. The target fluorophore includes a fluorophore which is of analytical, prognostic, diagnostic, physiological, pathological interest or a combination thereof. In various embodiments, the target fluorophore may be naturally occurring in the biological material (i.e., an endogenous fluorophore), externally administered into the biological material (i.e., an exogenous fluorophore) in a precursor or final form, or a combination thereof. Examples of naturally occurring or endogenous fluorophores include porphyrins, nicotinamide adenine dinucleotide (NAD), elastin, collagen, flavins, and amino acids. In embodiments where a porphyrin is the target fluorophore, the porphyrin includes a class of organic compounds that are in relevant biological systems and are formed as precursor intermediates in the biosynthesis of heme. For example, in humans and other mammals, porphyrins with 8-, 7-, 6-, 5- and 4-carboxyl groups are commonly formed in excess for heme synthesis, and thus are excreted in urine. In various embodiments, the term “porphyrin” includes, for example, porphyrin derivatives, coproporphyrin, uroporphyrin, protoporphyrin, porphyrin conjugates, liposomes, and nanovesicles.

[0025] Examples of exogenous fluorophores include various fluorescent probes or fluorescence inducing agents which may be used to augment (e.g., enhance) or provide fluorescent properties to a component of the biological material. For example, a fluorescent probe may associate with or attach to the component of the biological material to, for example, enhance fluorescence of an endogenous fluorophore in the component. Examples of exogenous fluorescent probes include fluorescein isothiocyanate (FITC), fluorescein, a fluorescent dye, 4′,6-diaminidino-2-phenylindole (DAPI), and eosin. An example of a fluorescence inducing agent includes a gene which may be inserted into a cell chromosome to induce the production of fluorescent proteins (e.g., green fluorescent protein). The fluorescence inducing agent may be an adjuvant that can augment the fluorescence response of the target fluorophore. For example, in embodiments where the target fluorophore is porphyrin, the adjuvant may be a selected food source (e.g., porphyrinogenic foods or chemicals), aminolevulinic acid or inhibitors of certain enzymes in the HEME pathway (e.g., ferrochelatease inhibitors) which when consumed or administered to the subject, increase the fluorescence response of porphyrin.

[0026] The biological material naturally fluoresces or “autofluoresces” in the absence of exogenously administered fluorophores due to the presence of various endogenous fluorophores in the biological material. Autofluorescence originates from various fluorophores in the biological material, including for example nicotinamide adenine dinucleotide (NAD), elastin, collagen, flavins, amino acids, lipofuscins, advanced glycation end-products, and porphyrins. The biological material includes a material that has been processed or otherwise treated prior to being used in the various embodiments of the method and system of the invention. For example, in certain embodiments, pre-treatment may involve photo bleaching of the biological material to reduce the autofluorescence of the biological material presumably by inactivating some of the autofluorescent endogenous fluorophores, and thus facilitating clearer subsequent resolution of the target fluorophore in cases where the target fluorophore is comparatively less susceptible to photobleaching or photobleaches at a slower rate than autofluorescent fluorophores in the biological material.

[0027] In accordance with the various embodiments, the method comprises illuminating the biological material with a first excitation light to induce a first fluorescence emission arising from both autofluorescence of the biological material and fluorescence of the target fluorophore, and with a second excitation light to induce a second fluorescence emission arising from the autofluorescence of the biological material. In various embodiments, the wavelength of the first excitation light is selected such that when the first excitation light illuminates the biological material, the fluorophores in the biological material which give rise to autofluorescence and the target fluorophore are both excited and emit a first fluorescence emission. In various embodiments, the wavelength of the second excitation light is selected such that only the fluorophores in the biological material giving rise to autofluorescence are excited and emit a second fluorescence emission. In various embodiments, for example, the first excitation light may have a wavelength ranging from about 350 nm to about 450 nm and the second excitation light may have a wavelength ranging from about 450 nm to about 700 nm. Illumination of the biological material with the first excitation light and the second excitation light includes intermittent illumination, continuous illumination or a combination thereof.

[0028] In the embodiment where the target fluorophore is porphyrin, the first excitation light has a wavelength of about 405 nm, and the second excitation light has a wavelength of about 450 nm. FIGS. 2A-2C (shaded areas) illustrates data obtained from porphyrin in urine. In particular, FIG. 2A is a first fluorescence emission spectrum arising from both autofluorescence and porphyrin in urine, and FIG. 2B is a second fluorescence emission spectrum arising from autofluorescence only. FIG. 2C is the differential spectrum corresponding to porphyrin only. In the examples illustrated in FIGS. 2A-2C, the urine was pretreated by photobleaching to facilitate a better discrimination of porphyrin from autofluorescence. In particular, photobleaching pre-treatment was conducted by illuminating the urine with the second excitation light of a wavelength of about 450 nm for about 3 minutes, which resulted in improved discrimination of the phorphyrin from autofluorescence of urine as compared with untreated urine (FIGS. 3A-3B). FIGS. 3A-3B shows fluorescence spectra from freshly obtained urine (FIG. 3A) and spectra obtained following an approximately 3-minute photobleaching exposure of the urine to light at about 450 nm (FIG. 3B).

[0029] In accordance with the various embodiments, the method comprises acquiring a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission, and processing the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore wherein the relative intensities of the first and second excitation light are modulated prior to acquiring the first and second fluorescence images. FIGS. 4A to 4D are images corresponding to the spectra in FIGS. 3A-3B acquired when the urine is freshly obtained and when the urine has been exposed to light at about 450 nm for about 3 minutes. FIGS. 4E and 4F are the differential images corresponding to porphyrin only resulting from the processing as described in connection with the various embodiments.

[0030] According to an embodiment, modulation of the relative intensities prior to image acquisition comprises identifying a wavelength region in the first and second fluorescence emissions, wherein the wavelength region is a region where emission arising from the fluorophore is present in the first fluorescence emission and absent in the second fluorescence emission, selecting a waveband outside the wavelength region, calculating at the selected waveband a ratio of relative intensities of the first and second fluorescence emissions, and adjusting the relative intensities of the first and second excitation lights to adjust the corresponding first fluorescence emission, second fluorescence emission or both until a suitable calculated ratio is achieved. According to various embodiments a waveband includes a wavelength. For example, in the embodiments where the target fluorophore is porphyrin, as is shown in FIG. 2C or FIG. 3B, the wavelength region where the emission arising from porphyrin is present in the first fluorescence emission and absent in the second fluorescence emission ranges, for example, from about 615 nm to about 625 nm and from about 660 nm to about 700 nm. Therefore, 600 nm was selected as the waveband outside this wavelength region and used as the waveband at which the ratio of the relative intensities at 405 nm and 450 nm was calculated for determining whether adjustment of the relative intensities is needed. In this example, the relative intensities were adjusted until the calculated ratio of about 1 was achieved within +/−2%. In this example, the ratio was calculated at the 600 nm waveband by dividing an area-under-the curve value corresponding to the first fluorescence emission (i.e., the emission arising from excitation at about 405 nm) by an area-under-the curve value corresponding to the second fluorescence emission (i.e., the emission arising from excitation at about 450 nm). In various embodiments, the ratio may be calculated by dividing the intensity at the selected waveband (e.g., a selected wavelength) of the first fluorescence emission by the intensity at the selected waveband (e.g., a selected wavelength) of the second fluorescence emission. In various embodiments, other methods may be used for calculation of the ratio. For example, one or more intensity points in the spectra arising from the respective emissions at 405 nm and 450 nm at the selected waveband (e.g., 600 nm) rather than areas may be used for such a calculation.

[0031] In various embodiments, processing comprises subtracting the second fluorescence image from the first fluorescence image to produce an autofluorescence-free image of the target fluorophore (e.g., FIGS. 4E and 4F).

[0032] The methods and systems according to the various embodiments may be used for detecting in situ fluorescence. Experimental data in FIGS. 5A-5C and 6A-6B illustrate example results where porphyrin was applied topically on the skin of a subject. In this example, a porphyrin solution was prepared by dissolving about 0.1 mg of coproporphyrin ester (Sigma-Aldrich) in about 10 mL of dimethyl sulfoxide (DMSO, Sigma Aldrich). The porphyrin solution was applied onto a small area of the subject's forearm using a Q-tip. The fluorescence imaging system used to acquire the data featured a dual-excitation capability at the porphyrin absorption maxima of about 405 nm and about 450 nm. The latter was chosen as the shortest wavelength outside the main porphyrin absorption band, and due to its property to induce high levels of tissue autofluorescence. To ensure that the reflected excitation light does not interfere with the fluorescence images, a 600 nm band pass filter (600 nm±5 nm) was placed in front of the detector in the imaging system, and the excitation intensities at 405 nm and 450 nm were modulated until the ratio of autofluorescence at 450 nm to autofluorescence at 405 nm reached about 1. FIG. 5A is an in vivo fluorescence image of the subject's forearm when excited with 405 nm light displaying autofluorescence from the forearm and porphyrin fluorescence. FIG. 5B is a fluorescence image of the same region of the forearm as in FIG. 5A upon excitation with 450 nm light. Since the autofluorescence induced by 450 nm excitation is greater than the autofluorescence induced by 405 nm excitation, the excitation light at 450 nm was modulated to produce autofluorescence at a level similar to the autofluorescence level in FIG 5A. FIG. 5C illustrates a fluorescence image of porphyrin with the autofluorescence removed in accordance with an embodiment.

[0033] FIG. 5A illustrates that the use of single excitation at 405 nm produces a well-localized fluorescence region arising from porphyrin fluorescence. High levels of background in surrounding areas arise from the presence of several endogenous fluorophores in skin (e.g., flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD) and lipids). A similar autofluorescence pattern can also be observed when the same region was excited with different excitation wavelength (450 nm) away from the absorption spectra of porphyrins (FIG. 5B). FIG. 5C shows the resultant image after the processing according to the methods of the present invention where the autofluorescence was successfully attenuated using the method.

[0034] FIG. 6A illustrates background intensity values at 405 nm and 450 nm after the excitation intensities were adjusted at 600 nm, and a lower than about 2% difference between background values was observed between excitations. FIG. 6B illustrates a comparison of the signal-to-noise ratio (S/N Ratio) from fluorescence images excited at 405 nm and after background was removed in accordance with an embodiment. An increase in the S/N Ratio can be achieved using this approach as compared to using single excitation at 405 nm (see FIG. 6B where S/N Ratios of about 28.5 and about 1.9, respectively, are shown).

[0035] Various conventional approaches involve simultaneous acquisition of the fluorescence images where all fluorescence species are illuminated, and their fluorescence emissions are collected at the same time. The acquired images are then processed using one or more autofluorescence removal models involving spectral unmixing or background subtraction. Although various conventional approaches have been proposed for removing autofluorescence such, as for example, spectral unmixing (linear decomposition) and digital background subtraction to reveal the differential, such approaches rely on manipulating the images post-acquisition and pre-characterized spectra for autofluorescence, require calibration, and are susceptible to changes in sensitivity based on the concentration of the target fluorophore. While these methods may be cost effective and applicable to both in vitro and in vivo imaging, they are not able to completely remove the autofluorescence component from fluorescence images, and further to account for an instrumental background signal. The modulation of relative intensities of the first and second excitation lights prior to image acquisition, as described in connection with the various embodiments, compensates for relative changes in emission with time due to, for example, different rates of photobleaching between the target fluorophore (e.g., porphyrin) and the fluorophores in the biological material giving rise to autofluorescence. If the modulation of intensities is performed digitally post-image acquisition, as is described in the prior art, the accuracy of the processing of the two images to derive the image of the target fluorophore (e.g., subtraction) is decreased, especially if the magnitude of the first and second fluorescence signals is significantly different. Prior art spectral unmixing methods often require prior knowledge of the amount of autofluorescence in the sample, which may not be constant. In addition, images of the biological material may also include a certain amount of noise or background contributed by the acquisition system itself. Therefore, in contrast to the present invention, normalization of intensities post-image acquisition, as taught in the prior art, is noisier and limited in signal quality especially when the target fluorophore has a low level signal as compared to the autofluorescence signal (e.g., endogenous fluorophores or fluorophores in low-concentration components of the biological material). Furthermore, post-image acquisition amplification of the low level signal of the target fluorophore, as taught in the prior art, also amplifies the instrumental background signal, which further negatively impacts the signal quality. Unlike the prior art approaches, the present invention facilitates dynamic real-time correction for changes in fluorescence in the biological material, and therefore enables a real time representation of the nature of the biological material.

[0036] The data generated according to the various embodiments demonstrates that the dual-excitation method of the present invention, as described in connection with the various embodiments, facilitates a reduction in or mitigates the fluorescence background signal during fluorescence imaging of biological tissue by modulating the autofluorescence intensities at a selected wavelength prior to acquisition of fluorescence images. According to the various embodiments, acquisition of spectral images is carried out by timed excitation and light collection from only a target fluorophore of interest or background at a time, This temporal separation of excitation and fluorescence collection minimizes cross-talk. Instead of collecting the emission signal under the same excitation source, the present method according to the various embodiments, induces equivalent background levels by means of a second excitation wavelength (which does not induce fluorescence from the target fluorophore of interest), and then can be subsequently subtracted without decreasing the fluorescence signal from the target fluorophore of interest.

[0037] The present method can be beneficial for fluorescence imaging applications where tissue autofluorescence affects fluorescence imaging. The detection of equivalent autofluorescence signatures from different excitation sources facilitates a more accurate molecular diagnosis than a single fluorescence excitation. Moreover, the dual fluorescence imaging approach in accordance with the various embodiments is more robust and accurate than other post-processing analysis techniques since the fluorescence intensity of the fluorophore of interest is not affected by digitally removing the background or modulating the background levels. As is illustrated by the experimental data collected according to an embodiment, this method may be used for the identification of malignant tissues in vivo by exploiting the preferential accumulation of fluorophores such as porphyrins.

[0038] In accordance with an aspect of the invention, there is provided a system for extracting an image of a target fluorophore in a biological material. The system comprises a light source configured to illuminate the biological material with a first excitation light to induce a first fluorescence emission arising from both autofluorescence of the biological material and fluorescence of the target fluorophore and with a second excitation light to induce a second fluorescence emission arising from the autofluorescence of the biological material, an image acquisition assembly configured to acquire first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission, a modulator configured to modulate relative intensities of the first and second excitation lights prior to acquisition of the first and second fluorescence images, and a processor assembly configured to process the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore.

[0039] Selected aspects relating to the system have been described above in connection with the various embodiments of the method of the present invention. Referring to FIG. 7, there is shown an exemplary embodiment of a system 10 for extracting the image of the target fluorophore 15 in the biological material 14. The system 10 comprises the means for illuminating 12 for illumination (e.g., a light source configured to illuminate the biological material) of the biological material 14 with dual fluorescence excitation light, means for acquiring 16 fluorescence images (e.g., an image acquisition assembly configured to acquire fluorescence images) arising from both the autofluorescence and the target fluorophore and from the autofluorescence alone, and means for processing 18 the acquired fluorescence images (e.g., a processor assembly configured to process the acquired images) to extract an image representing only the target fluorophore. In various embodiments, the means for illuminating 12 (e.g., the light source configured to illuminate the biological material) comprises, for example, an illumination module 20 shown in FIG. 8. The illumination module 20 comprises a fluorescence excitation source 22 operatively configured for providing fluorescence excitation having suitable intensities and suitable wavelengths for exciting the target fluorophore and the fluorophores giving rise to autofluorescence. In one embodiment, the fluorescence excitation source 22 may be a single excitation source having dual excitation capabilities for providing a first excitation light for inducing emission arising from both autofluorescence and fluorescence of the target fluorophore, and the second excitation light for inducing emission arising from the autofluoresenee only. In another embodiment, the fluorescence excitation source 22 may comprise two excitation sources (not shown), one for providing the first excitation light and the other for providing the second excitation light. In various embodiments, the fluorescence excitation source 22 includes, for example, a laser diode (which may comprise, for example, one or more fiber-coupled diode lasers), one or more LEDs, arc lamps, or other illuminant technologies of sufficient intensity and appropriate wavelength for providing the first and second excitation lights. In various embodiments, the first and second excitation light from the fluorescence excitation source 22 may be projected through an optical element (i.e., one or more optical elements) to shape and guide the output being used to illuminate the biological sample. The shaping optical element may consist, for example, of one or more lenses, light guides and diffusers. As is illustrated in FIG. 8, the output 24 from the fluorescence excitation source 22 is passed through one or more focusing lenses 26, and then through a homogenizing light pipe 28 such as, for example, light pipes commonly available from Newport Corporation, USA. Finally, the light is passed through an optical diffuser 30 (i.e., one or more optical diffusers or diffractive elements) such as, for example, ground glass diffusers also available from Newport Corporation, USA. Power to the fluorescence excitation source 22 itself is provided by, for example, a high-current laser driver such as those available from Lumina Power Inc., USA. In the embodiment where the fluorescence excitation source 22 is a laser, the laser may be operated in a pulsed mode during the image acquisition process. In this embodiment, an optical sensor such as a solid state photodiode 32 is incorporated into the illumination module 20 and samples the illumination intensity produced by the illumination module 20 via scattered or defuse reflections from the various optical elements.

[0040] In an alternative embodiment, the means for illuminating 12 (e.g., the light source) may also be configured to provide an additional functionality such as white light illumination. In another embodiment, the method and system of the present invention may further comprise acquiring and combining the third fluorescence image representing the target fluorophore with a white light image of the biological material. In this manner, the location of the targeted fluorophore can be visualized within the context of the biological material. This is useful in instances in which the biological material cannot be viewed directly with the human eye.

[0041] In various embodiments, the illumination module 20 in FIG. 8 comprises means for modulating (not shown) the relative intensities of the first and second excitation lights from the fluorescence excitation source 22 (e.g., a modulator configured to modulate the relative intensities of the first and second excitation lights from the fluorescence excitation source), so as to allow intensity adjustment. Such modulation may include modulation of the power to the light source, mechanical interruption of the light beam by shutters, apertures or choppers, optical, opto-mechanical or electro-optical diversion, filtering or blocking of the light beam or similar modulation.

[0042] Referring back to FIG. 7, the means for acquiring 16 (e.g., an image acquisition assembly) comprises, for example, a fluorescence emission acquisition module 30 (e.g., a camera module) shown in FIG. 9 for acquiring the first and second fluorescence images. As is shown in FIG. 9, the fluorescence emission 42 from the target fluorophore in the biological material and the fluorescence emission from other fluorophores giving rise to autofluorescence or both is collected and focused onto an image sensor 44 using an arrangement of various optical elements, e.g., 46a, 46b, 48 and 50. The charge that results from the optical signal transduced by the image sensor 44 is converted to a video signal by the appropriate read-out and amplification electronics in the fluorescence emission acquisition module 30.

[0043] Referring back to FIG. 7, in various embodiments, the means for processing 18 (e.g., a processor assembly) comprises, for example, a processor module (not shown) for analyzing the emission signals, performing calculations for subtracting the second fluorescence image from the first fluorescence image to output the calculated information to an appropriate display and/or recording device. In various embodiments, the processor module comprises any computer or computing means such as, for example, a tablet, laptop, desktop or networked computer. In various embodiments, the processor module may have a data storage module with the capability to save data (e.g., image sequences) to a tangible non-transitory computer readable medium such as, for example, internal memory, a hard disk, or flash memory, so as to enable recording and/or post-processing of acquired data. In various embodiments, the processor module may have an internal clock to enable control of the various elements and ensure correct timing of illumination and sensor shutters. In various other embodiments, the processor module may also provide user input and graphical display of outputs. The imaging system may optionally be configured with a video display (not shown) to display the images as they are being acquired or played back after recording, or further to visualize the data generated at various stages of the method. In various embodiments, the means for processing (e.g., the processor assembly) is in communication with an imaging system or is a component of the imaging system. An example of the imaging system in accordance with an embodiment is an endoscope.

[0044] In operation, and with continuing reference to the embodiments in FIGS. 7 to 9, the biological material is positioned in the illumination path of the means for illuminating 12 (e.g., the light source) of the system 10 comprising the illumination module 20, and such that, for example, the illumination module 20 produces a substantially uniform field of illumination across substantially the entire area of the biological material. The fluorescence excitation source 22 (e.g., the laser diode) is turned on and begins the shutter sequence for the image sensor (e.g., image sensor 44 of the fluorescence emission acquisition module 30). The fluorescence emission from the biological material is collected by the front imaging optics of the fluorescence emission acquisition module 30 such as optics 46a for example in FIG. 9 at the selected waveband (e.g., for porphyrin the selected wavelength is about 600 nm), and a ratio of the relative intensities is calculated. If the calculated ratio is suitable (e.g., for porphyrin, a suitable calculated ratio is in the range of about 0.98 to 1.02), the first and second fluorescence images are acquired. If the ratio is not suitable, the relative intensities of the first and second excitation lights are modulated and re-calculated until the suitable ration is achieved. The obtained first and second fluorescence images are then subtracted to extract a third fluorescence image representing only the target fluorophore.

[0045] According to another aspect of the invention, there is provided a tangible non-transitory computer readable medium having computer-executable (readable) program code embedded thereon comprising a method for extracting an image of a target fluorophore in a biological material wherein a waveband for the target fluorophore emission overlaps a waveband for autofluorescence emission in the biological material, the method comprising:

• • [0046] illuminating the biological material with a first excitation light to induce a first fluorescence emission arising from both autofluorescence of the biological material and fluorescence of the target fluorophore and with a second excitation light to induce a second fluorescence emission arising from the autofluorescence of the biological material;
  • [0047] acquiring a first fluorescence image from the first fluorescence emission and a second fluorescence image from the second fluorescence emission; and
  • [0048] processing the first and second fluorescence images to extract a third fluorescence image representing the target fluorophore, wherein relative intensities of the first and second excitation lights are modulated prior to acquiring the first and second fluorescence images.

[0049] One skilled in the art will appreciate that program code according to the various embodiments can be written in any appropriate programming language and delivered to the processor in many forms, including, for example, but not limited to information permanently stored on non-writeable storage media (e.g., read-only memory devices such as ROMs or CD-ROM disks), information alterably stored on writeable storage media (e.g., hard drives), information conveyed to the processor through communication media, such as a local area network, a public network such as the Internet, or any type of media suitable for storing electronic instruction. When carrying computer readable instructions that implement the various embodiments of the method of the present invention, such computer readable media represent examples of various embodiments of the present invention. In various embodiments, the tangible non-transitory computer readable medium comprises all computer-readable media, and the present invention scope is limited to computer readable media wherein the media is both tangible and non-transitory.

[0050] In yet further aspects, there is provided a kit including the system and the exogenous fluorophore as described in connection with the various embodiments.

[0051] Therefore, the various embodiments of the invention facilitate discrimination of the fluorescence of interest from an unknown combination of autofluorescence and fluorescence of interest. The present invention facilitates improvements in image quality for target fluorophores, preserves signal fluorescence while eliminating autofluorescence as well as background, and increases the resulting signal to autofluorescence ratio and the overall sensitivity of detection. The present invention is adaptable to a wide array of biological materials, and may be applied to any fluorescence imaging application. The present invention may be used to image and analyze a biological sample to discern the presence, absence, concentration, and/or spatial distribution of one of more fluorophore targets in the biological material. The present invention may be further used as a complementary tool for medical assessment or biological assessment (e.g., assessment of a biological phenomenon), diagnostic assessment, therapeutic assessment, physiological assessment, or a combination thereof.

[0052] While the present invention has been illustrated and described in connection with various embodiments shown and described in detail, it is not intended to be limited to the details shown, since various modifications and structural changes may be made without departing in any way from the scope of the present invention. Various modifications of form, arrangement of components, steps, details and order of operations of the embodiments illustrated, as well as other embodiments of the invention may be made without departing in any way from the scope of the present invention, and will be apparent to a person of skill in the art upon reference to this description. It is therefore contemplated that the appended claims will cover such modifications and embodiments as they fall within the true scope of the invention. For the terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.