Time-resolved laser-induced fluorescence spectroscopy systems and uses thereof

Abstract

To provide systems for characterizing a biological sample by analyzing emission of fluorescent light from the biological sample upon excitation and methods for using the same.SOLUTION: A biological sample 101 is projected with pulsed laser of an excitation source 100. A fluorescence signal from the biological sample is relayed through a collection fiber CF to a demultiplexer and resolved into a number of narrow spectral bands, each with a distinct central wavelength. Further, spectral bands time-delayed by a delay device 105 arrive at multi-channel plate photomultiplier tubes (MCP-PMTs) 106 at different time points, allowing detection of a decay profile of each spectral band.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] U.S. Government Rights This invention was made with Government support under Grant No. NS060685 awarded by the National Institutes of Neurological Disorders and Stroke. The United States Government has certain rights in the invention.

[0002] The present invention relates generally to techniques for characterizing biomaterials by analyzing laser-induced emission from labeled or unlabeled biomolecules. [Background technology]

[0003] All references cited herein are incorporated by reference in their entirety as if fully set forth herein. Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22 nd ed., Pharmaceutical Press (September 15, 2012) (Non-Patent Document 1); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008) (Non-Patent Document 2); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3 rd ed., revised ed., J. Wiley & Sons (New York, NY 2006) (Non-Patent Document 3); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 thed., J. Wiley & Sons (New York, NY 2013) (Non-Patent Document 4), Singleton, Dictionary of DNA and Genome Technology 3 rd ed., Wiley-Blackwell (November 28, 2012) (Non-Patent Document 5), and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) (Non-Patent Document 6) provide those of skill in the art with a general guide to many of the terms used in this application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2 nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013) (Non-Patent Document 7); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9 (Non-Patent Document 8); Queen and Selick, Humanized immunoglobulins, US Patent No. 5,585,089 (1996 Dec) (Patent Document 1); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7 (Non-Patent Document 9).

[0004] Laser-induced fluorescence spectroscopy (LIFS) has been widely applied to complex biological systems to diagnose diseases such as tumors or atherosclerotic plaques, and to analyze the chemical or biochemical composition of organic matter. The advantages of LIFS include its non-invasive approach to obtain both qualitative and quantitative information of biological systems in vivo. Further advantages of LIFS include tunable wavelength, narrow bandwidth excitation, directionality, and short pulse excitation. Moreover, LIFS can selectively and efficiently excite fluorophores in organic matter, greatly improving the selectivity and detectability of fluorescence.

[0005] Time-resolved techniques allow the direct recording of the real-time occurrence of laser-induced luminescence, made possible by the availability of short (nanosecond) and ultrashort (picosecond) pulsed lasers, as well as advances in high-speed electronics. Because the luminescence process occurs in a very short time interval after the stimulating event (e.g., fluorescence decay times are on the order of nanoseconds), time-resolved measurements can provide information about the molecular species and protein structure of the sample. For example, time-resolved techniques allow "fast" processes (usually direct excitation of a short-lived state or very rapid subsequent reactions) and "slow" processes (usually delayed excitation from a long-lived state, due to persistence of electron clusters or reactions subsequent to the original electronic process) to be separated in the measured data.

[0006] Time-resolved measurements obtain only the integrated contributions from a wide range of wavelengths, which can be supplemented by the spectral information of the laser-induced emission to reveal further characteristics of the sample. To resolve the laser-induced emission into its component wavelengths while still being able to perform time-resolved measurements, some existing LIFS techniques use a scanning monochromator to select one wavelength at a time from the broadband emission and direct the selected wavelength component to a photodetector. However, to resolve another wavelength from the emission spectrum, the sample must be excited again to produce another re-emission while the monochromator is adjusted to select the new wavelength. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Queen and Selick, Humanized immunoglobulins, US Patent No. 5,585,089 (1996 Dec) [Non-patent literature]

[0008] [Non-Patent Document 1] Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012) [Non-Patent Document 2] Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008) [Non-Patent Document 3] Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J.Wiley & Sons (New York, NY 2006) [Non-Patent Document 4] Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J.Wiley & Sons (New York, NY 2013) [Non-Patent Document 5] Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (November 28, 2012) [Non-Patent Document 6] Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) [Non-Patent Document 7] Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013) [Non-Patent Document 8] Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur.J.Immunol.1976 Jul, 6(7):511-9 [Non-Patent Document 9] Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7 Summary of the Invention [Problem to be solved by the invention]

[0009] These existing techniques can take a significant amount of time to resolve multiple spectral components from a broadband emission. Although each wavelength component can be recorded in real time, the transition time when using a monochromator to select a different wavelength can take several seconds, which is a limiting factor in making real-time measurements. Furthermore, the entire measurement can take a lot of time if multiple stimulus positions on the sample must be measured. Thus, there is a need for a system and method that facilitates near real-time recording of both time-resolved and wavelength-resolved information from the emission resulting from a single excitation of the sample. [Means for solving the problem]

[0010] Summary of the Invention The present invention provides a system for characterizing a biological sample by analyzing the emission from the biological sample in response to an excitation signal. The system first emits a laser impulse into the biological sample to generate a response emission in the biological sample. The system then splits the response emission into a set of spectral bands with different center wavelengths using a wavelength splitting device. A time delay is then applied to the set of spectral bands such that each spectral band arrives at an optical detector at a different time, which allows the optical detector to resolve the response emission of each spectral band separately in time. The delayed spectral bands are then captured by the system within a single detection window of the optical detector. The captured spectral bands are then processed. [The present invention 1001] 1. A system for characterizing a biological sample by analyzing fluorescent emissions from the biological sample upon excitation, comprising: (i) a laser light source connected to a biological sample via an excitation fiber (ExF), the laser being configured to emit laser pulses of a predetermined wavelength into the biological sample to produce a responsive fluorescent signal in the biological sample; (ii) a collection fiber (CF) that collects the fluorescent signal from the sample and relays the signal to a demultiplexer; (iii) a demultiplexer comprising a wavelength division filter for dividing the signal from the CF at predetermined wavelengths to obtain spectral bands; (iv) an optical delay device; A system comprising: [The present invention 1002] The system of claim 1001, further comprising a photomultiplier tube with a preamplifier to amplify the signal after it passes through the photomultiplier tube before it is digitized. [The present invention 1003] The system of the present invention 1002 further comprising a digitizer for digitizing signals received from said photomultiplier tube, and a computer system for processing and displaying said signals. [The present invention 1004] The system of the present invention 1001, wherein the optical delay device is adapted to couple the spectral band from the demultiplexer to the delay device, enabling the spectral band to pass through the delay device and to introduce a controlled time delay as the spectral band passes through the delay device so as to capture multiple wavelengths in a single shot. [The present invention 1005] The system of the present invention 1001, wherein the collection fibers form a single bundle. [The present invention 1006] The system of the present invention 1001, wherein the demultiplexer splits the incoming signal at wavelengths of 355 nm (less than 360), 365-410 nm, 410-450 nm, 450-480 nm, 500-560 nm, 560-600 nm, and greater than 600 nm. [The present invention 1007] 1. A method for characterizing a biological sample by analyzing emission of a fluorescent signal from the biological sample upon excitation, comprising: (i) irradiating a biological sample with a laser pulse of a predetermined wavelength to produce a responsive fluorescent signal in the biological sample; (ii) collecting the fluorescent signal from the sample; and (iii) splitting the signal at predetermined wavelengths to obtain spectral bands; (iv) passing the spectral band through a time delay mechanism; (v) obtaining time delayed spectral bands; and (vi) processing the time-delayed spectral band signals; and A method comprising: [The present invention 1008] The method of claim 1007, wherein processing the signal includes passing the signal received from the photomultiplier tube through a digitizer to digitize the signal and passing the signal to a computer system for processing and displaying. [The present invention 1009] 1007. The method of claim 1007, wherein splitting the signal comprises splitting the incoming signal with a demultiplexer at wavelengths of 355 nm (less than 365 nm), 365-410, 410-450 nm, 450-480 nm, 500-560 nm, 560-600 nm, and greater than 600 nm. [The present invention 1010] The method of claim 1007, wherein said fluorescent signal is emitted by a biological molecule. [The present invention 1011] The method of the present invention 1010, wherein the biological molecule is any one or more of PLP-GAD (pyridoxal-5'-phosphate (PLP) glutamic acid decarboxylase (GAD)), bound NADH, free NADH, flavin mononucleotide (FMN) riboflavin, flavin adenine dinucleotide (FAD) riboflavin, lipopigments, endogenous porphyrins, or combinations thereof. [The present invention 1012] A method for determining tissue viability comprising analyzing the emission of a fluorescent signal from a biomolecule in the tissue by the method of the present invention 1007, wherein an increase in fluorescence of the biomolecule in the subject compared to a normal subject indicates low tissue viability. [The present invention 1013] A method for continuous monitoring of a cell's metabolism, comprising analyzing the emission of a fluorescent signal by the method of the invention 1007. [The present invention 1014] A method for determining drug or metabolite levels in plasma comprising analyzing the emission of a fluorescent signal from a biomolecule by the method of the present invention 1007. [The present invention 1015] The method of claim 1014, wherein the biomolecule is NADH. [The present invention 1016] The method of the present invention, wherein the NADH is free, bound, or a combination thereof. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1 shows a schematic of (A) multi-excitation time-resolved laser-induced fluorescence spectroscopy according to various embodiments of the present invention. BS: beam splitter, FB: fiber bundle, OD: optical density, LPFW: long-pass filter wheel, ExF: excitation fiber, CF: collection fiber, PMT: photomultiplier tube. [Figure 1B] FIG. 1B illustrates trigger synchronization according to various embodiments of the present invention. [Diagram 2] FIG. 1 illustrates a schematic diagram of an exemplary demultiplexer design in accordance with various embodiments of the present invention. [Diagram 3] FIG. 1 shows a schematic diagram of a probe according to various embodiments of the present invention. [Figure 4] FIG. 1 illustrates the fluorescence emission of various exemplary biomolecules according to various embodiments of the present invention. [Diagram 5] FIG. 1 shows a schematic illustrating the use of continuous NADH monitoring of ex-vivo brain samples according to various embodiments of the present invention. [Figure 6] FIG. 13 shows data demonstrating the ability of the TRLIFS device to continuously monitor NADH levels while cells according to various embodiments of the present invention are exposed to rotenone, a compound that inhibits NADH-dependent ATP production. [Figure 7]FIG. 1 shows (A) the regions observed by the TRLIFS system, (B) an overlay of samples from (A) after treatment with TTC, and (C) the fluorescence intensity plotted for each region (spot), according to various embodiments of the present invention. [Figure 8] FIG. 1 shows agar / gels with different concentrations of methotrexate according to various embodiments of the present invention. [Figure 9] FIG. 1 shows (A) fluorescence of MTX at various concentrations after 20 minutes exposure to 350 nm wavelength light, and (B) a plot of the fluorescence time course over 20 minutes showing the increase in MTX fluorescence due to generation of the active fluorescent form, according to various embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22 nd ed., Pharmaceutical Press (September 15, 2012), Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008), Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3 rd ed., revised ed., J.Wiley & Sons (New York, NY 2006), Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 thed., J.Wiley & Sons (New York, NY 2013), Singleton, Dictionary of DNA and Genome Technology 3 rd ed., Wiley-Blackwell (November 28, 2012), and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provide those of skill in the art with a general guide to many of the terms used in this application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2 nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, US Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

[0013] The following description is presented to enable any person skilled in the art to make and use the invention and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the claims.

[0014] The present invention relates to techniques for characterizing biomaterials by analyzing laser-induced emission from biomolecules (labeled or unlabeled). More particularly, the present invention relates to methods and apparatus for characterizing biomaterials by performing time- and wavelength-resolved analysis of laser-induced fluorescent emission from the biomaterials.

[0015] The systems described herein may be used to characterize various physiological and disease states, including but not limited to, assessment of tissue viability after injury, detection of tumors and tumor margins, continuous monitoring of cell metabolism, monitoring of plasma to optimize drug therapy. The systems can be adapted for various applications / uses depending on the substrates / markers being analyzed.

[0016] system The excitation light source is a pulsed laser 100. Output pulses from the pulsed laser are emitted onto the biological sample 101 at a predetermined wavelength and power level suitable for exciting the biological sample without damaging the sample. The pulsed laser is controlled by an internal or external pulse controller device or digital delay or trigger device 102 that provides precise timing to each laser impulse output. This precise timing is checked pulse by pulse using a photodiode and updated using an analog-to-digital converter device, e.g., NI PCIe-6320. In one embodiment, the pulsed laser emits ultraviolet (UV) light pulses to excite the biological sample. In another embodiment, the pulsed laser emits visible or near-infrared light pulses to excite the biological sample.

[0017] Laser emission from a pulsed laser can be coupled / focused into an optical fiber and directed to a specific location on the biological sample through either an optical fiber 103 (FIG. 3) or a lens system. Laser impulse excitation causes the biological sample to emit a response light, such as a fluorescent emission, that typically has a broad spectrum containing many wavelengths. This laser-induced emission is then collected by one or more collection fibers or lenses. In one embodiment of the present invention, the collection fiber is a bundle of multimode fibers 103. In another embodiment, collection is achieved using an objective lens.

[0018] The collection fiber then carries the broadband emission light to a wavelength splitting device 104 (FIG. 2), which may comprise one or more wavelength splitting stages. The broadband emission light undergoes a series of wavelength splitting processes so that the broadband signal can be resolved into a number of narrow spectral bands, each with a distinct center wavelength. The wavelength resolved spectral bands are coupled to corresponding delay devices 105, which apply a predetermined time delay to each spectral band as it travels towards the photodetector 106. The time delayed spectral bands exiting the delay devices are arranged on a fast response photomultiplier tube so that the fluorescence decay profile of each wavelength resolved spectral band, including the laser light, can be individually recorded and time resolved. The delays applied to these spectral bands allow each light signal to arrive at a multichannel plate photomultiplier tube (MCP-PMT) at different times, which allows the decay profile of each spectral band to be detected by the MCP-PMT separately along with the laser light. In one embodiment, a high speed digitizer 107 can be used to record and display the output from the MCP-PMT. In another embodiment, an oscilloscope can be used to record and display the output from the MCP-PMT. In one embodiment, the MCP-PMT is a gated MCP-PMT controlled by a gate control circuit, so that the MCP-PMT only responds to light signals during a narrow detection window when the MCP-PMT is open. In one embodiment, the gate control circuit and the pulse control are synchronized so that all the fluorescence decay profiles associated with a single laser-induced excitation can be recorded within a single MCP-PMT detection window. In one embodiment, the timing of the MCP-PMT gate opening is synchronized with the laser pulse by varying the delay between the laser trigger and the MCP-PMT gate with a correction based on the previous delay. The laser trigger delay (the delay between the trigger signal and the actual firing of the laser light) is recorded using a photodiode. The measured trigger delay is used to correct the synchronization between the laser trigger and the MCP-PMT gate trigger. Other photodetectors may be used instead of or in addition to the MCP-PMT, including but not limited to avalanche photodiodes (APDs), silicon PMTs.The gain of the MCP-PMT can be automatically controlled. In one embodiment of the present invention, the voltage of the MCP-PMT can be dynamically changed based on the fluorescent signal. In one embodiment of the present invention, the change in voltage can be determined by analyzing the fluorescent signal and determining the amount of change before recording the signal.

[0019] The pulsed laser 103 has an inherent delay in generating laser light after the unit is externally triggered. In an exemplary embodiment, the delay in generating laser light after the external delay can be, but is not limited to, up to 85 microseconds. The delay in triggering the signal, hereafter referred to as the "trigger delay", can vary between each pulse of the laser. To synchronize the laser light with the opening of the PMT gate, we use a photodiode to detect the timing of the laser pulse, compare it to the external trigger, and then correct the timing of the next trigger based on the previous trigger delay (FIG. 1B). In FIG. 1B, t0 is the time the laser is triggered, t1 is the time the laser fires, t2 is the time the PMT is triggered, and t3 is the time the PMT gate turns on. By enabling this feedback-based trigger synchronization, t2 is dynamically set to ensure that the voltage gain on the MCP-PMT is "on" when the fluorescent signal arrives at the MCP-PMT. The digitizer is triggered "on" using a second photodiode to ensure a smaller data size.

[0020] A schematic diagram of a TRLIFS system is depicted in Figure 1. In various embodiments, the system includes (i) an excitation fiber (ExF), (ii) a collection fiber (CF), (iii) a demultiplexer (demuxer), a wavelength division device that provides an infinitesimal measurement of the lifetime of the fluorescent signal (i.e., the exponential decay of the fluorescent signal), (iv) a photomultiplier tube (MCP-PMT, e.g., a high gain (10) such as a Photek 210, (v) a 1000 Hz wavelength splitter, and (vi) a 1000 Hz wavelength splitter. 6), a low noise, and fast rise time (~80 ps) detector), (v) an optional preamplifier to provide additional gain after the photomultiplier tube before the signal is digitized, (vi) a digitizer (e.g., SP Devices: 108ADQ Tiger) to digitize the signal received from the photomultiplier tube (e.g., at 6.4 Gsamples / sec) for data analysis, and (vii) a computer system to process and display the signal.

[0021] Fluorescence signals from biological tissues can be very high or low based on the fluorophores in the biological system. Fluorophores emit fluorescence emission intensities based on quantum efficiency and / or absorption of excitation light, which may be hindered due to certain conditions such as the type of sample (e.g., tissue, blood, plasma). To properly record the fluorescence spectrum, the PMT gain needs to be adjusted so that increased fluorescence emission does not cause saturation of the signal and low fluorescence emission does not lead to a very low signal-to-noise ratio. This can be achieved by rapidly changing the voltage across the MCP-PMT based on previously recorded data. In one embodiment, the fluorescence from two pulses of the laser is averaged and analyzed (e.g., using software) to determine whether the fluorescence signal is too high or too low, and then the voltage across the MCP-PMT (responsible for controlling the gain of the PMT) is changed via communication with a high-voltage power supply and a computer. If the fluorescence emission is too high, the voltage is repeatedly lowered until the correct amount of signal-to-noise ratio is achieved, and vice versa. Only after the correct SNR is achieved is the true signal stored and analyzed.

[0022] In some embodiments, an excitation fiber (e.g., 600 μm diameter, 0.12 NA UV grade silica core fiber) connects the laser source to the sample to excite the sample at the desired wavelength. Collection fibers (e.g., 12 fibers of 200 μm diameter, 0.22 NA UV grade silica core fiber) are packaged into a single bundle, which leads to a demultiplexer (FIG. 3). The 12 fibers can be combined into a single fiber using a technique that combines multimode fibers into a single fiber. (http: / / www.ofsoptics.com / ). Upon excitation of the sample with a laser of a given wavelength, the collection fibers collect the fluorescent signal from the sample and relay the signal to the demultiplexer. Various wavelength splitting filters in the demultiplexer split the incoming signal based on wavelength into a beam splitting device such as, but not limited to, a filter or a prism. The fluorescence signal pulse (after pulse excitation) is relayed via a photomultiplier tube, preamplifier, and digitizer to a computer system, and the fluorescence decay in this case is calculated by deconvolving the laser pulse (previously recorded) from the recorded fluorescence pulse.

[0023] wavelength division device A schematic of a wavelength division multiplexer is shown in Figure 2. Laser-induced luminescence signals (containing a wide range of wavelengths) from a biological sample are collected by a collection fiber, which carries the emitted signals towards the wavelength division multiplexer.

[0024] In an exemplary embodiment of the invention, the biological sample is excited with wavelengths between about 337-350 nm. In one embodiment, a wavelength splitting device (demultiplexer) depicted in Figures 1 and 2 separates the incoming signal into wavelengths below 365 nm (excitation wavelength), 365-410 nm, 410-450 nm, 450-480 nm, 500-550 nm, 550-600 nm, and greater than 600 nm. As shown in Figure 1, the incoming optical signal is directed onto a first beam splitting device of the wavelength splitting device which separates the incoming signal into wavelengths above about 495 nm and wavelengths below about 495 nm. After passing through the first beam splitting device, signals with wavelengths greater than 495 nm are focused using a lens with a focal length of 60 mm, then pass through a second beam splitting device that splits the signal at wavelengths between 500 and 560 nm and wavelengths greater than 560 nm, and finally, a third beam splitter splits the light at wavelengths between 560 and 600 nm and wavelengths greater than 600 nm. Signals with wavelengths less than 495 nm also pass through a lens with a focal length of 60 mm and are focused before passing through a fourth beam splitting device that splits the 495 nm optical signal into wavelengths between about 410 and 480 nm and wavelengths less than 410 nm. Optical signals with wavelengths between 410 and 450 nm pass through a fifth beam splitting device that splits the signal into wavelengths between about 415 and 450 nm and wavelengths between 450 and 495 nm. The optical signal from wavelengths less than 410 nm passes through a sixth beam splitter and is split into wavelengths between 365 and 410 nm and wavelengths less than 365 nm, including the laser excitation signal. It is possible to ensure accurate deconvolution by recording the laser simultaneously with the fluorescence. This demultiplexer design allows for the detection of biomolecules including, but not limited to, flavin mononucleotide (FMN) riboflavin, flavin adenine dinucleotide (FAD) riboflavin, lipopigments, endogenous porphyrins, as well as the fluorescence of molecules such as NADH and PLP-GAD in the incident signal. The beam splitting devices described herein can be, but are not limited to, dichroic filters, prisms, and diffraction gratings.

[0025] In another exemplary embodiment of the present invention, the biological sample is excited with a wavelength of about 337-350 nm. In this embodiment, the wavelength splitting device splits the incoming signal into wavelengths below 400 nm, 415-450 nm, 455-480 nm, 400-600 nm, and greater than 500 nm. Before exiting the collection fiber and entering the wavelength splitting device, the emitted light is first collimated using a collimating lens. The collimating lens may include, but is not limited to, a gradient index (GRIN) lens or an aspheric lens. The collimated light beam is directed onto a first beam splitting device of the wavelength splitting device, which splits the incoming signal into wavelengths above about 400 nm and wavelengths below about 400 nm. After passing through the first beam splitting device, the signal with wavelengths above 400 nm passes through a second beam splitting device, which splits the signal into wavelengths between 400-500 nm and wavelengths above 500 nm. The signals with wavelengths in the range of 400-500 nm pass through a third beam splitting device that separates the light signals into those with wavelengths greater than about 450 nm and those with wavelengths less than 450 nm. In various embodiments, the signals with wavelengths less than 450 nm are analyzed for biomolecular activity. These wavelengths are of interest for measuring biomolecules including, but not limited to, free and bound NADH, PLP-GAD, or combinations thereof.

[0026] By varying the configuration of the beam splitting device, various spectral bands of wavelengths can be detected. Other wavelength ranges can be achieved using different sets of filters, as will be apparent to one skilled in the art.

[0027] Time Delay Optical Device As shown in FIG. 1, each resolved wavelength component from the wavelength splitting device is coupled to a corresponding delay device and then experiences a predetermined amount of delay in the corresponding delay device. In various embodiments, the delay devices are optical fibers having different lengths L1, L2, L3, L4, etc. In certain embodiments, the lengths of the optical fibers may be approximately 5 feet, 55 feet, 115 feet, 165 feet, 215 feet, 265 feet, and 315 feet. Other lengths of optical fibers may be selected based on the delay required, which will be apparent to those skilled in the art. In order to separate each of the wavelength components in time at the same optical detector, each of the wavelength components travels through different lengths of optical fiber, thereby experiencing a different amount of delay. Ultimately, each of the wavelength components arrives at the optical detector at a different time, allowing each component to be detected separately.

[0028] In addition to the length of the optical fiber, other physical properties of the optical fiber, including but not limited to the refractive index of the fiber, are also used to determine the length of the fiber to achieve a particular amount of delay. In the time domain, since each spectral component has an attenuation profile that lasts for a particular amount of time (e.g., tens of nanoseconds), the time delay between two adjacent spectral components can be designed to be long enough to separate the two attenuation profiles in time.

[0029] In one embodiment of the present invention, the optical detector is a gated MCP-MCP-PMT that responds only to the incident optical signal within a short detection window controlled by a gate control circuit. This gate window can be designed to be long enough so that all resolved and time-separated wavelength components will reach the MCP-PMT within the gate window. Thus, the gated MCP-PMT can capture all wavelength components generated by a single laser-induced emission within one detection window. The delay device used to time-separate the resolved spectral bands is not limited to optical fibers, and any delay device can be generally used.

[0030] In various embodiments, the sample is a solid, semi-solid, or liquid biological sample, hi various embodiments, the sample is any one or more of blood, plasma, urine, tissue, microorganisms, parasites, saliva, vomit, cerebrospinal fluid, or any other biological sample from which a chemical signature can be detected.

[0031] In various embodiments, the tissue can be any one or more of the prostate, lung, kidney, brain, mucosa, skin, liver, gastrointestinal tract, colon, bladder, muscle, breast, and / or cervix.

[0032] The systems described herein may be used to detect any molecule that has a detectable (e.g., emitted) characteristic. In some embodiments, the emitted characteristic is a fluorescent emission. In some embodiments, the characteristic is a decay of the fluorescent emission.

[0033] The demultiplexer designs described herein allow for the detection of, for example, therapeutic agents (labeled or unlabeled), antibodies (labeled or unlabeled), toxins (labeled or unlabeled), endotoxins (labeled or unlabeled), exotoxins (labeled or unlabeled), tumor markers, and / or combinations thereof. In various embodiments, unlabeled biomolecules have inherent fluorescence.

[0034] The systems described herein allow for the detection of biomolecules including, but not limited to, flavin mononucleotide (FMN) riboflavin, flavin adenine dinucleotide (FAD) riboflavin, lipopigments, endogenous porphyrins, and the fluorescence of molecules such as NADH and PLP-GAD in the incident signal.

[0035] In various embodiments, the therapeutic agent comprises a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, albumin-bound paclitaxel (nab-paclitaxel), actinomycin, alitretinoin, all-trans retinoic acid, azacitidine, azathioprine, bevacizumab, bexatotene, bleomycin, bortezomib, carboplatin, capecitabine, cetuximab, cisplatin, chlorambucil, ribavirin ... Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Ipilimumab, Irinotecan, Mechlorethamine, Melphalan , Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab, Ofatumumab, Oxaliplatin, Paclitaxel, Panitumab, Pemetrexed, Rituximab,Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vorinostat, The chemotherapeutic agents include Romidepsin, 5-Fluorouracil (5-FU), 6-Mercaptopurine (6-MP), Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin, Mitomycin, Ixabepilone, Estramustine, or combinations thereof. As described herein, the chemotherapeutic agents may be labeled or unlabeled (e.g., agents with inherent fluorescence). In some embodiments, the label is a fluorescent label. Examples of fluorescent labels that may be used with the systems, devices, and methods described herein to label therapeutic agents include, but are not limited to, indocyanine green (ICG), curcumin, rhodamine (such as rhodamine B, rhodamine 123, rhodamine 6G, or variants thereof), green fluorescent protein (GFP), luciferin, fluorescein, quantum dots, or combinations thereof.

[0036] In various embodiments, the antibody, including the therapeutic antibody, is selected from the group consisting of 3F8, 8H9, Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, and the like. Mafenatox, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab, Atorolimumab, Bapineuzumab, Basiliximab, Bavitximab Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab-mertansine mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansineravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, cBR96-doxorubicin immunoconjugate, Cedelizumab, Certolizumab pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetane Tetraxetan, Conatumumab, Concizumab, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab Aritox, Drozitumab, Durigotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab Pegol pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomab-SituxetanCituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab-Ozogamicin ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, IMAB362, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, infliximab, inolimomab, inotuzumab ozogamicinozogamicin, Intetumumab, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Rodelcizumab, Lorvotuzumab-mertansine Mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatoxEstaphenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentane merpentan, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pancomab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumabvedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romo Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab Iplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomabaritox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, tildrakizumab, TNX-650, tocilizumab (atrix) Tocilizumab (atlizumab), Toralizumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab-Celmoleukin Celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vantictumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab-Mafodotin mafodotin), Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, Zolimomabaritox). As described herein, the antibody may be labeled or unlabeled. In some embodiments, the label is a fluorescent label. Examples of fluorescent labels that may be used with the systems, devices, and methods described herein to label a therapeutic agent include, but are not limited to, indocyanine green (ICG), curcumin, rhodamine (such as rhodamine B, rhodamine 123, rhodamine 6G, or variants thereof), green fluorescent protein (GFP), luciferin, fluorescein, quantum dots, or combinations thereof.

[0037] In various embodiments, the toxin includes, but is not limited to, alpha toxin, anthrax toxin, bacterial toxin, diphtheria toxin, exotoxin, pertussis toxin, shiga toxin, shiga-like toxin, heat-stable enterotoxin, channel-forming toxin, mycotoxin, cholera toxin, scorpion toxin, chlorotoxin, and / or tetanus toxin. As described herein, the toxin may be labeled or unlabeled. In some embodiments, the label is a fluorescent label. Examples of fluorescent labels that may be used with the systems, devices, and methods described herein to label therapeutic agents include, but are not limited to, indocyanine green (ICG), curcumin, rhodamine (such as rhodamine B, rhodamine 123, rhodamine 6G, or variants thereof), green fluorescent protein (GFP), luciferin, fluorescein, quantum dots, or combinations thereof.

[0038] In some embodiments, proteins (e.g., cell surface proteins) may be detected using the systems described herein. In some embodiments, proteins may be detected using an antibody (e.g., a labeled or unlabeled antibody) that binds to a cell surface marker. In some embodiments, proteins may be detected using an siRNA (e.g., a labeled or unlabeled siRNA) that binds to a protein of interest. Examples of proteins that may be detected using the systems described herein include 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, annexins (e.g., annexins A1, A2, A5), BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R alpha, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGF beta 2, TGF-beta, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, or vimentin.Further examples include AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (alpha chain of the IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, Lama glama, LFA-1 (CD11a), MEDI-528, myostatin, OX-40, rhuMAb These include, but are not limited to, β7, scleroscin, SOST, TGF beta 1, TNF-α, VEGF-A, beta amyloid, MABT5102A, L-1β, CD3, C5, cardiac myosin, CD41 (integrin α-IIb), fibrin II, beta chain, ITGB2 (CD18), sphingosine-1-phosphate, anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, E. coli proteins, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-α, Lewis Y and CEA antigens, Tag72, folate binding protein, or combinations thereof. In some embodiments, the protein is labeled. In some embodiments, the label is a fluorescent label. Examples of fluorescent labels that may be used with the systems, devices, and methods described herein to label therapeutic agents include, but are not limited to, indocyanine green (ICG), curcumin, rhodamine (such as rhodamine B, rhodamine 123, rhodamine 6G, or variants thereof), green fluorescent protein (GFP), luciferin, fluorescein, quantum dots, or combinations thereof.

[0039] method Based on the combination of excitation wavelength and Demaxa's wavelength-splitting beam splitter, the fluorescence of various molecules may be analyzed (Figure 4). For example, with excitation of the sample at a wavelength of 350 nm and a suitable wavelength-splitting beam splitter of Demaxa, the fluorescence of biomolecules including, but not limited to, PLG-GAD (pyridoxal-5'-phosphate (PLP) glutamic acid decarboxylase (GAD)), bound NADH, and free NADH may be analyzed. That is, with excitation of the sample at a wavelength of 440 nm and a suitable wavelength-splitting beam splitter of Demaxa, the fluorescence of biomolecules such as FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and porphyrins may be analyzed.

[0040] The present invention provides a method for determining tissue viability following injury in a subject in need thereof using the TRLIFS system described herein. The method includes using the system described herein to measure fluorescence emitted from a biomolecule (e.g., NADH redox state), where a change in the fluorescence signal is indicative of tissue viability. In some embodiments, the change in the fluorescence signal of the biomolecule is an increase in the fluorescence signal from the biomolecule of the subject compared to a control (normal) subject. In some embodiments, the change in the fluorescence signal of the biomolecule is a decrease in the fluorescence signal from the biomolecule of the subject compared to a control (normal) subject. In one embodiment, the change in the NADH redox state is indicative of tissue viability. In one embodiment, an increase in the NADH fluorescence of the subject is indicative of NADH accumulation and reduced tissue viability.

[0041] The present invention also provides a method for monitoring cellular metabolism in a subject in need thereof using the system described herein. The method includes using the TRLIFS system described herein to measure fluorescence emitted from a biomolecule (e.g., NADH redox state), where a change in the fluorescence signal is indicative of cellular metabolism. In some embodiments, the change in the fluorescence signal of the biomolecule is an increase in the fluorescence signal from the biomolecule of the subject compared to a control (normal) subject. In some embodiments, the change in the fluorescence signal of the biomolecule is a decrease in the fluorescence signal from the biomolecule of the subject compared to a control (normal) subject. In one embodiment, NADH fluorescence may be used to monitor cellular metabolism. Cellular metabolism may be monitored continuously or periodically. In various embodiments, continuous monitoring of cellular metabolism allows for, for example, assessment of cell viability and vulnerability under ischemic conditions, effects of drugs on cellular metabolism (e.g., during drug development or to optimize therapeutic windows), and / or simultaneous monitoring of pH and oxygen levels to determine the metabolic state of the cell.

[0042] As described herein, the present invention also provides methods for detecting tumors using the TRLIFS system described herein. EXAMPLES

[0043] Example 1 Continuous monitoring of cell metabolism The system described herein allows for continuous monitoring of changes in NADH levels on a very fine scale to determine changes in metabolic state in response to oxygen deprivation, the effects of neuroprotective drugs, etc. (Figures 1 and 5).

[0044] Nicotinamide adenosine dinucleotide (NADH) is involved in the redox reactions involved in the production of ATP in aerobic respiration. NADH is produced in mitochondria during glycolysis and the citric acid (TCA) cycle. NADH is oxidized to NAD+ at the mitochondrial membrane to produce ATP in the process. This process is disrupted in conditions including, but not limited to, ischemia due to stroke. In hypoxic conditions, intracellular NADH accumulation and persistent oxygen deprivation may lead to complete degradation of NADH, eventually causing cell death. These variations in NADH levels allow for the assessment of cell viability and vulnerability in ischemic conditions. The variations in NADH levels may be assessed by measuring the fluorescence emission from NADH. Although both NAD+ and NADH have strong absorption in the UV spectrum, they have different fluorescence characteristics. NADH exhibits strong fluorescence in the violet / blue band around wavelengths 440 / 460 nm depending on its free versus its bound state (to cytochromes). Real-time measurement of this fluorescence allows monitoring of changes in NADH levels, assessment of the metabolic state of NADH, and thus monitoring of cellular metabolism.

[0045] A Q-switched Nd:YaG laser (Teem Photonics PNVM02510) emitting at a wavelength of 350 nm, operating at 1 KHz with a pulse width (FWHM) of 400 ps, ​​was used to excite the tissue. The total energy per pulse did not exceed 5 μJ to prevent photobleaching of NADH. Excitation light was delivered to the tissue using a custom-made three-pronged optical probe. The probe had a 600-micron central fiber for the delivery of excitation light surrounded by twelve 200-micron fibers for collecting the fluorescence (Figure 3). Every other fiber from the twelve collecting fibers was bundled together to form two channels. One collecting channel / bundle was connected to a spectrometer (Ocean Optics, Maya) that measured the fluorescence spectrum every 100 ms, and the other channel / bundle was connected to a beam splitter (demultiplexer). The beam splitter significantly separated the free and bound fluorescence at a wavelength of 452 nm, which was recorded by both the MCP-PMT and the spectrometer.

[0046] After sacrificing the animals in the OR, the rabbit brains were removed and transported to the laboratory in cold, oxygen-rich Cleverin gel solution. The cortices were isolated and stored in 95% O2 to maintain tissue viability. 2 and 5% CO 2 The tissue was placed in Cleverin's gel with a mixture of NADH and NADH gas continuously bubbled through it. The probe was adjusted onto the tissue to record the fluorescence as shown in FIG. 5. Baseline NADH (bound and free) was recorded until the fluorescence from the tissue had equilibrated and plateaued. After approximately 30 minutes, a measured amount of 50 nM rotenone was added, which blocks the binding of NADH to the cytochromes in the mitochondria. Additional concentrations of rotenone were added every 10 minutes.

[0047] The effect of various concentrations of rotenone on rabbit brain tissue was recorded (Figure 6). The results showed that it was possible to map the concentrations of both free and bound NADH in real time (every ∼100 ms) and record the response to external stimuli. Figure 6 shows a continuous plot of NADH fluorescence levels over a period of more than 2 hours. When rotenone at a concentration of 50 nM was added to the solution, an increase in NADH levels was observed as expected due to the inhibition of NADH consumption and subsequent accumulation. As the concentration of rotenone increased, NADH fluorescence increased as expected. At 80 minutes, gas was continuously bubbled through the liquid, which was then turned off and then resumed, allowing the evaluation of the effect of hypoxia on the accumulation of NADH in the tissue and its subsequent consumption after oxygen supply was restored. This demonstrated that the TRLIFS system described herein can monitor metabolic status in real time.

[0048] Example 2 Determining tissue viability after injury Recording NADH levels over a large area of ​​the brain after ischemic stroke allows for the assessment of the number of viable cells that may be in shock due to lack of oxygen but have not undergone apoptosis and can therefore be rescued. These cells make up the majority of the area known as the penumbra, and an important goal of stroke treatment is to reduce the size of the penumbra while rescuing as many neurons as possible. Monitoring NADH over the entire penumbra area allows for the assessment of the effectiveness of various interventions designed for this purpose.

[0049] A Q-switched Nd:YaG laser (Teem Photonics PNVM02510) emitting at a wavelength of 350 nm, operating at 1 KHz with a pulse width (FWHM) of 400 ps, ​​was used to excite the tissue. The total energy per pulse did not exceed 5 μJ to prevent photobleaching of NADH. Excitation light was delivered to the tissue using a custom-made three-pronged optical probe. The probe had a 600-micron central fiber for delivery of excitation light surrounded by twelve 200-micron fibers for collecting the fluorescence. Every other fiber from the twelve collecting fibers was bundled together to form two channels. One collecting channel / bundle was connected to a spectrometer (Ocean Optics, Maya) that measured the fluorescence spectrum every 100 ms, and the other channel / bundle was connected to a beam splitter (demultiplexer).

[0050] A rabbit stroke model was used in which stroke was induced in rabbit brains by injecting a blood clot into the cerebral arteries. The rabbits were sacrificed after testing for neurological damage. The brains were removed and placed in cryo-O 2 The infarcted cortex was separated from the rest of the brain and transported to the laboratory in saturated Cleverin gel solution. 2 and 5% CO 2The tissue samples were placed in Cleverin's gel with a mixture of acetaldehyde and ethyl acetate bubbling through it. A single reading from the edge of the cortex was recorded and the probe was moved over the surface of the cortex as shown in Figure 7. Fluorescence intensity was recorded from the tissue samples. The tissue samples were immersed in a solution of TTC (2,3,5-triphenyltetrazolium), which then turned red when absorbed by viable cells. TTC is currently the gold standard test for testing cell viability. Tissue stained with TTC was compared with the recorded fluorescence intensity.

[0051] A smooth gradient of NADH autofluorescence was observed from healthy tissue (red stained areas in Figure 7) to dead tissue (unstained areas in Figure 7). It was also noted that rather than an abrupt change from viable to dead brain tissue, as seen with TTC staining, the fluorescence intensity (Figure 7) changed gradually, indicating the presence of viable cells in the areas shown to be dead.

[0052] Example 3 Using fluorescence to determine drug / metabolite levels in plasma Some anti-cancer drugs are toxic at high doses and lose their effectiveness at lower doses. This optimal plasma concentration of a drug (therapeutic window), where the drug is most effective, varies between patients due to variability in height, weight, metabolism, and ethnicity. Despite these variabilities, drug dosages are currently calculated based on the patient's weight and standardized pharmacokinetic profile. A fast and inexpensive method to determine plasma drug levels would allow for optimization of dosage for individual patients. Plasma levels of drugs may be detected using fluorescence spectroscopy. Some anti-cancer drugs, such as methotrexate, are known to have fluorescent properties. Applicants herein have shown, using the TRLIFS system described herein, that varying the concentration of methotrexate (MTX) in agar (FIG. 8) resulted in a corresponding change in the fluorescence of MTX.

[0053] A Q-switched Nd:YaG laser (Teem Photonics PNVM02510) emitting at a wavelength of 350 nm, operating at 1 KHz with a pulse width (FWHM) of 400 ps, ​​was used to excite the agar gel. The total energy per pulse did not exceed 5 μJ to prevent photobleaching of NADH. Excitation light was delivered to the gel using a custom-made three-pronged optical probe. The probe had a 600-micron central fiber for the delivery of excitation light surrounded by twelve 200-micron fibers for collecting the fluorescence. Every other fiber from the twelve collecting fibers was bundled together to form two channels. One collecting channel / bundle was connected to a spectrometer (Ocean Optics, Maya) that measured the fluorescence spectrum every 100 ms, and the other channel / bundle was connected to a beam splitter (demultiplexer).

[0054] A serial dilution of MTX (from 25 μg / ml to 25 ng / ml) was prepared in the agar gel. MTX when exposed to UV light is converted to a more fluorescent form. Upon exposure to UV light, the fluorescent form accumulates. To detect the fluorescent form, conversion from the low-fluorescent form to the fluorescent form was allowed to occur until a saturation level was reached. The final fluorescence intensity was recorded and compared with the concentration. The fluorescence intensity of MTX after 20 min of UV exposure is a good indicator of the concentration of MTX in the agar gel as shown in Figure 9.

[0055] Example 4 Tumor detection Laser-induced fluorescence spectroscopy (LIFS) represents a promising new additive technique for in vivo diagnostics. Fluorescence spectroscopy involves exciting endogenous fluorophores (without labels) in tissues and recording the emission. Fluorescence spectroscopy is employed in two ways: steady-state or time-resolved fluorescence spectroscopy. Time-resolved measurements resolve the decay of fluorescence intensity in terms of lifetime, thus providing additional information about the kinetics underlying the decay of fluorescence intensity. Time-resolved measurements are also independent of factors such as absorption by tissue endogenous fluorophores (e.g., blood), photobleaching, or any other conditions that may affect the fluorescence intensity. By measuring fluorescence decay features that reflect differences in the relaxation kinetics of distinct fluorescent molecules, time-resolved measurements have the ability to resolve overlapping spectra, improving the specificity of fluorescence measurements.

[0056] Applicants demonstrate that in patients, the TR-LIFS system described herein can differentiate gliomas (both high and low grade) from surrounding normal brain tissue during surgery. This study is to establish the potential of TR-LIFS to enhance the ability of neurosurgeon-neuropathologist teams to rapidly distinguish between tumors and normal brain during surgery.

[0057] Instrumentation: Experiments were performed with an instrumental configuration that allows for spectrally resolved fluorescence lifetime measurements. A schematic of the instrument optics and electronics layout is shown in Figure 1. Briefly, this consisted of a) a pulsed Q-switched Nd:YaG laser (Teem Photonics, model Teem Photonics PNVM02510, λ=350 nm, pulse width=400 ps FWHM, pulse rate=1 KHz) used as the excitation light source, b) a custom sterilizable three-pronged fiber optic probe (Fiberguide, NJ), c) a gated multichannel plate photomultiplier tube (MCP-PMT, Photek, UK, model 210, rise time=80 ps) with optional high speed preamplifier (Photek, UK, model PA200-10, 2 GHz), e) a digitizer (ADQ-108, SPDevices, Sweden, 7 Gsamples / sec), and f) a laptop computer, g) a custom demultiplexer and peripheral electronics as shown in FIG. 1. The equipment was housed in a standard endoscopy cart (70 x 70 x 150 cm3) with internal modifications to accommodate the individual devices, allowing for mobility. All equipment was shielded from the mains power supply using medical grade isolation transformers (Toroid® ISB-170A) to ensure very low noise levels from the electronics used, such as the high voltage supplies and preamplifier power supplies.

[0058] Delivery Catheter: Light delivery and collection was achieved with a custom-made, bifurcated, sterilizable probe. The probe consisted of a non-solarizing silica / silica step-index fiber with a numerical aperture (NA) of 0.11 (Fiberguide, New Jersey, NJ). It had a central excitation fiber with a core diameter of 600 μm surrounded by a collection ring of 12 core diameter fibers with a core diameter of 200 μm. All collection fibers were bundled together and combined into a single 600 micron fiber. The center-to-center separation between the excitation and collection fibers was 480 μm. The probe was flexible throughout its entire length (3 meters) except for a distal section of 7 cm, which was made of rigid stainless steel tubing. This facilitated probe placement and micromanipulation. A spacer with two slits on either side was added before the distal end of the probe. This allowed the probe to make contact with the tissue while maintaining a constant distance from the tissue. The two slits on the spacer allowed the surgeon to apply suction to maintain a clear view. The laser light was coupled to the illumination channel of the probe with a standard SMA connector, while the distal end of the collection channel was straightened to facilitate coupling to the spectrometer. After tissue excitation, the emitted fluorescence was collected and guided to the entrance slit of the demaxer by bundle 1 and to the spectrometer via bundle 2. The signal was then detected by the MCP-PMT, amplified by a high-speed preamplifier, and finally digitized by a digital oscilloscope with 8-bit resolution. The overall time resolution of the system was approximately 150 ps.

[0059] The fiber optic probe was positioned 3 mm above the exposed brain tissue specimen with the aid of a spacer to optimize the light collection efficiency of the probe as described above and to stabilize the probe on the tissue. The time-resolved emission of each sample was recorded in seven distinct wavelength ranges (355 (<365 nm)), 365-410 nm, 415-450 nm, 450-490 nm, 500-560 nm, 560-600 nm, and >600 nm) spectral ranges. The energy output (at the tip of the fiber) of the laser for exciting the samples was adjusted to 5.0 μJ / pulse. After spectroscopic analysis, tissues were biopsied at the exact site and sent for pathology examination.

[0060] Each biopsy sample was fixed in 10% buffered formalin. Tissue samples were fixed on slides and stained with H&E. All biopsy specimens were examined by a pathologist and correlated with the original fluorescence spectroscopy measurements. Histologically, gliomas were classified based on WHO grading as low grade: oligodendroglioma, oligodendroglioma, diffuse astrocytoma (WHO grade II), intermediate grade: anaplastic astrocytoma (WHO grade III), and high grade: anaplastic oligodendroglioma, anaplastic oligoastrocytoma, and glioblastoma multiforme (grades III-IV). For the purposes of spectroscopic classification in this study, gliomas were grouped as low grade glioma (LGG) (grades I and II) and high grade glioma (HGG) (grades III and IV).

[0061] TR-LIFS Data Analysis: In the context of TR-LIFS, the intrinsic fluorescence impulse response function (IRF), h(n), describes the true kinetics of the fluorescence decay. The IRF was recovered by numerical deconvolution of the measured input laser pulse from the measured fluorescence transient. The Laguerre expansion technique was used for the deconvolution. The Laguerre expansion technique was chosen over more conventional multi-exponential curve fitting for a series of reasons. It allows for faster deconvolution of the fluorescence IR. Since the Laguerre basis is orthonormal, it provides a unique complete expansion of the decay function. This technique is also non-parametric and therefore does not require a priori assumptions of the functional expression of the decay. As a result, this allows for the approximation of fluorescent systems with unknown complex relaxation kinetics such as biological tissues. This method allows for the direct recovery of the intrinsic properties of a dynamic system from experimental input-output data. The technique uses orthonormal Laguerre functions to expand the IRF and estimate the Laguerre expansion coefficients (LECs). Normalized fluorescence spectra were obtained by dividing the discrete intensity values ​​by the intensity values ​​at the peak emission. Furthermore, to characterize the temporal kinetics of the fluorescence decay, two sets of parameters were calculated: 1) the mean lifetime (τ ), calculated as the interpolated time for the IRF to decay to its maximum value; λ ) and 2) the corresponding normalized value of LEC. Therefore, the emission wavelength λ E A complete description of the fluorescence from each sample as a function of was given by the variation of a set of spectroscopic parameters at discrete wavelengths (emission intensity I λ , the mean lifetime of fluorescence emission τ fλ , and the Laguerre coefficient LEC f ). Analytical techniques for characterizing this fluorescence decay have been recently developed by our research group and described in detail elsewhere. Applicants were able to retrieve lifetime and Laguerre coefficient values.

[0062] The various methods and techniques described above provide many ways of implementing the present application. Of course, it is understood that not all of the described objectives or advantages can be achieved in accordance with any particular embodiment described herein. Thus, for example, one skilled in the art will recognize that a method can be performed in a manner that achieves or optimizes one or a group of advantages taught herein without necessarily achieving other objectives or advantages taught or suggested herein. Various alternatives are described herein. It is understood that some preferred embodiments specifically include one, another, or several features, while other embodiments specifically exclude one, another, or several features, while still other embodiments relax a particular feature by including one, another, or several advantageous features.

[0063] Moreover, those skilled in the art will recognize the applicability of various features from different embodiments. Similarly, the various elements, features, and steps described above, as well as other known equivalents for each of such elements, features, or steps, can be used in various combinations by those skilled in the art to implement methods according to the principles described herein. Some of the various elements, features, and steps will be specifically included and others will be specifically excluded in various embodiments.

[0064] Although the present application has been disclosed in connection with particular embodiments and examples, it will be understood by those skilled in the art that the embodiments of the present application extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications thereof and equivalents.

[0065] In some embodiments, the articles "a," "an," and "the" and similar references used in connection with the description of certain embodiments of the present application (particularly in connection with some of the claims below) can be construed to cover both the singular and the plural. The recitation of ranges of values ​​herein is intended only to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise specified herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein with respect to specific embodiments is intended only to better describe the application and does not impose limitations on the scope of the application as otherwise claimed. No language herein should be construed as indicating any non-claimed element essential to the practice of the application.

[0066] Preferred embodiments of the present application are described herein, including the best mode known to the inventors for carrying out the present application. Variations of these preferred embodiments will become apparent to those of ordinary skill in the art upon reading the above description. It is contemplated that such variations may be employed by those of ordinary skill in the art, and the present application may be practiced in other ways than as specifically described herein. Accordingly, many embodiments of the present application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by this application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0067] All patents, patent applications, patent application publications, and other materials, such as literature, books, specifications, publications, documents, articles, and / or the like, cited herein are hereby incorporated by reference in their entirety for all purposes, except for any executable file history related thereto, any of which is inconsistent or contradictory with the present specification, or any of which may have a limiting effect as to the broadest scope of any claims now or later related hereto. By way of example only, to the extent that any inconsistency or contradiction may exist between the explanations, definitions, and / or usage of terms related to any of the incorporated materials and related herein, the explanations, definitions, and / or usage of terms in this specification shall control.

[0068] It is understood that the embodiments of the present application disclosed herein are illustrative of the principles of the embodiments of the present application. Other modifications that can be adopted are within the scope of the present application. Thus, by way of example only, and not of limitation, alternative configurations of the embodiments of the present application can be used in accordance with the teachings herein. Thus, the embodiments of the present application are not limited to those precisely as shown and described.

[0069] References Butte, PV, Fang, Q., Jo, JA, Yong, WH, Pikul, BK, Black, KL, & Marcu, L.(nd).Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy.Journal of Biomedical Optics, 15(2), 027008.doi:10.1117 / 1.3374049 Butte, P.V, Pikul, B.K., Hever, A., Yong, W.H., Black, K.L., & Marcu, L.(2005).Diagnosis of meningioma by time-resolved fluorescence spectroscopy.Journal of Biomedical Optics, 10(6), 064026.doi:10.1117 / 1.2141624 Butte, P.V., Mamelak, A.N., Nuno, M., Bannykh, S.I., Black, K.L., & Marcu, L.(2010).Fluorescence lifetime spectroscopy for guided therapy of brain tumors.NeuroImage, 54, S125-S135.doi:10.1016 / j.neuroimage.2010.11.001 Marcu, L., Jo, J.a, Butte, P.V, Yong, W.H., Pikul, B.K., Black, K.L., & Thompson, R.C.(2004).Fluorescence lifetime spectroscopy of glioblastoma multiforme.Photochemistry and Photobiology, 80, 98-103.doi:10.1562 / 2003-12-09-RA-023.1 Yong, W.H., Butte, P.V, Pikul, B.K., Jo, J.A., Fang, Q., Papaioannou, T., ... Marcu, L.(2006).Distinction of brain tissue, low grade and high grade glioma with time-resolved fluorescence spectroscopy.Frontiers in Bioscience: A Journal and Virtual Library, 11(4), 1255-63.Retrieved from http: / / www.ncbi.nlm.nih.gov / pubmed / 16368511 Jo, J.a, Fang, Q., Papaioannou, T., & Marcu, L.(2004).Fast model-free deconvolution of fluorescence decay for analysis of biological systems.Journal of Biomedical Optics, 9(4), 743-52.doi:10.1117 / 1.1752919 Lakowicz, J.R.(2006).Principles of fluorescence spectroscopy (3rd ed., p.xxvi, 954 p.).New York: Springer.Retrieved from http: / / www.loc.gov / catdir / enhancements / fy0824 / 2006920796-b.html Pogue, B.W., Pitts, J.D., Mycek, M.a, Sloboda, R.D., Wilmot, C.M., Brandsema, J.F., & O'Hara, J.a.(2001).In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy.Photochemistry and Photobiology, 74(6), 817-24.Retrieved from http: / / www.ncbi.nlm.nih.gov / pubmed / 11783938 Schneckenburger, H.(1992).Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators.Optical Engineering, 31(7), 1447.doi:10.1117 / 12.57704 Sun, Y., Phipps, J., Elson, D.S., Stoy, H., Tinling, S., Meier, J., ... Marcu, L.(2009).Fluorescence lifetime imaging microscopy: in vivo application to diagnosis of oral carcinoma.Opt Lett, 34(13), 2081-2083.doi:183277 [pii]

Claims

1. 1. A method for characterizing an ex-vivo biological sample by analyzing the emission of a fluorescent signal from said ex-vivo biological sample upon excitation, comprising: (a) illuminating the ex-vitro biological sample with a predetermined wavelength using a laser light source to produce a responsive fluorescent signal in the ex-vitro biological sample; (b) collecting the response fluorescent signal from the in vitro biological sample, wherein the response fluorescent signal is split based on wavelength with one or more wavelength splitting devices and a delay is provided to the response fluorescent signal with a delay device; (c) detecting the response fluorescent signal with a detector, wherein a gain of the detector is automatically adjusted based on the response fluorescent signal; (d) characterizing the ex vivo biological sample by at least the fluorescence lifetime of the response fluorescent signal. A method comprising:

2. The method of claim 1 , wherein the response fluorescent signal is split by a beam splitting device.

3. The method of claim 2 , wherein the beam splitting device comprises a plurality of filters.

4. The method of claim 3 , wherein the plurality of filters comprises at least three filters.

5. The method of claim 1 , wherein the responsive fluorescent signal is emitted by a biomolecule.

6. The method of claim 1 , wherein the gain of the detector is automatically adjusted by adjusting a voltage provided to the detector.

7. 10. The method of claim 1, wherein the gain of the detector is automatically adjusted so that the detected response fluorescent signal does not saturate the detector.

8. 10. The method of claim 1, wherein the ex vivo biological sample comprises human tissue.

9. 10. The method of claim 1, wherein the ex vivo biological sample comprises human tissue excised during surgery.

10. The method of claim 1 , wherein the delay device comprises a plurality of optical fibers.

11. 1. A system for characterizing a biological sample by analyzing a fluorescent signal emitted from the biological sample upon excitation, comprising: (a) a laser light source configured to illuminate a biological sample with a predetermined wavelength to produce a responsive fluorescent signal in the biological sample; (b) one or more wavelength splitting devices optically coupled to the biological sample, the one or more wavelength splitting devices configured to collect the response fluorescent signals from the biological sample and split the response fluorescent signals based on wavelengths of the one or more wavelength splitting devices, the one or more wavelength splitting devices including a delay device that provides a delay to the response fluorescent signals; (c) a detector optically coupled to the one or more wavelength splitting devices and configured to detect the response fluorescent signal, wherein a gain of the detector is automatically adjusted based on the response fluorescent signal; and (d) a processor configured to characterize the biological sample by at least the fluorescence lifetime of the response fluorescent signal; and A system comprising:

12. 12. The system of claim 11, wherein the response fluorescent signal is split by a beam splitting device.

13. The system of claim 12 , wherein the beamsplitting device comprises a plurality of filters.

14. 14. The system of claim 13, wherein the plurality of filters comprises at least three filters.

15. 12. The system of claim 11, wherein the responsive fluorescent signal is emitted by a biomolecule.

16. 12. The system of claim 11, wherein the gain of the detector is adjusted automatically by adjusting a voltage provided to the detector.

17. 12. The system of claim 11, wherein the gain of the detector is automatically adjusted so that the detected response fluorescent signal does not saturate the detector.

18. 12. The system of claim 11, wherein the biological sample comprises human tissue.

19. 12. The system of claim 11, wherein the biological sample comprises human tissue removed during surgery.

20. The system of claim 11 , wherein the delay device comprises a plurality of optical fibers.

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