Method and system for raman spectroscopy

Abstract

Systems and methods for Raman spectroscopy. In one example, a method for analysing a sample (404, 530) includes irradiating the sample (404, 530) with a second light and acquiring a fluorescence signal (504), and adjusting a first light (305) based on the fluorescence signal (504). The method includes irradiating the sample (404, 530) with the adjusted first light (305) and acquiring a Raman signal, and analysing a sample composition based on the Raman signal.

Description

METHOD AND SYSTEM FOR RAMAN SPECTROSCOPYFIELD

[0001] The present disclosure generally relates to systems and methods for conducting spectroscopic analytical techniques, such as Raman spectroscopy.BACKGROUND

[0002] Raman spectroscopy is an effective tool for identifying and characterizing various sample compounds and substances. In Raman spectroscopy, light, typically from a laser and of a known wavelength, is directed at a sample compound or substance (referred to herein as a “sample”). The laser photons (also sometimes referred to as a Raman pump) inelastically scatter, or “Raman scatter,” off the molecules in the sample and experience wavelength shifting to new frequencies given by bond vibrational frequencies present in the molecules of the sample. The precise nature of this wavelength shifting depends upon the materials present in the sample. A unique wavelength signature (typically called the Raman signature) is produced by each sample. This unique Raman signature permits the sample to be identified and characterized. More specifically, the spectrum of light returning from the sample is analyzed with a spectrometer so as to identify the Raman-induced wavelength shifting from the Raman pump light, and this wavelength signature is compared (e.g., by a computing device) with a library of known Raman signatures to identify characteristics of the sample.SUMMARY

[0003] In one aspect, a method for analyzing a sample includes irradiating the sample with a second light and acquiring a fluorescence signal, and adjusting a first light based on the fluorescence signal. The method includes irradiating the sample with the adjusted first light and acquiring a Raman signal, and analyzing a sample composition based on the Raman signal.

[0004] In another aspect, a method for analyzing a sample includes repetitively irradiating the sample with a first light and a second light, and acquiring fluorescence signals from the sample responsive to the irradiation by the second light. The method includes acquiring a plurality of spectra from the sample responsive to the irradiation by the first light. The first light is adjusted based on the fluorescence signal. The method includes analyzing a sample composition based on the plurality of spectra.

[0005] In another aspect, a system for performing spectroscopic measurement on a sample includes at least one light source for generating a first light and a second light, a first detector for acquiring a Raman signal responsive to irradiating the sample with the first light, a second detector for acquiring a fluorescence signal responsive to irradiating the sample with the second light, and a controller including an electronic processor and a memory for storing computer readable programs. By executing the computer readable programs in the electronic processor, the controller is configured to receive, via the second detector, the fluorescence signal from the sample and adjust the first light, via the at least one light source, based on the fluorescence signal. The controller is configured to receive, via the first detector, a Raman signal from the sample responsive to irradiating the sample with the adjusted first light and analyze a sample composition based on the Raman signal.

[0006] There is no specific requirement that a system, method, or technique relating to determination-based spectroscopy include all of the details characterized herein to obtain some benefit according to the present disclosure. Thus, the specific examples characterized herein are meant to be example applications of the techniques described and alternatives are possible.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Features and advantages of the present technology will become more apparent from the following detailed description of example embodiments thereof taken in conjunction with the accompanying drawings in which:

[0008] FIG. 1 is a block diagram of an example Raman spectroscopy system, according to some implementations of the present disclosure.

[0009] FIG. 2 is a block diagram of a controller included in the example Raman spectroscopy system of FIG. 1, according to some implementations of the present disclosure.

[0010] FIG. 3 is a perspective view of a Raman probe tip, according to some implementations of the present disclosure.

[0011] FIG. 4 illustrates an example Raman optical configuration, according to some implementations of the present disclosure.

[0012] FIGS. 5A-5C are schematic diagrams of example optical configurations of the Raman spectroscopy system of FIG. 1, according to some implementations of the present disclosure.

[0013] FIG. 6 is a block diagram of an example bioreactor control system, according to some implementations of the present disclosure.

[0014] FIG. 7 is a block diagram of a method performed by the controller of FIG. 2, according to some implementations of the present disclosure.

[0015] While the present technology is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.DETAILED DESCRIPTION

[0016] Raman spectroscopy may be used to identify characteristics of compounds and substances of samples. Performing Raman spectroscopy includes exciting the sample with a laser and obtaining a spectrum of the scattered light from the sample. The spectrum may be correlated with known spectrums of molecules to determine the sample composition. For example, Raman spectroscopy may be used to measure a glucose concentration within a bioreactor. In some instances, the glucose concentrations are estimated using the Raman spectrum with a trained machine learning model or a chemometric model.

[0017] Fluorescence generated responsive to the laser irradiation may interfere with the Raman measurement. Accordingly, the fluorescence signals (i.e., fluorescence baseline) may need to be removed from the acquired spectrum before comparing the acquired spectrum with known spectrum. Also, in some use cases, the fluorescence signals change over time. For example, as cells within a bioreactor are developed and / or die, the cells autofluorescence signal increases, resulting in varied noise in the acquired Raman signal and a change in the fluorescence baseline over time. Increases in the fluorescence baseline cause data degradation and saturate the spectrometer.

[0018] To address these and other issues, methods and systems are provided for monitoring and removing the fluorescence baseline while performing Raman spectroscopy.

[0019] In one example, a first light and a second light are provided. Responsive to irradiating the sample with a first light, a Raman signal may be acquired from the scattered light.Responsive to irradiating the sample with a second light, a fluorescence signal may be acquired. The first light is adjusted based on the fluorescence light. Raman signal acquired from the sample responsive to irradiating the sample with the adjusted first light is used for determining the sample composition.

[0020] As such, the fluorescence signal captured can be used as a feedback control variable to adjust the first light (such as the Raman excitation laser). For example, the fluorescence signal intensity may be used to automate tuning of the Raman excitation laser power as well as provide an estimation of the fluorescence baseline in analysis of the second Raman signal. Tuning of the Raman excitation laser power may be performed by subtracting the value of the fluorescence signal from the Raman signal. The fluorescence-based tuning may be used over long period of time while monitoring sample changes using Raman spectroscopy. Further, saturation of the detector for Raman signal detection can be avoided while enabling detecting signals with a large dynamic range provided by the Raman detector. The Raman signal may be acquired in the form of a spectrum, and the fluorescence signal may be acquired in the form of a spectrum or a single value. In some examples, the first light is generated by a first light source, such as a laser. The second light is generated by a second, different, light source, such as a LED.

[0021] In some aspects, different sample locations are simultaneously irradiated with the first light and the second light. In some aspects, the first light and the second light irradiate the sample at different times. As such, influence of the fluorescence signal acquisition on the Raman signal acquisition can be minimized.

[0022] Responsive to irradiating the sample with the first light, the scattered light (photons) from the sample is directed to a first detector and a Raman signal in the form of a spectrum is acquired. Responsive to irradiating the sample with the second light, fluorescence signal from the sample is directed to a second detector. The fluorescence intensity may be calculated using the detected fluorescence signal.

[0023] In one example, the second detector is the same as the first detector. The fluorescence signal is acquired as a fluorescence spectrum. The fluorescence intensity may be calculated from the fluorescence spectrum by integrating signal amplitude in a wavelength range.

[0024] In another example, the second detector is different from the first detector. The second detector, such as a photodiode, may directly read the fluorescence intensity. As such, the fluorescence signal is a single value representing the fluorescence intensity.

[0025] In some examples, the Raman spectroscopy may be performed using a probe. The first light and the second light may be delivered to the sample via the probe. A first end of the probe may be optically coupled to the Raman excitation light source (such as a laser) for generating the first light. The first end of the probe may also be optically couped to a spectrometer. A second end of the probe has one or more optical openings for delivering the first and second lights to the sample and receiving the light from the sample. The second end of the probe may be immersed in the sample.

[0026] In some examples, the first light and the second light are delivered to the sample via different optical openings (i.e., output ports) on the probe, so that the fluorescence signals and the Raman signals are collected from different sample locations. In some examples, the first light and the second light are delivered via the same optical opening of the probe, and the fluorescence signals and the Raman signals are collected at different times.

[0027] In one example, a fluorescence detector, such as a photodiode, may be embedded within the probe housing for detecting the fluorescence signal. In some examples, both the light source for generating the second light and the fluorescence detector are embedded within the probe housing.

[0028] In some aspects, adjusting the first light based on the fluorescence signal includes adjusting an intensity of the first light based on the fluorescence signal. The intensity of the first light may be adjusted based on the fluorescence intensity. For example, responsive to the fluorescence intensity being higher than a first threshold level, the output power of the source for generating the first light is decreased. The amount of the decrease in power may be determined based on the value of the fluorescence intensity. In another example, the first light may be adjusted based on the change or average of the fluorescence intensities over time. After adjusting the first light, a Raman spectrum is acquired for sample composition analysis. For example, if the fluorescence intensity is not higher than the first threshold level, the sample composition may be analyzed based on the most recently acquired Raman spectrum.

[0029] In some aspects, if the fluorescence intensity is lower than a second, lower, threshold level and the amplitude of the Raman spectrum is low, the power of the light source for generating the first light may be increased to take advantage of the full dynamic range of the Raman detector.

[0030] In some aspects, analyzing the sample composition based on the second Raman signal includes determining the sample composition by comparing the spectrum of the second Raman signal with a library of Raman spectrum of known composition.

[0031] In some aspects, the sample includes a plurality of cells within a bioreactor, and the method includes determining, based on the fluorescence signal, a density of dead cells to live cells within the plurality of cells.

[0032] In some aspects, analyzing sample composition includes determining, based on the Raman signal, a first analyte level of the sample and adding, based on the first analyte level, a first analyte or a second analyte to the sample. The first analyte or second analyte or both may be, for example, glucose, lactate, and constituents of biological cell culture, such as ions, nutrients, and the like. However, other types of analytes may be monitored and controlled to provide, among other things, quality control.

[0033] In some aspects, analyzing sample composition includes determining chemical structure and / or concentration of the sample. For example, the chemical structure is determined by providing the Raman signal and the fluorescence signal to a chemical analysis model, altering, with the chemical analysis model, the Raman measurement using the fluorescence measurement to obtain an altered Raman measurement, and identifying the chemical structure and / or concentration of the sample using the altered Raman measurement.

[0034] In some aspects, analyzing the sample composition further includes determining a change in the sample composition over time based on the change of the fluorescence intensity over time.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Example methods and systems are described below, although methods and systems similar or equivalent to those described herein can be used in practice or testing of the present disclosure. The systems, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0036] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise.

[0037] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0038] As used herein, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless specified otherwise, or clear from context, “X employs A or B” is intended to mean any of the natural inclusive permutations. That is, if X employs A, X employs B, or X employs both A and B, then “X employs A or B” is satisfied under any of the foregoing instances. Moreover, articles “a” and “an” as used in the subject specification and annexed drawings should generally be construed to mean “one or more” unless specified otherwise or clear from context to be directed to a singular form.

[0039] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0040] The present disclosure is now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numbers of specific details are set forth in order to provide an improved understanding of the present disclosure. It may be evident, however, that the systems and methods of the present disclosure may be practiced without one or more of these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing the systems and methods of the present disclosure.

[0041] It should be understood that although implementations are described herein as being used with a spectrometer or other optical instrument, implementations can be constructed as stand-alone devices for measuring a characteristic of a sample compound or substance. Furthermore, although some implementations are described herein with respect to measuring aparticular characteristic of a sample compound or substance, example methods and systems described herein can be used to measure other characteristics, such as, for example a Raman spectrum of the sample compound or substance.

[0042] FIG. 1 illustrates an example Raman spectroscopy system 100 according to some aspects. As illustrated in FIG. 1, the example Raman spectroscopy system 100 includes a Raman spectrometer 110 and, optionally, a computing device 120, a Raman probe 300, and a network 130. The system 100 may be used to determine sample composition via one or more of the methods described herein.

[0043] As illustrated in FIG. 1, the Raman spectrometer 110 includes a Raman laser 119 to provide an excitation beam (e.g., an excitation laser providing a variety of several different visible wavelengths), a Raman detector 117 (e.g., a CCD detector or a PMT detector), a fluorescence light source 121, and a fluorescence detector 123, all of which may be enclosed in a housing 122. The Raman spectrometer 110 also includes (e.g., within the housing 122) a controller 111 for performing operations described herein or a portion thereof. The operations described herein as being performed by the controller 111 may be distributed among multiple controllers. As described in further detail herein, the controller 111 may be configured to process signals from the fluorescence detector 123 and the Raman detector 117.

[0044] As illustrated in FIG. 1, the Raman spectroscopy system 100 may also include a human machine interface 114, which may include one or more input / output devices, such as, for example, a display 112, input devices 109, such as, for example, a keyboard, a mouse, a touch screen, buttons, dials, or the like, or a combination thereof. The Raman spectrometer 110 may also include additional components not illustrated in FIG. 1 for sake of brevity, such as, for example, power components, optical components (e.g., mirrors, lens, filters, etc.).

[0045] In some implementations, example methods of the present disclosure (or portions thereof) may be implemented on the optional computing device 120, which may include one or more electronic processors. The computing device 120 may be a standalone device, a server, internet of things (loT), a laptop computer, a tablet computer, a netbook computer, a personal computer (PC), a personal digital assistant (PDA), a desktop computer, or any programmable electronic device capable of receiving, sending, and processing data. In some implementations, the computing device 120 is remote from the Raman spectrometer 110 and, as illustrated in FIG. 1, communicates with the Raman spectrometer 110 via the network 130. Various intermediatedevices may also be used to establish communication between the Raman spectrometer 110 and the computing device 120.

[0046] The Raman spectroscopy system 100 may be operated by a user to take Raman spectroscopy measurements (i.e., Raman spectrum). In some implementations, the Raman spectrometer 110 and the computing device 120 are packaged together as a portable system that can be operated on battery power for use outside a laboratory setting. In some implementations, the Raman spectrometer 110 and the computing device 120 are separate systems that are communicatively connected (e.g., wirelessly, electrically, mechanical, and the like) and can be operated on battery power, operated using alternating power from a wall outlet, or a combination thereof for use inside or outside of a laboratory setting. For example, in some aspects, the computing device 120 may be located separate from Raman spectrometer 110 to provide the opportunity for increased computing power at a central location or across multiple locations. One skilled in the art can envision various interconnections, both physical and wireless, between the components of the system 100. Also, in some embodiments, the functionality described herein is performed at the Raman spectrometer 110 without using a separate computing device, such as the computing device 120. Accordingly, it should be understood that the example methods of the present disclosure may be implemented at last partially on the Raman spectrometer 110.

[0047] Referring now to FIG. 2, the controller 111 may include an electronic processor 250, an input / output (I / O) interface 252, and a data storage device 254; however, it should be understood that the controller 111 may have additional or fewer components. The controller 111 is suitable for the application and setting, and can include, for example, multiple electronic processors, multiple I / O interfaces, multiple data storage devices, or combinations thereof. In some implementations, some or all of the components included in the controller 111 may be attached to one or more mother boards and enclosed in a housing (e.g., including plastic, metal and / or other materials). In some implementations, some of these components may be fabricated onto a single system-on-a-chip, or SoC (e.g., an SoC may include one or more processing devices and one or more storage devices).

[0048] As used herein, “processors” or “electronic processor” refers to any device(s) or portion(s) of a device that process electronic data from registers and / or memory to transform that electronic data that may be stored in registers and / or memory. The electronic processor 250 may include one or more digital signal processors (DSPs), application-specific integrated circuits(ASICs), central processing units (CPUs), graphics processing units (GPUs), cryptoprocessors (specialized processors that execute cryptographic algorithms within hardware), server processors, or any other suitable processing devices.

[0049] The data storage device 254 may include one or more local or remote memory devices such as random-access memory (RAM) devices (e.g., static RAM (SRAM) devices, magnetic RAM (MRAM) devices, dynamic RAM (DRAM) devices, resistive RAM (RRAM) devices, or conductive-bridging RAM (CBRAM) devices), hard drive-based memory devices, solid-state memory devices, networked drives, cloud drives, or any combination of memory devices. In some implementations, the data storage device 254 may include memory that shares a die with a processor. In such an embodiment, the memory may be used as a cache memory and may include embedded dynamic random-access memory (eDRAM) or spin transfer torque magnetic random-access memory (STT-MRAM), for example. In some implementations, the data storage device 254 may include non-transitory computer readable media having instructions thereon that, when executed by one or more processors (e.g., the electronic processor 250), causes the controller 111 to store various applications and data for performing one or more of the methods described herein or portions described herein. For example, the data storage device 254 may store a sample identification program 260 and a fluorescence detection program 265. It should be understood that each method described herein may be implemented via one application or multiple applications and, in some embodiments, the data storage device 254 stores additional data in various configurations.

[0050] The fluorescence detection program 265 may be, for example, software configured to (through execution by the electronic processor 250) determine a fluorescence baseline based on the fluorescence signal. The fluorescence intensity is determined from the fluorescence signal acquired responsive to the irradiations of the fluorescence light source. The fluorescence baseline may be determined based on the fluorescence intensity, and one or more of the radiation intensity of the fluorescence light source, the radiation intensity of the Raman laser, and the characteristics of the Raman detector. The characteristics of the Raman detector may include the CCD bias of the detector. The fluorescence baseline may be removed, such as by subtraction, from the acquired Raman signal.

[0051] The sample identification program 260 may be, for example, software configured to (executable by the electronic processor 250) analyze provided Raman spectra and identifycharacteristics of a sample based on the Raman spectra, such as, for example, by comparing provided Raman spectra to a library of known spectra. In some instances, the sample identification program 260 may use a model, such as a machine learning model and / or a chemical analysis model, to analyze the sample. Example machine learning models may include support vector machines, convolutional neural networks, deep learning models, regression models, and the like.

[0052] The I / O interface 252 of controller 111 may include one or more communication chips, connectors, and / or other hardware and software to govern communications between the controller 111 and other components. For example, the I / O interface 252 may include circuitry for managing wireless communications for the transfer of data to and from the controller 111. In some implementations, the I / O interface 252 may include one or more antennas (e.g., one or more antenna arrays) for receipt and / or transmission of wire communications.

[0053] While illustrated as being enclosed in the housing 122 of the Raman spectrometer 110, in some implementations, the Rama spectrometer 110 may be used with a Raman probe positioned external to the housing 122. For example, referring to FIG. 3, an example Raman probe 300 is illustrated that includes a probe housing 301 having a second end 302. The Raman excitation optics 303 on the second end project light from the Raman laser 119 towards the sample. The second end 302 may also include fluorescence excitation optics 304 that project light from the fluorescence light source 121 towards the sample. In some instances, the Raman excitation optics 303 and / or the fluorescence excitation optics 304 may include optical components to focus the respective light onto a sample (for example, sample 404 shown in FIG. 4). In other instances, the Raman excitation optics 303 and the fluorescence excitation optics 304 are positioned within the housing 301 of the Raman probe 300 and ports (e.g., optical openings or optically transparent windows) in the second end 302 of the Raman probe 300 are configured to pass light provided by the Raman excitation optics 303 and the fluorescence excitation optics 304. In other instances, the Raman excitation optics 303 and / or the fluorescence excitation optics 304 includes optical fibers for delivering and / or receiving light. At least a portion of the Raman excitation optics forms a first output port to project the first light towards the sample. At least a portion of the fluorescence excitation optics forms a second output port to project the second light towards the sample.

[0054] The first end 307 of probe housing 301 illustrated in FIG. 3 is coupled to the Raman spectrometer 110. The probe housing 301 may be optically coupled with the Raman spectrometer 110 via one or more cables, such as an input (e.g., fiber optic) cable 305 and an output (e.g., fiber optic) cable 306. The Raman laser 119 and / or the fluorescence light source 121 may be situated within the Raman spectrometer 110, and light from the Raman laser 119 and the fluorescence light source 121 may be provided to the probe housing 301 via the input cable 305. After the sample is excited, the light emitted from the sample is received by the Raman spectrometer 110 via the output cable 306. In some aspects, a single cable may be used for both input to the Raman probe 300 and output from the Raman probe 300. In one example, the fluorescence light source may be outside of the Raman spectrometer 110. In another example, the fluorescence light source may be positioned within the housing 301 of the Raman probe 300.

[0055] Referring to FIG. 4, an example Raman optical components 400 for delivering the Raman excitation and receiving light responsive to the excitation is illustrated. The Raman optical components 400 may be positioned within the housing 301. In some examples, the Raman optical components 400 may be integrated with the Raman spectrometer 110. The Raman optical components 400 is merely an example, and additional or fewer optical components may be provided to provide light from the Raman laser 119 to, as well as receive light from, a location of a sample 404. For example, the Raman optical components 400 can include one or more collimating lens and mirrors, filters, such as, for example, a notch filter, diffraction gratings, mirror relays, or a combination thereof in addition to, or in place of, components shown in the Raman optical components 400.

[0056] The optical components 400 includes collimating lens 401, mirror 402, dichroic beam splitter 403, and focusing lens 405. The collimating lens 401 collimates the light provided by the Raman laser 119. The mirror 402 directs the collimated light to a dichroic beam splitter 403. The dichroic beam splitter 403 reflects the light to the Raman excitation optics 303. The Raman excitation optics 303 focus the light onto the sample 404 (e.g., sample of the compound or substance), exciting the sample 404 and thereby generating scattered or excited light. In some instances, the second end 302 of the probe housing 301, and therefore the Raman excitation optics 303, are placed within the sample.

[0057] The excited light emitted from sample is received by the Raman excitation optics 303 and directed to the dichroic beam splitter 403. The dichroic beam splitter 403 transmits theexcited light to the focusing lens 405. The focusing lens 405 focuses the excited light to the output cable 306, where the excited light is transmitted to the Raman detector 117 in the spectrometer 110.

[0058] The Raman laser 119 may emits light at, for example, between about 400 nm to about 1064 nm. In some implementations, the Raman laser 119 may include a laser power of 0.1 mW to about 500 mW. The laser power of the Raman laser 119 may be continuously variable between 0.1 mW to about 500 mW.

[0059] FIGS. 5A-5C are schematic diagrams of optical components for the Raman spectrometer 110 and the Raman probe 300, and each configuration of optical components are referred to herein as an optical system 500. As described in more detail below, the optical systems 500A-500C illustrated in FIGS. 5A-5C (and referred to commonly herein as the “optical system 500”) may include similar components but may be configured differently (e.g., communicate differently). For example, the optical systems 500 illustrated in FIGS. 5A-5B include the Raman laser 119 directing the light to Raman optical components 116, and the Raman optical components 116 directing the light to the Raman excitation optics 303. The light from the Raman excitation optics 303 is emitted onto sample 530. The scattered light from the sample 530 is received by Raman optical components 116 and directed to the Raman detector 117. In the example of FIGS. 5A-5C, the Raman optical components 116 are internal to the probe housing 301, and the Raman laser 119 and Raman detector 117 are external to the probe housing 301. For example, the Raman laser 119 and the Raman detector 117 may be situated within the Raman spectrometer 110 or another device. An example of the Raman optical components is shown in FIG. 4.

[0060] The optical systems 500 illustrated in FIGS. 5A-5C include the fluorescence light source 121 projecting light to fluorescence optical components 118. The fluorescence light source may be LED or lamp that projects light of a particular wavelength, optical power, and / or bandwidth for generating a fluorescent light from a sample. The fluorescence optical components 118 can include one or more collimating lens and mirrors, beam splitters (such as beam splitter 118b), filters, such as, for example, a notch filter, diffraction gratings, mirror relays, or a combination thereof. In the example of FIGS. 5A-5C, the fluorescence optical components 118 include a collimating lens 118a that collimates light from the fluorescence light source 121 and directs the collimated light to beam splitter 118b, such as a dichroic mirror. The beam splitter118b directs the light to the fluorescence excitation optics 304. The light from the fluorescence excitation optics 304 is emitted onto the sample 530. Similar to the Raman optical components 116, the fluorescence optical components 118 direct the light from the fluorescence light source 121 to the sample 530. In some embodiments, the light from the Raman laser 119 may be directed to the same or a different portion (e.g., adjacent portions) of the sample 530 than the light from the fluorescence light source 121. Fluorescence from the sample 530 is received by the fluorescence excitation optics 304 and travels back to the beam splitter 118b. The fluorescence light passes through the beam splitter 118b and is directed by lens 118c to the fluorescence detector 123 external to the probe housing 301 (for example, via the output cable 306).

[0061] In the example of FIG. 5 A, lights from Raman optical components 116 and fluorescence optical components 118 are provided to the Raman detector 117 and fluorescence detector 123, respectively, via separate optical paths. The output from the Raman optical components 116 are provided to the Raman detector 117 via a Raman optical path 502, and output from the fluorescence optical components 118 is provided to the fluorescence detector 124 via a fluorescence optical path 504. The results of the Raman detector 117 and fluorescence detector 123 (signals output via the detectors 117 and 123) are processed via the controller 111, which may be configured to analyze the results (signals). The fluorescence detector 123 may be a photodiode or a spectrometer.

[0062] In the example of FIG. 5B, output from the Raman optical components 116 and output from the fluorescence optical components 118 is provided to the Raman detector 117 via a single, shared optical path 506. The output from the detectors 117 can then be processed via the controller 111 as described herein. In both implementations (e.g., the separate communication lines and the shared communication line), the Raman laser 119 and the fluorescence light source 121 may be controlled such that the sample 530 is excited by only a single light source at a given time.

[0063] In the example of FIG. 5C, a photodiode 510, acted as a fluorescence detector, is positioned between the fluorescence optical components 118 and the controller 111. As illustrated in FIG. 5C, in this implementation, the photodiode 510 is positioned within the probe housing 301. The fluorescence may be focused onto the photodiode 510 by a focusing lens 118c,and the photodiode sends a signal to the controller 111 indicative of the intensity of the fluorescence from the sample 530.

[0064] The fluorescence light source 121 may emit a light of a different wavelength than the Raman laser 119 or a light of the same wavelength as the Raman laser 119. For example, in implementations where separate optical paths are provided (FIG. 5 A), the wavelength of light emitted by the fluorescence light source 121 may be different than the wavelength of light emitted by the Raman laser 119. In implementations where a shared optical path is provided (FIG. 5B), the wavelength of light emitted by the fluorescence light source 121 may be the same as the wavelength of light emitted by the Raman laser 119. The fluorescence light source 121 may be, for example, the same light source as the Raman laser 119, a separate light emitting diode (LED), or the like. A wavelength of the light emitted by the fluorescence light source 121 may be known by the controller 111. In some instances, the Raman detector 117 and the fluorescence detector 123 both output light spectrums (e.g., a Raman spectrum and a fluorescence spectrum, respectively). In other instances, the Raman detector 117 outputs a Raman spectrum and the fluorescence detector 123 measures a single light intensity. The Raman optical path 502, the fluorescence optical path 504, and / or the shared optical path 506 may be fiber optic cables.

[0065] FIG. 6 illustrates a block diagram of a bioreactor system 600 according to one example implementation. The bioreactor system 600 of FIG. 6 includes the Raman probe 300, the Raman spectrometer 110, a bioreactor control system 602, and a bioreactor 608. The Raman probe 300 and Raman spectrometer 110 monitor the bioreactions in the bioreactor 608 over time. The controller 111 may adjust operating parameters of the Raman spectrometer 110 based on the detected Raman signal and detected fluorescence signal, as described herein. The controller 111 may also provide the detected Raman signal and / or the detected fluorescence signal to the bioreactor control system 602. In some instances, the controller 111 sends control signals to the bioreactor control system 602 based on the detected Raman and fluorescence signals, as described below in more detail. In some implementations, the Raman spectrometer 110 (or portions thereof) may be integrated with the bioreactor control system 602 such that the Raman probe 300 may be directly coupled to the bioreactor control system (rather than through a separate spectrometer) and output from the Raman probe 300 (e.g., Raman signals) may be directly sent to and processed in the bioreactor control system 602.

[0066] The bioreactor control system 602 includes an electronic processor 604 and a memory 606. The electronic processor 604 may include one or more digital signal processors (DSPs), application-specific integrated circuits (ASICs), central processing units (CPUs), graphics processing units (GPUs), cryptoprocessors (specialized processors that execute cryptographic algorithms within hardware), server processors, or any other suitable processing devices.

[0067] The memory 606 may include one or more local or remote memory devices such as random-access memory (RAM) devices, hard drive-based memory devices, solid-state memory devices, networked drives, cloud drives, or any combination of memory devices. In some implementations, the memory 606 may include non-transitory computer readable media having instructions thereon that, when executed by one or more processors (e.g., the electronic processor 604), causes the bioreactor control system 602 to store various applications and data for performing one or more of the methods described herein or portions described herein.

[0068] FIG. 7 illustrates a block diagram of an example method 700 for monitoring a sample using Raman spectroscopy. The method 700 may be performed by the controller 111, the bioreactor control system 602, or a combination thereof. The steps of the method 700 are described in iterative manner for descriptive purposes. Various steps described herein with respect to the method 700 are capable of being executed simultaneously, in parallel, or in an order that differs from the illustrated serial and iterative manner of execution. In the example of method 700, the sample may be held within the bioreactor 608.

[0069] In some instances, prior to performing the method 700, the Raman probe 300 is calibrated. For example, the x-axis of the fluorescence measurement may be calibrated, and the relationship between the fluorescence y-ais and the Raman y-axis is calibrated. In examples where a single photodiode is used (see, e.g., photodiode 510 in FIG. 5C), a relationship between the fluorescence measurement and the saturation of the Raman detector may also be calibrated.

[0070] At block 702, the method 700 includes irradiating a sample with a first light. For example, the controller 111 controls the Raman laser 119 to emit a monochromatic light (i.e., a first light). The light emitted by the Raman laser 119 travels through the input cable 305 to the Raman probe 300. The light is directed by the Raman optical components 116 within the Raman probe 300 onto the sample. In some examples, the Raman probe 300 is immersed within a liquid or powdered sample for performing the measurement (e.g., within the bioreactor 608). The first light may be generated by a laser power of 0.1 mW to about 500 mW.

[0071] At block 704, the method 700 includes obtaining, via a first detector, a first Raman signal from the sample. The Raman signal is in the form of a spectrum, herein also referred to as Raman spectrum. For example, the light scattered by the sample is directed by the Raman optical components 116 to the output cable 306. The scattered light is received by the Raman detector 117 within the Raman spectroscopy system 100. The Raman detector 117 detects the light from the output cable and outputs a spectrum to the controller 111.

[0072] The first Raman signal may be used to adjust parameters of the Raman spectrometer. For example, the gain of the Raman detector and / or the intensity of the first light may be increased responsive to the amplitude of the Raman signal smaller than a threshold value. As such, the full dynamic range of the Raman detector may be utilized. In some examples, sample composition can be analyzed based on the first Raman signal.

[0073] In some embodiments, blocks 702 and 704 may be omitted. That is, the fluorescence signal may first be acquired before acquiring the Raman signal from the sample for composition analysis.

[0074] At block 706, the method 700 includes irradiating the sample with a second light. The second light may irradiate a different location of the sample from the first light. For example, the controller 111 controls the fluorescence light source 121 to emit a light (i.e., a second light). The light emitted by the fluorescence light source 121 is directed to the sample 404 via the Raman probe 300 (e.g., traveling through the input cable 305 to the probe housing 301).

[0075] At block 708, the method 700 includes obtaining, via a second detector, a fluorescence signal from the sample. For example, the light scattered by the sample 404 is directed by the fluorescence optical components 118 to the output cable 306. The scattered light is received by the fluorescence detector 123. The fluorescence detector 123 detects the light and outputs an electrical signal, which includes the fluorescence signal, to the controller 111 (and, therefore, the controller 111). The fluorescence signal may be a spectrum or an intensity value.

[0076] In one embodiment, the second light is directed to the sample at a different time from the first light. The first and second lights may be of different wavelengths or substantially the same wavelength. In other embodiments, the sample may be irradiated with the first and second light substantially simultaneously. The first and second light may be of different wavelength ranges.

[0077] At block 710, the method 700 includes adjusting one or more parameters of the Raman spectrometer based on the fluorescence signal. In particular, one or more operation parameters of the first light source (i.e., Raman laser 119) are adjusted based on the fluorescence measurement. For example, the controller 111 may adjust an output power level of the Raman laser 119 based on the fluorescence signal. Additionally, one or more other parameters of the Raman spectrometer may be adjusted. For example, an integration time and / or gain of the Raman detector is adjusted based on the fluorescence signal. By adjusting the Raman spectrometer, saturation of the Raman detector 117 is avoided while at the same time ensuring the large dynamic range of the received Raman signal.

[0078] Adjusting the parameters of the Raman spectrometer based on the fluorescence signal includes adjusting the parameters based the fluorescence intensity. In some examples, fluorescence intensity is determined from the fluorescence signal. The fluorescence intensity may be directly obtained from the fluorescence detector (such as the photodiode) or calculated from the fluorescence spectrum. The fluorescence intensity may be the maximum intensity of the fluorescence spectrum. For example, as the fluorescence intensity increases, the intensity level of the Raman laser 119 may be decreased. In some examples, the integration time of the Raman detector may decrease with increased fluorescence strength.

[0079] At block 712, the method 700 includes irradiating a sample with the first light. The first light is adjusted based on the fluorescence signal. For example, the controller 111 controls the Raman laser 119 to emit a light (i.e., a first light), as described with respect to block 702.

[0080] At block 714, the method 700 includes obtaining, via the first detector, a second Raman signal from the sample with the adjusted Raman spectrometer.

[0081] At block 716, the method 700 includes analyzing a sample composition based on the second Raman signal. The sample composition may include one or more of chemical structure, sample identification, composition concentration, and a concentration of analytes within the sample 404. The second Raman signal may be processed based on the fluorescence signal obtained at 708. For example, the baseline fluorescence may be calculated based on the fluorescence signal. The baseline fluorescence may then be subtracted from the second Raman signal before analyzing the spectrum for sample composition. If the fluorescence signal is a fluorescence spectrum, the baseline fluorescence may be determined by scaling the fluorescence spectrum. The fluorescence spectrum may be scaled based on one or more of the fluorescencelight intensity and the Raman laser intensity. The fluorescence spectrum may also be scaled based on calibrated scaling factors. If the fluorescence signal is a single value fluorescence intensity, the fluorescence baseline may be determined by scaled by the fluorescence intensity. As one example, the controller 111 may determine an analyte level (for example, a glucose level) of the sample within the bioreactor. In some embodiments, the sample condition may optionally be adjusted based on the sample composition. For example, analyte may be added to the sample based on the analyte level determined from the Raman measurement. In one example, the analyte may be glucose. For example, the controller 111 may compare the determined glucose level to a threshold. When the glucose level is below the threshold, the controller 111 transmits a signal to the bioreactor control system 602 to add glucose to the bioreactor 608.

[0082] In another example, the controller 111 identifies a chemical structure of the sample 404 based on the second Raman signal. In such an instance, the controller 111 may provide the Raman signal to a chemical analysis model, which may be stored in the data storage device 254. The chemical analysis model may include a principal component analysis (PCA) model or a partial least squares (PLS) model, wherein a full spectrum is provided to the model to determine the concentration of an analyte.

[0083] In some aspects, the chemical structure of the sample is determined based on the Raman signal and the fluorescence signal. The chemical analysis model may include a two-stage model. The first stage modifies the Raman spectrum based on the fluorescence signal. The chemical analysis model in the first stage may include a regression model. The first stage adjusts performance class data and / or device characteristics data based on the fluorescence level, thereby selecting a particular chemical analysis model. The output of the first stage is then passed (as an altered spectrum) to a second stage including the PCA or PLS model. In some aspects, if identification or composition concentration is needed, the PCA or PLS models may be replaced with a partial least squares discriminate analysis (PLS-DA) model, a boosted decision tree, or a neural network.

[0084] In yet another example, the controller 111 determines, based on the fluorescence measurement at 708, a density of dead cells to live cells, the ratio of the dead cells to the live cells within the bioreactor, the ratio of the dead cells to a volume of the bioreactor, the ratio of the live cells to the volume of the bioreactor, the ratio of dead cells to total cells, the ratio of live cells to total cells, or combinations thereof. As the number of dead cells increase, the amplitudeor intensity of the fluorescence signal may increase. In one example, the density of the dead cells to live cells may be estimated by comparing the fluorescence signal with calibrated or library data stored in the data storage device. In some examples, the density of the dead cells may be estimated further based on the change of the fluorescence signal over time. In some examples, a viability dye is added to the sample 404 within the bioreactor, providing a density of the live cells. The ratio of the live cells to dead cells may then be determined from the background fluorescence indicated by the fluorescence measurement. Fluorescence signals may be acquired periodically over time, for example every 5 minutes. The fluorescence intensity is feedback to the controller for adjusting the Raman excitation.

[0085] In some implementations, the density of the dead cells (or the ratio of the dead cells to the live cells) may be determined based on the accumulated fluorescence signals over time. For example, the controller 111 determines an integral of the fluorescence value (e.g., an integrated intensity) over repeated measurements. In such an implementation, the controller 111 may determine the density of dead cells to live cells based on the integral of the fluorescence value. In some instances, the controller 111 determines an average fluorescence value over time based on the change in fluorescence of a sample over repeated measurements.

[0086] In some implementations, after block 714, the method 700 returns to block 706 and continues irradiating the sample with the second light source to obtain fluorescence signals. In this manner, the controller 111 continuously monitor the fluorescence level of the sample and adjusts the Raman laser 119 based on the fluorescence value. The controller 111 may periodically irradiate the sample with either the Raman laser 119 or the fluorescence light source 121.

[0087] The Raman signal and the fluorescence signal may be periodically acquired at different frequency. That is, the sample is irradiated with the first light and the second light at different frequencies. For example, the Raman signal may be acquired at a higher frequency comparing to the fluorescence signal.

[0088] In some instances, the controller 111 provides a pulse width modulated (PWM) signal to the Raman laser 119 and the fluorescence light source 121. In such an instance, the PWM signal to the Raman laser 119 is offset from the fluorescence light source 121 such that only one of the Raman laser 119 and the fluorescence light source 121 are on (active) at a given moment.

[0089] Alternatively, in another implementation, a sample is simultaneously irradiated by the Raman laser 119 and the fluorescence light source 121. However, in such an implementation, the different locations on the sample are irradiated by the light sources such that the fluorescence light source 121 does not interfere with the Raman laser 119 and the Raman laser 119 does not interfere with the fluorescence light source 121.

[0090] Additionally, in some implementations, rather than two separate detectors (e.g., the Raman detector 117 and the fluorescence detector 123), a single detector, such as a spectrometer, is implemented to receive and process scattered light.

[0091] Accordingly, implementations described herein provide systems, methods, computing and storage devices, and computer-readable media for performing analysis of samples, such as for example, detecting the fluorescence value of samples using a Raman probe. As discussed above, the implementations described herein provide for adjusting a Raman laser based on the fluorescence value, adjusting analysis of obtained Raman spectrum using the fluorescence value, and the like. Implementations described herein provide for an increased accuracy and precision of sample analysis and a faster adjustment in the environment of a bioreactor. Thus, implementations disclosed herein provide improvements to Raman spectroscopy.

[0092] As described above in the detailed description, reference is made to the accompanying drawings that form a part hereof wherein like numerals designate like parts throughout, and in which is shown, by way of illustration, implementations that may be practiced. It is to be understood that other implementations may be utilized, and structured or logical changes may be made, without departing from the scope of the present disclosure. Therefore, the detailed description as described above is not to be taken in a limiting sense. For example, while implementations described herein may describe a Raman spectroscopy system including a spectrometer, a Raman probe, and an optional remote computing device, the methods described herein may also be used with handheld instruments implementing Raman technology, such as, for example, the Rarnina Process Analyzer provided by Thermo Scientific™.

[0093] Various operations may be described as multiple discrete actions or operations in turn, in a manner that is most helpful in understanding the subject matter disclosed herein. However, the order of description should be construed as to imply that these operations are necessarily order dependent. In particular, these operations may not be performed in the order of presentation. Operations described may be performed in a different order from the describedimplementation. Various additional operations may be performed, and / or described operations may be omitted in additional implementations.Clauses

[0094] Implementations of the present disclosure are disclosed in the following clauses:

[0095] Clause 1 : A method for analyzing a sample, comprising: irradiating the sample with a second light and acquiring a fluorescence signal; adjusting a first light based on the fluorescence signal; irradiating the sample with the adjusted first light and acquiring a Raman signal; and analyzing a sample composition based on the Raman signal.

[0096] Clause 2: The method according to clause 1, wherein irradiating the sample with the first light and the second light includes simultaneously irradiating different sample locations with the first light and the second light.

[0097] Clause 3: The method according to any of the preceding clauses, wherein irradiating the sample with the first light and the second light includes irradiating the sample with the first light and the second light at different time points.

[0098] Clause 4: The method according to any of the preceding clauses, wherein the sample includes a plurality of cells within a bioreactor, and wherein the method further includes: determining, based on the fluorescence signal, a ratio of dead cells to live cells within the plurality of cells or a density of dead cells to live cells within the plurality of cells.

[0099] Clause 5: The method according to any of the preceding clauses, further including: determining, based on the Raman signal, an analyte level of the sample; and adding an analyte to the sample based on the determined analyte level.

[0100] Clause 6: The method according to any of the preceding clauses, further including: providing the Raman signal and the fluorescence signal to a chemical analysis model; selecting the chemical analysis model based on the fluorescence signal; altering, with the chemical analysis model, the Raman signal using the fluorescence signal to obtain an altered first Raman signal; and identifying a chemical structure of the sample using the altered first Raman signal.

[0101] Clause 7: The method according to any of the preceding clauses, wherein adjusting the first light based on the fluorescence signal includes adjusting an intensity of the first light based on the fluorescence signal.

[0102] Clause 8: The method according to any of the preceding clauses, wherein the Raman signal include one or more spectra, and wherein the fluorescence signal includes a single value.

[0103] Clause 9: The method according to any of the preceding clauses, wherein irradiating the sample with the first light and / or the second light includes periodically irradiating the sample with the first light and / or the second light.

[0104] Clause 10: A method for analyzing a sample, comprising: repetitively irradiating the sample with a first light and a second light; acquiring fluorescence signals from the sample responsive to the irradiation by the second light; acquiring a plurality of spectra from the sample responsive to the irradiation by the first light, wherein the first light is adjusted based on the fluorescence signal; and analyzing a sample composition based on the plurality of spectra.

[0105] Clause 11 : The method according to clause 10, wherein analyzing the sample composition based on the plurality of spectra includes analyzing the sample composition further based on the fluorescence signal.

[0106] Clause 12: A system for performing spectroscopic measurement on a sample, including: at least one light source for generating a first light and a second light; a first detector for acquiring a Raman signal responsive to irradiating the sample with the first light; a second detector for acquiring a fluorescence signal responsive to irradiating the sample with the second light; and a controller including an electronic processor and a memory for storing computer readable programs, by executing the computer readable programs in the electronic processor, the controller is configured to: receive, via the second detector, the fluorescence signal from the sample, adjust the first light, via the at least one light source, based on the fluorescence signal, receive, via the first detector, a Raman signal from the sample responsive to irradiating the sample with the adjusted first light, and analyze a sample composition based on the Raman signal.

[0107] Clause 13: The system according to clause 12, wherein the first detector and the second detector are the same detector.

[0108] Clause 14: The system according to any one of clauses 12 to 13, wherein the first light is generated by a first light source and the second light is generated by a second, different, light source.

[0109] Clause 15: The system according to clause 14: wherein adjusting the first light includes decreasing a power level of the first fight source responsive to an intensity of the fluorescence signal higher than a previously measured intensity of the fluorescence signal.

[0110] Clause 16: The system according to any one of clauses 14 to 15, wherein the second light source is a LED and the second detector is a photodiode.

[0111] Clause 17: The system according to any one of clauses 12 to 16, further comprising: a probe for delivering the first and second light to the sample, the probe including a first end optically coupled with the first detector and / or the second detector and a second end in direct contact with the sample.

[0112] Clause 18: The system according to clause 17, wherein the second end of the probe includes: a first output port configured to project the first light towards the sample at a first sample location; and a second output port configured to project the second light towards the sample at a second, different, sample location.

[0113] Clause 19: The system according to clause 18, wherein the second detector is a photodiode, the photodiode is enclosed within a probe housing.

[0114] Clause 20: The system according to clause 19, wherein the at least one light source for generating the first light and the second light includes a first light source for generating the first light and the second light source for generating the second light, and wherein the second light source is a LED positioned within the probe housing.

Claims

CLAIMSWhat is claimed is:

1. A method for analyzing a sample, comprising: irradiating the sample with a second light and acquiring a fluorescence signal; adjusting a first light based on the fluorescence signal; irradiating the sample with the adjusted first light and acquiring a Raman signal; and analyzing a sample composition based on the Raman signal.

2. The method of claim 1, wherein irradiating the sample with the first light and the second light includes simultaneously irradiating different sample locations with the first light and the second light.

3. The method of claim 1, wherein irradiating the sample with the first light and the second light includes irradiating the sample with the first light and the second light at different time points.

4. The method of any of claims 1-3, wherein the sample includes a plurality of cells within a bioreactor, and wherein the method further includes: determining, based on the fluorescence signal, a ratio of dead cells to live cells within the plurality of cells or a density of dead cells to live cells within the plurality of cells.

5. The method of any of claims 1-4, further including: determining, based on the Raman signal, an analyte level of the sample; and adding an analyte to the sample based on the determined analyte level.

6. The method of any of claims 1-5, further including: providing the Raman signal and the fluorescence signal to a chemical analysis model; selecting the chemical analysis model based on the fluorescence signal; altering, with the chemical analysis model, the Raman signal using the fluorescence signal to obtain an altered first Raman signal; andidentifying a chemical structure of the sample using the altered first Raman signal.

7. The method of any of claims 1-6, wherein adjusting the first light based on the fluorescence signal includes adjusting an intensity of the first light based on the fluorescence signal.

8. The method of any of claims 1-7, wherein the Raman signal include one or more spectra, and wherein the fluorescence signal includes a single value.

9. The method of any of claims 1-8, wherein irradiating the sample with the first light and / or the second light includes periodically irradiating the sample with the first light and / or the second light.

10. A method for analyzing a sample, comprising: repetitively irradiating the sample with a first light and a second light; acquiring fluorescence signals from the sample responsive to the irradiation by the second light; acquiring a plurality of spectra from the sample responsive to the irradiation by the first light, wherein the first light is adjusted based on the fluorescence signal; and analyzing a sample composition based on the plurality of spectra.

11. The method of claim 10, wherein analyzing the sample composition based on the plurality of spectra includes analyzing the sample composition further based on the fluorescence signal.

12. A system for performing spectroscopic measurement on a sample, including: at least one light source for generating a first light and a second light; a first detector for acquiring a Raman signal responsive to irradiating the sample with the first light; a second detector for acquiring a fluorescence signal responsive to irradiating the sample with the second light; anda controller including an electronic processor and a memory for storing computer readable programs, by executing the computer readable programs in the electronic processor, the controller is configured to: receive, via the second detector, the fluorescence signal from the sample, adjust the first light, via the at least one light source, based on the fluorescence signal, receive, via the first detector, a Raman signal from the sample responsive to irradiating the sample with the adjusted first light, and analyze a sample composition based on the Raman signal.

13. The system of claim 12, wherein the first detector and the second detector are the same detector.

14. The system of claim 12, wherein the first light is generated by a first light source and the second light is generated by a second, different, light source.

15. The system of claim 14, wherein adjusting the first light includes decreasing a power level of the first light source responsive to an intensity of the fluorescence signal higher than a previously measured intensity of the fluorescence signal.

16. The system of claim 14, wherein the second light source is a LED and the second detector is a photodiode.

17. The system of any of claims 12-16, further comprising: a probe for delivering the first and second light to the sample, the probe including a first end optically coupled with the first detector and / or the second detector and a second end in direct contact with the sample.

18. The system of claim 17, wherein the second end of the probe includes: a first output port configured to project the first light towards the sample at a first sample location; anda second output port configured to project the second light towards the sample at a second, different, sample location.

19. The system of claim 18, wherein the second detector is a photodiode, the photodiode is enclosed within a probe housing.

20. The system of claim 19, wherein the at least one light source for generating the first light and the second light includes a first light source for generating the first light and the second light source for generating the second light, and wherein the second light source is a LED positioned within the probe housing.

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