Reduction of stray light noise in optical Raman probe sensors

Abstract

A system and method are disclosed for quantifying and removing stray light incident on a Raman optical sensor from a Raman spectrum. One or more background noise measurements are performed by a Raman analyzer, with the laser disabled during the background noise measurements. One or more conventional signal acquisition measurements are then performed. The background noise measurements and the conventional signal acquisition measurements are then processed, resulting in a clean spectrum with significantly reduced background noise. In some embodiments, background noise measurements are performed before and after the conventional signal acquisition measurements, and these noise measurements are averaged.

Description

[Technical Field]

[0001] Embodiments of the present disclosure relate to systems and methods for reducing stray light noise when using optical Raman probe sensors. [Background technology]

[0002] In many applications, such as bioprocessing, it is important to carefully and accurately monitor the composition of materials. For example, in bioreactors, it can be important to monitor the amounts of various molecules such as glucose, lactate, glutamine, ammonium, etc.

[0003] This monitoring is often done using Raman spectroscopy. In Raman spectroscopy, a laser is used to shine a specific wavelength of light towards the target molecule. The photons reach the molecule and excite it. When a photon excites a molecule, several outcomes are possible. The most common is that the excitation is temporary, causing the molecule to return to its initial energy state. In this mode, the photon is scattered or redirected upon interaction with the molecule. Furthermore, because the photon's energy was not absorbed by the molecule, the wavelength of the photon remains unchanged. This phenomenon is called Rayleigh scattering, and does not provide any information about the molecule being analyzed.

[0004] In another mode, a molecule is excited by a photon and moves into a different vibrational or rotational state. If the new state is higher in energy than the original, the photon loses energy and becomes lower in frequency. In this way, the total energy is conserved. This mode is called Stokes Raman scattering.

[0005] If the new state is lower in energy than the original, the photon will gain energy and therefore increase in frequency, a mode called anti-Stokes Raman scattering.

[0006] Stokes and anti-Stokes Raman scattering can be used to generate a spectrum, which usually has wavenumbers on the horizontal axis and is typically defined as: 1 / λ0-1 / λ1 where λ is the wavelength of the laser and λ is the wavelength of the Raman scattered light. The vertical axis is used to represent intensity.

[0007] Importantly, when excited, each molecule produces a unique spectrum that can be used to identify the molecule, and therefore the presence of different molecules can be determined using this technique. Summary of the Invention [Problem to be solved by the invention]

[0008] Compared to Rayleigh scattering, the rate of Stokes Raman scattering is very low and it is very sensitive to noise: for example, ambient light from the sun or room lighting can alter the Raman spectrum.

[0009] It would be advantageous to have a system and method that reduces the effect on the Raman spectrum of stray light noise entering an optical Raman probe sensor. [Means for solving the problem]

[0010] Systems and methods are disclosed for quantifying the effect of stray light incident on a Raman optical sensor and removing this stray light from a Raman spectrum. One or more background noise measurements are performed by a Raman analyzer, with the laser disabled during the background noise measurements. One or more conventional signal acquisition measurements are then performed. The background noise measurements and the conventional signal acquisition measurements are then processed, resulting in a clean spectrum with significantly reduced background noise. In some embodiments, background noise measurements are performed before and after the conventional signal acquisition measurements, and these background noise measurements are averaged.

[0011] According to some embodiments, a method for measuring Raman scattering is disclosed. The method includes performing one or more noise acquisition measurements using a Raman analyzer, where the one or more noise acquisition measurements are performed with a laser in the Raman analyzer disabled; performing one or more Raman signal acquisition measurements to generate a Raman spectrum, where the Raman signal acquisition measurements are performed with a laser in the Raman analyzer enabled; averaging the one or more noise acquisition measurements to generate an average background noise spectrum; and processing the average background noise spectrum and the Raman spectrum to generate a clean Raman spectrum. In some embodiments, the clean Raman spectrum is generated by subtracting the average background noise spectrum from the Raman spectrum. In some embodiments, multiple Raman signal acquisition measurements are performed and results of the multiple Raman signal acquisition measurements are averaged to generate the Raman spectrum. In some embodiments, at least one of the one or more noise acquisition measurements is performed before the Raman signal acquisition measurement. In some embodiments, at least one of the one or more noise acquisition measurements is performed after the Raman signal acquisition measurement. In some embodiments, multiple noise acquisition measurements are performed, with at least one of the noise acquisition measurements being performed before the Raman signal acquisition measurement and at least one of the noise acquisition measurements being performed after the Raman signal acquisition measurement.

[0012] According to some other embodiments, a system for measuring Raman scattering is disclosed. The system includes an optical Raman probe sensor, a Raman analyzer including an optical detector and a laser, the Raman analyzer communicating with the optical Raman probe sensor via a conduit, and a controller, wherein the controller disables the laser and performs one or more noise acquisition measurements; enables the laser and performs one or more Raman signal acquisition measurements to generate a Raman spectrum; averages the one or more noise acquisition measurements to generate the noise spectrum; and processes the noise spectrum and the Raman spectrum to generate a clean Raman spectrum. In some embodiments, the controller subtracts the noise spectrum from the Raman spectrum to generate the clean Raman spectrum. In some embodiments, multiple Raman signal acquisition measurements are performed, and the controller averages results of the multiple Raman signal acquisition measurements to generate the Raman spectrum. In some embodiments, the controller performs at least one of the one or more noise acquisition measurements before the Raman signal acquisition measurement. In some embodiments, the controller performs at least one of the one or more noise acquisition measurements after the Raman signal acquisition measurement. In some embodiments, the controller performs multiple noise acquisition measurements, with at least one of the noise acquisition measurements being performed before the Raman signal acquisition measurement and at least one of the noise acquisition measurements being performed after the Raman signal acquisition measurement.

[0013] For a better understanding of the present disclosure, reference is made to the accompanying drawings, which are incorporated herein by reference. [Brief explanation of the drawings]

[0014] [Figure 1A] 1 illustrates a bioreactor with a Raman probe sensor and a noise reduction cap according to some embodiments according to the present disclosure. [Figure 1B] 1 illustrates a bioreactor with a Raman probe sensor and a noise reduction cap according to some embodiments according to the present disclosure. [Figure 1C]1 illustrates a bioreactor with a Raman probe sensor and a noise reduction cap according to some embodiments according to the present disclosure. [Figure 2] 1 illustrates a sequence of operations for generating a clean Raman spectrum according to some embodiments of the present disclosure. [Figure 3] 1 is a graph illustrating the effect of the disclosed method on the resulting Raman spectrum, according to some embodiments of the present disclosure. [Figure 4] 1 is a graph illustrating the effect of the disclosed method on measuring stable glucose concentration in a light environment compared to standard Raman, according to some embodiments of the present disclosure. [Figure 5] 1 is a graph illustrating the effect of the disclosed method on measuring stable lactate concentration in a light environment compared to standard Raman, according to some embodiments of the present disclosure. [Figure 6A] Included are graphs showing the effect of the disclosed methods on the measurement of stable glucose concentrations in cell cultures performed in a typical laboratory environment, compared to standard Raman and with reference to offline reference values, according to some embodiments of the present disclosure. [Figure 6B] According to some embodiments of the present disclosure, graphs are included showing the effect of the disclosed methods on the measurement of stable lactate concentrations in cell cultures performed in a typical laboratory setting, compared to standard Raman and with reference to offline reference values. [Figure 6C] Included are graphs showing the effect of the disclosed methods on stable viable cell density (VCD) measurements in cell cultures performed in a typical laboratory environment, compared to standard Raman and with reference to offline reference values, according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0015] Embodiments of the present disclosure describe systems and methods for reducing stray light noise used in generating Raman spectra.

[0016] In many applications, such as bioprocessing, careful and accurate monitoring of materials within a bioreactor is important.

[0017] Figure 1 illustrates a bioreactor with a Raman probe sensor and a noise reduction cap according to some embodiments of the present disclosure. Figure 1A illustrates an exemplary bioreactor 1. The bioreactor 1 may be controlled by a bioreactor controller 2. The bioreactor controller 2 may control various valves, spargers, impellers, and other functions of the bioreactor 1.

[0018] The bioreactor bag 10 is typically inserted into the bioreactor 1. The bioreactor bag 10 can have multiple ports to allow for the introduction of various sensors, actuators, spargers, and other mechanisms into the bioreactor bag 10. In this example, an optical Raman probe sensor 20 enters the bioreactor bag 10 through port 11. The optical Raman probe sensor 20 includes a tube with a window, e.g., a sapphire window. The optical Raman probe sensor 20 also optionally includes a connection thread compatible with a bioreactor, such as PG13.5. In some embodiments, the maximum outer diameter of the tube of the optical Raman probe sensor 20 is 12 mm. The optical Raman probe sensor 20 is immersed in the material contained within the bioreactor bag 10. The optical Raman probe sensor 20 emits laser light along an optical axis 31. The laser beam passes through the tube, the sapphire window, and enters the bioreactor bag 10. The optical Raman probe sensor 20 also receives scattered light from a target 30, which may be a molecule or group of molecules. The scattered light travels along the collection field of view and enters the tip of the optical Raman probe sensor 20. The optical Raman probe sensor 20 may include an optical assembly 25 of lenses and filters to collect the optical signal from the target 30.

[0019] The optical Raman probe sensor 20 is connected to a Raman analyzer 21 external to the bioreactor 1 using a conduit 23. The Raman analyzer 21 may include a laser 22 that generates a laser beam that travels through the conduit 23 to the optical Raman probe sensor 20. This conduit 23 may be a fiber optic cable. The Raman analyzer 21 includes an optical detector 26, which may include a CCD or a photodetector. The Raman analyzer 21 also includes a processing unit 24 that controls the laser 22 and interprets the output from the optical detector 26.

[0020] The controller 50 is in communication with the Raman analyzer 21. The controller 50 may include a processing unit and an associated memory device. The processing unit may be any suitable component, such as a microprocessor, an embedded processor, an application-specific circuit, a programmable circuit, a microcontroller, or other similar device. The memory device stores instructions that, when executed by the processing unit, enable the controller 50 to perform the functions described herein. The memory device may be a non-volatile memory, such as a flash ROM, an electrically erasable ROM, or other suitable device. In other embodiments, the memory device may be a volatile memory, such as a RAM or a DRAM, or any non-transitory computer-readable storage medium.

[0021] The controller 50 can receive data from the Raman analyzer 21 and provide commands or instructions to the Raman analyzer 21. In the embodiment shown in Figure 1, the controller 50 is separate from the Raman analyzer 21. However, in other embodiments, these two components may be combined. In other embodiments, the controller 50 can send processed data to the bioreactor controller 2.

[0022] As mentioned above, Stokes Raman scattering occurs much less frequently than Rayleigh scattering and is therefore highly susceptible to noise. Therefore, ambient light 40 incident on the optical Raman probe sensor 20 can adversely affect detection accuracy. This ambient light could be sunlight, moonlight, room lighting, or other types of lighting. One way to address this is to quantify the amount of ambient light incident on the optical Raman probe sensor 20 and remove this background noise component from the Raman spectrum.

[0023] In normal operation, the Raman analyzer 21 activates a laser 22 located within the Raman analyzer 21. Light from the laser 22 travels through a conduit 23 to the optical Raman probe sensor 20. The light from the laser 22 excites molecules located in the optical axis 31 of the laser 22, causing Rayleigh scattering, Stokes Raman scattering, and anti-Stokes Raman scattering. The light is then received by an optical assembly 25 of the optical Raman probe sensor 20. Data from the optical Raman probe sensor 20 can then be transmitted to an optical detector 26 located within the Raman analyzer 21 to generate a Raman spectrum. This process is sometimes referred to as a Raman signal acquisition measurement, and the result of this process is sometimes referred to as a Raman spectrum.

[0024] In another mode, the Raman analyzer 21 does not enable the laser 22. However, as described above, data from the optical Raman probe sensor 20 can be sent to the Raman analyzer 21 to generate a Raman spectrum. In this mode, any signal that the optical detector 26 of the Raman analyzer 21 receives via the optical Raman probe sensor 20 is the result of background noise. Therefore, this process is referred to as a background noise acquisition measurement, and the result of this process is sometimes referred to as a background noise spectrum.

[0025] 1A-1C illustrate at least one embodiment according to the present disclosure. FIG. 1B is an exploded perspective view of this embodiment, while FIG. 1C illustrates a cross section of the assembled sensor. In some embodiments, the optical Raman probe sensor 20 is enclosed in a tube 56 comprising two sections: a tube body 27 and a tube head 28. The tube body 27 may comprise a hollow tube. The tube head 28 is secured to the tube body 27, such as by welding. The tube head 28 comprises a sapphire window and an optical lens 29. Additionally, the exterior surface of the tube head 28 is threaded to receive a cap 50. In some embodiments, cleaning may be desirable, and it is useful to be able to remove the cap 50. Additionally, in some embodiments, the tube head 28 has threads on its exterior surface near its distal end.

[0026] The cap 50 includes threads on the interior surface of the cylindrical body 52. ​​In operation, the cap 50 is threaded onto the tube head 28. The cap 50 is removable for easy cleaning. Additionally, in some embodiments, the cap 50 may be considered a disposable item, such that a new cap 50 is attached to the tube head 28 before each use. Furthermore, this configuration allows for the design of the cap to be selected depending on the application, without modifying other portions of the optical Raman probe sensor 20.

[0027] In some embodiments, the length of the cap 50 may be a design choice. For example, the cap 50 may be designed so that the distance from the tip of the optical Raman probe sensor 20 to the closed end 54 is 1-10 cm, although other dimensions are possible. The openings 58 are shown as two circular openings. However, the disclosure is not limited to this embodiment. Rather, the openings 58 may be circular, elliptical, rectangular, or other shapes. These processes may be used in combination with each other to produce clean Raman spectra.

[0028] 2 shows a flow chart illustrating the sequence of operations of the controller 50 and Raman analyzer 21 to generate a clean Raman spectrum. First, as shown in box 100, one or more background noise acquisition measurements can be performed. In certain embodiments, as many as 100 background noise acquisition measurements can be performed. In other embodiments, fewer background noise acquisition measurements can be performed. As noted above, this process is performed with the laser 22 disabled.

[0029] Next, one or more Raman acquisition measurements are performed, as shown in box 110. As described above, this involves enabling laser 22 and detecting the emitted optical signal using optical detector 26. Raman analyzer 21 can generate a Raman spectrum from the output of optical detector 26. In some embodiments, multiple Raman acquisition measurements are performed to generate the Raman spectrum. In some embodiments, ten or more Raman acquisition measurements are performed.

[0030] Next, one or more background noise acquisition measurements may be performed after the Raman signal acquisition measurements are performed, as shown in box 120. In certain embodiments, the number of background noise acquisition measurements may be as many as 100, as was done with the background noise acquisition measurements performed before the Raman signal acquisition measurements. In other embodiments, fewer background noise acquisition measurements may be performed. In other embodiments, no background noise acquisition measurements may be performed after the Raman acquisition measurements.

[0031] The background noise spectra obtained from the background noise acquisition measurements can then be averaged together and used to generate an average background noise spectrum, as shown in box 130. In certain embodiments, the background noise spectrum is generated from a background noise acquisition measurement taken before the Raman signal acquisition measurement and a background noise acquisition measurement taken after the Raman signal acquisition measurement. In other embodiments, the background noise acquisition measurement is performed only before or only after the Raman signal acquisition measurement. In these embodiments, either box 100 or box 120 can be omitted.

[0032] In box 140, the average background noise spectrum and the Raman spectrum are processed to produce a clean Raman spectrum with reduced background noise. In certain embodiments, the average background noise spectrum is subtracted from the Raman spectrum to generate a clean Raman spectrum. In other embodiments, other denoising algorithms can utilize the background noise spectrum and the Raman spectrum to generate a clean Raman spectrum.

[0033] The operations described in boxes 130 and 140 can be performed using controller 50 or a separate controller. For example, these operations can be performed using a separate controller that is part of the same network as Raman analyzer 21.

[0034] If there is more than one channel being analyzed, a new channel is established as shown in box 150 and the sequence is repeated.

[0035] The clean Raman spectrum can be used in a variety of ways, for example, it can be processed and analyzed by advanced mathematical tools to determine the composition of the media contained in the bioreactor bag 10.

[0036] The embodiments described herein above can have many advantages: Figure 3 is a graph illustrating the effect of the disclosed method on the resulting Raman spectrum, according to some embodiments of the present disclosure.

[0037] In many applications, the optical Raman probe sensor 20 is placed in a glass bioreactor or a plastic bioreactor bag, which is not completely opaque to ambient light. This ambient light can affect the results when generating Raman spectra. In one experiment, glucose concentrations ranging from 0 to 13 g / L were added to the bioreactor. Raman measurements were performed at each concentration. The first set of Raman measurements was performed with ambient light entering the bioreactor. These Raman measurements were then processed using conventional methods to obtain the data points labeled "Values ​​from Raman Spectra" in Figure 3. These data points are predicted glucose concentrations based on the Raman spectra. Note that there is a significant discrepancy between the actual glucose concentrations and those predicted from the Raman spectra. Furthermore, the sequence shown in Figure 2 was performed at each concentration, resulting in clean Raman spectra. The glucose concentrations obtained from these clean Raman spectra are represented by the data points labeled "Values ​​from Clean Spectra." Note that the predicted glucose concentrations based on the clean spectra are nearly identical to the actual glucose concentrations. Therefore, this technique is effective in reducing the effects of background noise.

[0038] Stray light management, for example, as described above, can include a light-reducing cap with a closed end and one or more openings attached to the tip of an optical Raman probe sensor. This cap serves to block stray light noise from entering the sensor tip, thereby resulting in more accurate and consistent Raman spectra. Furthermore, the cap can be permanently or removably attached to the sensor. In some embodiments, the closed end of the cap can include a reflective surface on the inner surface. This reflective surface can reflect Raman scattered light back toward the tip, enhancing the received signal.

[0039] 4 is a graph illustrating the effect of the disclosed method on stable glucose concentration measurements in a light environment compared to standard Raman, according to some embodiments of the present disclosure. FIG. 4 compares various stray light conditions and their measurements, according to some embodiments of the present disclosure. The first graph is a curve showing glucose measurement data points using a standard Raman probe. The second curve shows glucose measurement data points using an advanced Raman probe with an attenuation cap. The third graph is a third curve showing glucose measurement data points using the progressive Raman probe with the attenuation cap and with the software noise reduction filter applied. The fourth curve shows the glucose measurement data points using the advanced Raman probe with only the software noise reduction filter. As can be seen, without the stray light management implemented in the advanced Raman, i.e., the light attenuation cap and subtraction of the averaged background signal, the noise is as high as 1.5 g / L. With stray light management, the noise is below 0.2 g / L. Stray light management can include, for example, a light-reducing cap with a closed end and one or more openings attached to the tip of an optical Raman probe sensor. This cap serves to block stray light noise from entering the sensor tip, thereby resulting in more accurate and consistent Raman spectra. Furthermore, the cap can be permanently or removably attached to the sensor. In some embodiments, the closed end of the cap can include a reflective surface on the inner surface. This reflective surface can reflect Raman scattered light back toward the tip, enhancing the received signal.

[0040] Figure 5 is a graph showing the effect of the disclosed method, according to some embodiments of the present disclosure, on the measurement of stable lactate concentration under light conditions compared to standard Raman. Figure 5 compares various stray light conditions and their measurements, according to some embodiments of the present disclosure. The first curve shows lactate measurement data points using an advanced Raman probe with a light-attenuating cap and a software noise reduction filter applied. The second curve shows only the lactate measurement data points using the advanced Raman probe with the light-attenuating cap. The third curve shows the lactate measurement data points using the standard Raman probe. As can be seen, without the stray light management implemented in the advanced Raman solution, i.e., without the cap and subtraction of the averaged background signal, the noise is as high as 4.0 g / L. With stray light control, the noise is again below 0.2 g / L. The data show that the noise is reduced by over 70% when the light reduction cap is used.

[0041] Figure 6 includes three graphs illustrating the effect of the disclosed method on the measurement of stable glucose and lactate concentrations and viable cell density (VCD) in cell cultures performed in a typical laboratory environment, compared to standard Raman and with reference to offline reference values, in accordance with some embodiments of the present disclosure. Figure 6 compares measurements from different Raman probes in a fed-batch cell culture performed for 8 days under various stray light conditions, as shown for measuring glucose, lactate, and viable cell density, in accordance with embodiments of the present disclosure.

[0042] FIG. 6A shows a first graph illustrating the measurement of glucose concentration using an advanced Raman probe equipped with a light-attenuating cap and software noise reduction filter compared to a standard Raman probe and referenced to an offline reference.

[0043] FIG. 6B shows a second graph showing the measurement of lactate concentration using the advanced Raman probe equipped with a light-attenuating cap and software noise reduction filter compared to a standard Raman probe and referenced to an offline reference value.

[0044] 6C shows a third graph illustrating viable cell density measurements using the advanced Raman probe equipped with a light-attenuating cap and software noise-reduction filters compared to a standard Raman probe and referenced to an offline reference value. The third graph demonstrates that the present disclosure applies not only to nutrients and VCD, but also to other important parameters of cell culture. For example, it is shown that without the stray light management disclosed herein, VCD predictions are erratic. Improved glucose control helps to better manage cell growth and cell viability (i.e., live / dead cells). More accurate glucose measurement can be, and often is, a key factor for improving process control. Initially, lactate is not a nutrient but a waste product of cells during antibody production, and it is also an indicator of cell metabolism. Therefore, improving lactate measurement accuracy helps to better understand whether cells are in the right medium to produce antibodies.

[0045] The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various other embodiments and modifications of the present disclosure, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Accordingly, such other embodiments and modifications are intended to be included within the scope of the present disclosure.

[0046] Furthermore, while the present disclosure has been described herein in the context of particular implementations in particular environments for particular purposes, those skilled in the art will recognize that its usefulness is not limited thereto and that the present disclosure may be beneficially implemented in any number of environments for any number of purposes. Accordingly, the following claims should be construed in view of the full breadth and spirit of the present disclosure as described herein.

Claims

1. A method for measuring Raman scattering, comprising: performing one or more noise acquisition measurements using a Raman analyzer, wherein the one or more noise acquisition measurements are performed with a laser in the Raman analyzer disabled; performing one or more Raman signal acquisition measurements to generate a Raman spectrum, wherein the Raman signal acquisition measurements are performed with a laser enabled in the Raman analyzer; Averaging one or more noise acquisition measurements to generate an average background noise spectrum; and processing the average background noise spectrum and the Raman spectrum to generate a clean Raman spectrum; A method comprising:

2. 2. The method of claim 1, wherein the average background noise spectrum is subtracted from the Raman spectrum to produce the clean Raman spectrum.

3. The method of claim 1 or 2, wherein a plurality of Raman signal acquisition measurements are performed, and the results of the plurality of Raman signal acquisition measurements are averaged to generate the Raman spectrum.

4. The method of any one of claims 1 to 3, wherein at least one of the one or more noise acquisition measurements is performed before the Raman signal acquisition measurement.

5. The method of any one of claims 1 to 3, wherein at least one of the one or more noise acquisition measurements is performed after the Raman signal acquisition measurement.

6. The method according to any one of claims 1 to 3, wherein a plurality of noise acquisition measurements are performed, at least one of the noise acquisition measurements is performed before the Raman signal acquisition measurement, and at least one of the noise acquisition measurements is performed after the Raman signal acquisition measurement.

7. The method of any preceding claim, wherein the Raman analyzer includes having a light-reducing cap thereon.

8. 1. A system for measuring Raman scattering, comprising: an optical Raman probe sensor; a Raman analyzer comprising an optical detector and a laser, the Raman analyzer communicating with the optical Raman probe sensor via a conduit; Controller and wherein the controller Disabling the laser and performing one or more noise acquisition measurements; enabling the laser and performing one or more Raman signal acquisition measurements to generate a Raman spectrum; Averaging one or more noise acquisition measurements to generate a noise spectrum; and A system that processes the noise spectrum and the Raman spectrum to generate a clean Raman spectrum.

9. The system of claim 8 , wherein the controller subtracts the noise spectrum from the Raman spectrum to produce a clean Raman spectrum.

10. 10. The system of claim 8 or 9, wherein a plurality of Raman signal acquisition measurements are performed, and the controller averages results of the plurality of Raman signal acquisition measurements to generate the Raman spectrum.

11. The system of any one of claims 8 to 10, wherein the controller performs at least one of the one or more noise acquisition measurements before the Raman signal acquisition measurement.

12. The system of any one of claims 8 to 10, wherein the controller performs at least one of the one or more noise acquisition measurements after the Raman signal acquisition measurement.

13. The system of any one of claims 8 to 10, wherein the controller performs a plurality of noise acquisition measurements, at least one of the noise acquisition measurements being performed before the Raman signal acquisition measurement, and at least one of the noise acquisition measurements being performed after the Raman signal acquisition measurement.

14. The system of any of claims 8 to 13, further comprising a light-attenuating cap over the Raman probe sensor.

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