Stray light noise reduction in optical Raman probe sensors

Abstract

A system and method for quantifying stray light entering a Raman optical sensor and removing it from the Raman spectrum is disclosed. One or more background noise measurements are performed by the Raman analyzer, wherein the laser is disabled during these background noise measurements. One or more legacy signal acquisition measurements are then performed. The results from the background noise measurements and the traditional signal acquisition measurements are then processed. This results in a clean spectrum in which background noise is significantly reduced. In some embodiments, background noise measurements are performed before and after 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 during use of optical Raman probe sensors. Background Art

[0002] In many applications, such as bioprocessing applications, it is important to carefully and accurately monitor the composition of a material. For example, in a bioreactor, it may be important to monitor the amount of various molecules such as glucose, lactate, glutamine, ammonium, etc.

[0003] In many cases, this monitoring can be accomplished using Raman spectroscopy. In Raman spectroscopy, a laser is used to direct light of a specific wavelength toward the target molecule. The photon reaches the molecule and excites it. Once the photon excites the molecule, several possible outcomes are possible. The most common is that the excitation is temporary, and the molecule returns to its initial energy state. In this mode, the photon is scattered or redirected due to its interaction with the molecule. Furthermore, the photon's wavelength does not change because its energy is not absorbed by the molecule. 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 to a different vibrational or rotational state. If the new state is higher in energy than the original, the photon loses energy, resulting in a lower frequency. In this way, the total amount of energy is conserved. This mode is known as Stokes Raman scattering.

[0005] If the new state is a lower energy state than the original energy state, then the photon gains energy, which results in a higher frequency. This pattern is called anti-Stokes Raman scattering.

[0006] Stokes Raman scattering and anti-Stokes Raman scattering can be used to generate a spectrum. The spectrum is usually displayed with a horizontal axis corresponding to wave number, which is typically defined as: Where λ0 is the wavelength of the laser light, and λ1 is the wavelength of the Raman scattered light. The vertical axis is used to represent intensity.

[0007] Importantly, each molecule produces a unique spectrum when excited, which can be used to identify it. Therefore, this method can be used to determine the presence of different molecules.

[0008] Compared to Rayleigh scattering, the percentage of Stokes Raman scattering is very low and is highly sensitive to noise. For example, ambient light from the sun or indoor lighting can alter the Raman spectrum.

[0009] It would be advantageous if there were a system and method for reducing the effect of stray light noise entering an optical Raman probe sensor on the Raman spectrum. Summary of the Invention

[0010] A system and method for quantifying the impact of stray light entering a Raman optical sensor and removing it from a Raman spectrum is disclosed. A Raman analyzer performs one or more background noise measurements, during which the laser is disabled. One or more conventional signal acquisition measurements are then performed. The results from the background noise and conventional signal acquisition measurements are then processed. This results in a clean spectrum in which the background noise has been significantly reduced. In some embodiments, background noise measurements are performed before and after the conventional signal acquisition measurements, and the 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, wherein the one or more noise acquisition measurements are performed while a laser within the Raman analyzer is disabled; performing one or more Raman signal acquisition measurements to create a Raman spectrum, wherein the Raman signal acquisition measurements are performed while the laser within the Raman analyzer is enabled; averaging the one or more noise acquisition measurements to create an average background noise spectrum; and processing the average background noise spectrum and the Raman spectrum to create a clean Raman spectrum. In some embodiments, the average background noise spectrum is subtracted from the Raman spectrum to create the clean Raman spectrum. In some embodiments, more than one Raman signal acquisition measurement is performed, and the results from the more than one Raman signal acquisition measurement are averaged to create 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, more than one noise acquisition measurement is performed, and 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.

[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 a noise spectrum; and processes the noise spectrum and the Raman spectrum to create a clean Raman spectrum. In some embodiments, the controller subtracts the noise spectrum from the Raman spectrum to create the clean Raman spectrum. In some embodiments, more than one Raman signal acquisition measurement is performed, and the controller averages the results from the more than one Raman signal acquisition measurements to create 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 more than one noise acquisition measurement, and performs at least one of the noise acquisition measurements before and at least one of the noise acquisition measurements after the Raman signal acquisition measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For a better understanding of the present disclosure, reference is made to the accompanying drawings, which are incorporated herein by reference, and wherein:

[0014] FIG1 illustrates a bioreactor including a Raman probe sensor and a noise reduction cap according to some embodiments of the present disclosure;

[0015] Figure 2 shows a sequence of operations to create a clean Raman spectrum according to some embodiments of the present disclosure;

[0016] Figure 3 is a graph showing the effect of the disclosed method on the resulting Raman spectrum according to some embodiments of the present disclosure;

[0017] Figure 4 including a graph showing the effect of the disclosed method on stable glucose concentration measurement in a light environment compared to standard Raman according to some embodiments of the present disclosure;

[0018] Figure 5 is a graph showing the effect of the disclosed method on stable lactate concentration measurements in a light environment compared to standard Raman according to some embodiments of the present disclosure; and

[0019] 6 includes three graphs showing the effect of the disclosed methods on the measurement of stable glucose and lactate concentrations, and viable cell density (VCD) in cell cultures run in a normal laboratory environment, compared to standard Raman spectroscopy and referenced to offline reference values, according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0020] Embodiments of the present disclosure describe systems and methods for reducing stray light noise used in Raman spectrum generation.

[0021] In many applications, such as bioprocessing applications, it is important to carefully and accurately monitor the materials within the bioreactor.

[0022] 1 illustrates a bioreactor including a Raman probe sensor and a noise reduction cap according to some embodiments of the present disclosure. Figure 1A A representative bioreactor 1 is shown. The bioreactor 1 may be controlled by a bioreactor controller 2. The bioreactor controller 2 may control various valves, spargers, impellers, and other functional components of the bioreactor 1.

[0023] The bioreactor bag 10 is typically inserted into the bioreactor 1. The bioreactor bag 10 may have multiple ports to allow for the introduction of various sensors, actuators, sprayers, or other mechanisms into the interior of the bioreactor bag 10. In this illustration, an optical Raman probe sensor 20 enters the interior of the bioreactor bag 10 through port 11. The optical Raman probe sensor 20 includes tubing with a window (e.g., a sapphire window). The optical Raman probe sensor 20 may also optionally include a bioreactor-compatible connecting thread, such as PG13.5. In some embodiments, the tubing of the optical Raman probe sensor 20 has a maximum outer diameter of 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 projects a laser beam along an optical axis 31. The laser beam passes through the tubing, the sapphire window, and into 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 a 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 optical signals from a target 30 .

[0024] The optical Raman probe sensor 20 is connected to a Raman analyzer 21 outside 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. The conduit 23 may be a fiber optic cable. The Raman analyzer 21 may include an optical detector 26, which may include a CCD or a photodetector. The Raman analyzer 21 also includes a processing unit 24 to control the laser 22 and interpret the output from the optical detector 26.

[0025] The controller 50 can communicate 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, a dedicated circuit, a programmable circuit, a microcontroller, or another similar device. The memory device contains 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 DRAM, or any non-transitory computer-readable storage medium.

[0026] 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 FIG1 , the controller 50 is separate from the Raman analyzer 21. However, in other embodiments, the two components can be combined. In other embodiments, the controller 50 can transmit processed data to the bioreactor controller 2.

[0027] As mentioned above, Stokes Raman scattering occurs at a much lower frequency than Rayleigh scattering and is therefore very sensitive to noise. Therefore, ambient light 40 entering the optical Raman probe sensor 20 may adversely affect detection accuracy. This ambient light can be sunlight, moonlight, room lighting, or other types of illumination. One way to address this issue is to quantify the amount of ambient light entering the optical Raman probe sensor 20 and remove this background noise component from the Raman spectrum.

[0028] In normal operation, the Raman analyzer 21 activates the laser 22 disposed in the Raman analyzer 21. Light from the laser 22 travels via the conduit 23 to the optical Raman probe sensor 20. The light from the laser 22 excites molecules in the optical axis 31 of the laser 22, generating Rayleigh scattering, Stokes Raman scattering, and anti-Stokes Raman scattering. The light is then received by the optical assembly 25 of the optical Raman probe sensor 20. The data from the optical Raman probe sensor 20 is then transmitted to the optical detector 26 disposed within the Raman analyzer 21, which can generate a Raman spectrum. This process can be referred to as a Raman signal acquisition measurement, and the result of this process can be referred to as a Raman spectrum.

[0029] In another mode, Raman analyzer 21 does not activate laser 22. However, as previously described, data from optical Raman probe sensor 20 is transmitted to Raman analyzer 21, where it can create a Raman spectrum. In this mode, any signal received by optical detector 26 in Raman analyzer 21 via 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 can be referred to as a background noise spectrum.

[0030] Figure 1A-Figure 1C At least one embodiment according to the present disclosure is shown. Figure 1B shows an exploded view of this embodiment, and Figure 1C A cross-section of the assembled sensor is shown. In some embodiments, the optical Raman probe sensor 20 is enclosed in a tubing 56 that includes two parts: a tubing body 27 and a tubing head 28. The tubing body 27 can include a hollow tube. The tubing head 28 is attached to the tubing body 27, such as by welding. The tubing head 28 includes a sapphire window and an optical lens 29. Further, the outer surface of the tubing head 28 includes threads to accommodate a cap 50. In some embodiments, cleaning may be desirable, and the ability to remove the cap 50 is useful. Additionally, in some embodiments, the tubing head 28 has threads on its outer surface near its distal end.

[0031] The cap 50 includes threads on the inner surface of the cylindrical body 52. ​​In operation, the cap 50 is screwed onto the tube head 28. The cap 50 can be removed for easier cleaning. Furthermore, in some embodiments, the cap 50 can be treated as a disposable component, such that a new cap 50 is installed on the tube head 28 before each use. Furthermore, this configuration allows the cap design to be selected according to the application without changing other parts of the optical Raman probe sensor 20.

[0032] In some embodiments, the length of cap 50 can be a design decision. For example, cap 50 can be designed so that the distance from the tip of optical Raman probe sensor 20 to closed end 54 is between 1 and 10 cm, although other dimensions are possible. Opening 58 is shown as two circular holes. However, the present disclosure is not limited to this embodiment. Rather, opening 58 can be circular, elliptical, rectangular, or any other shape. These processes can be used in conjunction with each other to generate clean Raman spectra.

[0033] Specifically, Figure 2 A flow chart illustrating the sequence of operations of the controller 50 and the Raman analyzer 21 to create a clean Raman spectrum is shown. First, as indicated by block 100, one or more background noise acquisition measurements may be performed. In some embodiments, the number of background noise acquisition measurements may be as large as 100. In other embodiments, a smaller number of background noise acquisition measurements may be performed. As described above, this process is performed while the laser 22 is disabled.

[0034] Next, as shown in block 110, one or more Raman acquisition measurements are performed. As described above, this involves activating the laser 22 and detecting the emitted optical signal using the optical detector 26. The Raman analyzer 21 can then create a Raman spectrum based on the output of the optical detector 26. In some embodiments, multiple Raman acquisition measurements are performed to create the Raman spectrum. In some embodiments, ten or more Raman acquisition measurements are performed.

[0035] Next, as shown in block 120, after performing the Raman signal acquisition measurement, one or more background noise acquisition measurements may be performed. In some embodiments, the number of background noise acquisition measurements may be as high as one hundred, as with the background noise acquisition measurements performed before the Raman signal acquisition measurement. In other embodiments, a smaller number of background noise acquisition measurements may be performed. In other embodiments, no background noise acquisition measurement may be performed after the Raman acquisition measurement.

[0036] Next, as shown in block 130, the background noise spectra obtained from the background noise acquisition measurements can be averaged together and used to create an average background noise spectrum. In some embodiments, the background noise spectrum is generated based on background noise acquisition measurements performed before and after the Raman signal acquisition measurements. In other embodiments, background noise acquisition measurements are performed only before or only after the Raman signal acquisition measurements. In these embodiments, either block 100 or block 120 can be omitted.

[0037] In block 140, the average background noise spectrum and the Raman spectrum are processed to create a clean Raman spectrum with reduced background noise. In some embodiments, the average background noise spectrum is subtracted from the Raman spectrum to create the clean Raman spectrum. In other embodiments, other denoising algorithms may utilize the background noise spectrum and the Raman spectrum to create the clean Raman spectrum.

[0038] The operations described in blocks 130 and 140 may be performed using the controller 50 or a different controller. For example, the operations may be performed using a different controller that is part of the same network as the Raman analyzer 21.

[0039] If multiple channels are being analyzed, a new channel is set, as shown in block 150, and the sequence is repeated.

[0040] The clean Raman spectrum can be used in a variety of ways. For example, the clean Raman spectrum can be processed and analyzed by mathematically advanced tools to determine the composition of the medium contained in the bioreactor bag 10.

[0041] The above-described embodiments in the present application may have many advantages. Figure 3 is a graph showing the effect of the disclosed method on the resulting Raman spectrum according to some embodiments of the present disclosure.

[0042] In many applications, the optical Raman probe sensor 20 is deployed in a glass bioreactor or in a plastic bioreactor bag that is not completely opaque to ambient light. Therefore, when generating Raman spectra, ambient light may affect the results. In one experiment, different concentrations of glucose (from 0g / L to 13g / L) were added to the bioreactor. At each concentration, Raman measurements were performed. The first set of Raman measurements was performed with ambient light entering the bioreactor. These Raman measurements were then processed in a conventional manner to obtain Figure 3 The data points are labeled "Values ​​from Raman Spectroscopy" in Figure 2. These data points are the predicted glucose concentrations based on Raman spectroscopy. Note that there is a large offset between the actual glucose concentration and the predicted glucose concentration based on Raman spectroscopy. Additionally, at each concentration, Figure 2 Clean Raman spectra were obtained using the sequence shown. The glucose concentrations derived from these clean Raman spectra are represented by the data points labeled "Value from Clean Spectrum." Note that the predicted glucose concentration based on the clean spectra is nearly identical to the actual glucose concentration. Therefore, this technique is effective in reducing the effects of background noise.

[0043] Stray light management includes, for example, attaching a light attenuator cap having a closed end and one or more openings, as described above, to the tip of an optical Raman probe sensor. The cap serves to block stray light noise from entering the sensor tip. This allows for more accurate and consistent Raman spectroscopy. Furthermore, the cap can be permanently or removably attached to the sensor. In some embodiments, a reflective surface can be included on the inner surface of the closed end of the cap. This reflective surface can reflect Raman scattered light toward the tip, thereby enhancing the received signal.

[0044] Figure 4 Included are graphs showing the impact 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. Figure 4 Various stray light conditions and their measurements according to some embodiments of the present disclosure are compared. The first figure shows a graph showing glucose measurement data points using a standard Raman probe. The second graph shows glucose measurement data points using an advanced Raman probe with its light dimmer cap. The third figure shows a third graph showing glucose measurement data points using the advanced Raman probe with its light dimmer cap and a software noise reduction filter applied. The fourth graph shows glucose measurement data points using the advanced Raman probe with only the software noise reduction filter applied. As demonstrated, without implementing stray light management on the advanced Raman probe, namely, the light dimmer cap and subtracting the average background signal, the noise is as high as 1.5 g / L. With stray light management, the noise is 0.2 g / L or less. Stray light management includes, for example, attaching a light dimmer cap having a closed end and one or more openings to the tip of the optical Raman probe sensor. The cap serves to block stray light noise from entering the sensor tip. This results in more accurate and consistent Raman spectra. Furthermore, the cap can be permanently or removably attached to the sensor. In some embodiments, a reflective surface can be included on the inner surface of the closed end of the cap. The reflective surface can reflect Raman scattered light toward the tip, thereby enhancing the received signal.

[0045] Figure 5 is a graph showing the effect of the disclosed method on stable lactate concentration measurements in a light environment compared to standard Raman, according to some embodiments of the present disclosure. Figure 5Various stray light conditions and their measurements according to some embodiments of the present disclosure are compared. The first curve shows lactate measurement data points using the advanced Raman probe with its light-reducing cap and applying a software noise reduction filter. The second curve shows lactate measurement data points using only the advanced Raman probe with its light-reducing cap. The third curve shows lactate measurement data points using a standard Raman probe. As demonstrated, without implementing stray light management on the advanced Raman solution, i.e., capping and subtracting the average background signal, the noise is as high as 4.0 g / L. With stray light management, the noise is again 0.2 g / L or less. The data demonstrates that the noise is reduced by over 70% when the light-reducing cap is used.

[0046] Figure 6 includes three graphs showing the effects of the disclosed methods on the measurement of stable glucose and lactate concentrations, viable cell density (VCD) in cell cultures run in a normal laboratory environment, compared to standard Raman spectroscopy and referenced to offline reference values, according to some embodiments of the present disclosure. Figure 6 compares measurements from different Raman probes thereof in fed-batch cell cultures run over eight days under various stray light conditions, as described for measuring glucose, lactate, and viable cell density, according to embodiments of the present disclosure.

[0047] Figure 6A A first graph is shown depicting glucose concentration measurements using the advanced Raman probe with its dimmer cap and software noise reduction filter compared to a standard Raman probe and referenced to an offline reference value.

[0048] Figure 6B A second graph is shown depicting lactate concentration measurements using the advanced Raman probe with its dimmer cap and software noise reduction filter compared to a standard Raman probe and referenced to an offline reference value.

[0049] Figure 6C A third figure is shown depicting live cell density measurements using the advanced Raman probe with its dimmer cap and software noise reduction filter compared to a standard Raman probe and referenced to an offline reference value. The third figure demonstrates that the present invention is applicable not only to nutrients and VCD, but also to other key parameters of cell culture. For example, it is shown that without the stray light management disclosed herein, VCD predictions are abnormal. Improved glucose control helps to better manage cell growth as well as cell viability (~live cells / dead cells). More accurate glucose measurements can be and often are a key factor in achieving improved process control. Lactate is not originally a nutrient, but rather a waste product of cells when they are producing antibodies and is also an indicator of cellular metabolism. Therefore, the improved accuracy of lactate measurements helps to better understand whether the cells have a good culture medium to produce antibodies.

[0050] The scope of the present disclosure is not limited by the specific embodiments described herein. In fact, 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 above description and accompanying drawings. Therefore, these other embodiments and modifications are intended to fall within the scope of the present disclosure. Furthermore, although this document has been described in the context of specific embodiments in a specific environment for a specific purpose,

[0051] Although the present disclosure is provided, persons of ordinary skill in the art will recognize that its usefulness is not limited thereto and

[0052] Rather, the present disclosure can be beneficially implemented in any number of environments for any number of purposes.

[0053] Accordingly, the following claims should be taken into account insofar as the full breadth of the disclosure as described herein is concerned.

[0054] and spiritual explanation.

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 while a laser within the Raman analyzer is disabled; performing one or more Raman signal acquisition measurements to create a Raman spectrum, wherein the Raman signal acquisition measurements are performed while the laser within the Raman analyzer is enabled; averaging the one or more noise acquisition measurements to create an average background noise spectrum; as well as The average background noise spectrum and the Raman spectrum are processed to create a clean Raman spectrum.

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

3. The method according to claim 1 or 2, wherein: More than one Raman signal acquisition measurements are performed, and results from the more than one Raman signal acquisition measurements are averaged to create the Raman spectrum.

4. A method according to any one of the preceding claims, wherein At least one of the one or more noise acquisition measurements is performed before the Raman signal acquisition measurement.

5. The method according to 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 More than one noise acquisition measurement is performed, and 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. A method according to any one of the preceding claims, wherein The Raman analyzer includes a light-reducing cap thereon.

8. A system for measuring Raman scattering, comprising: 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; as well as A controller, 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 the one or more noise acquisition measurements to generate a noise spectrum; as well as The noisy spectrum and the Raman spectrum are processed to create a clean Raman spectrum.

9. The system according to claim 8, wherein: The controller subtracts the noise spectrum from the Raman spectrum to create the clean Raman spectrum.

10. The system according to claim 8 or 9, wherein: More than one Raman signal acquisition measurement is performed, and the controller averages results from the more than one Raman signal acquisition measurement to create the Raman spectrum.

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

12. The system according to 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 according to any one of claims 8 to 10, wherein: The controller performs more than one noise acquisition measurement, and 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.

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

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