Fiber ripple detection and monitoring system and method - Patent Application 20070122967

Abstract

A bioreactor monitoring device and method configured for in-situ or ex-situ analysis is provided. The monitoring system (5) includes a tunable laser spectrometer (8) that generates a swept wavelength signal and transmits it through an optical fiber (110), a reference light detection means (158) that detects the swept wavelength signal after transmission from the tunable laser spectrometer (8) through the optical fiber (110), and a sample light detection means (132) that detects the swept wavelength signal after transmission through a sample detection region (12). The polarization means (152) and the reference light detection means (158) that detects the swept wavelength signal before transmission through the sample detection region (12) from the tunable laser spectrometer (8) through the optical fiber (110) address noise resulting from polarization dependent loss.

Description

Related Applications

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 414,296, filed October 7, 2022, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Many processes in industries such as the chemical, biochemical, pharmaceutical, food and beverage industries require some type of monitoring.

[0003] Sensors have been developed and marketed for in-situ and real-time measurement of pH, dissolved oxygen (DO), temperature, or pressure. Common techniques used to detect chemical components include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GCMS), or enzyme- or reagent-based electrochemical methods.

[0004] While considered accurate, many existing techniques used to monitor materials in reactors are performed offline, tend to be destructive to the sample, often require expensive consumables, and / or take a long time to complete. The equipment required to perform such analyses is often not only expensive, but also requires complex calibration and skilled operators. The procedures can be time-consuming and labor-intensive, and often the solution is to reduce the frequency of sampling from the process of interest, resulting in fewer data points. Batch processing of samples after the process has finished is also an option, but provides little or no feedback for continuously adjusting conditions. These drawbacks can persist even when the sampling process is automated.

[0005] A variety of optical spectroscopic techniques are available for characterizing components (also called analytes) in a sample. Absorption spectroscopy is perhaps the most common. Incident light can excite the analyte's electrons from a low-energy ground state to a higher-energy excited state, and the energy can be absorbed by non-bonding n- and π-electrons in the molecular orbitals. Absorption spectroscopy can be performed in the ultraviolet, visible, and / or infrared regions, allowing analytes of various material phases and compositions to be probed with specific wavelengths or wavelength bands of light. The resulting transmitted light can then be used to resolve the absorption spectrum to determine one or more specific properties of the analyte or sample, such as composition, temperature, or pH, for applications in medical diagnostics, pharmaceutical development, and food and beverage quality control.

[0006] One area of ​​particular interest is the online and offline monitoring of bioreactors, which are widely used in various fields, including biotechnology and chemical engineering. Bioreactors are essentially vessels or systems configured to support the growth of microorganisms or cells, enabling controlled biological processes. Several common examples exist. Fermentation is used to produce a variety of products, including pharmaceuticals, biofuels, and food. Microorganisms, such as bacteria, yeast, and fungi, are cultivated in bioreactors to produce desired substances, such as antibiotics, ethanol, and enzymes. In biopharmaceutical manufacturing, bioreactors are used to culture mammalian cells to produce therapeutic proteins, monoclonal antibodies, and vaccines. These systems require continuous, strict control of environmental factors, such as temperature, pH, and oxygen levels, to ensure the quality and yield of the final product. In tissue engineering, bioreactors provide a controlled environment for the growth and maturation of artificial tissues and organs. In bioreactors, cells are cultured on scaffolds to create functional tissues. Other areas of application include algae cultivation, wastewater treatment, bioremediation, research and development, the food and beverage industry, and pharmaceutical screening.

[0007] US Pat. Pub. No. 2021 / 0088433 by Hassell et al. (issued November 15, 2022 as US Patent No. 11,499,903 B2), incorporated herein by reference in its entirety, describes a robust, hands-free, non-destructive, real-time technique for identifying and / or quantifying components within a process of interest. Typically, the process is carried out in a vessel such as a bioreactor. The contents of the bioreactor may change as the process progresses, and these changes are tracked using near-infrared (NIR) spectroscopy. Substances present within the bioreactor are identified using in-situ probes inserted and / or maintained within the bioreactor. This allows for analysis to be performed non-destructively and in real time.

[0008] Typically, a tunable laser spectrometer is equipped with a wavelength reference detector and a power reference detector. The wavelength reference allows the device to track the wavelength sweep of the tunable laser within the spectral scan band of the tunable laser. This ensures highly accurate wavelength resolution for absorption spectrum resolution. The power reference detector detects the instantaneous power during the sweep, allowing compensation for any changes in the laser power during the sweep. This ensures accurate resolution of the absorption spectrum of the substance of interest. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent Application Publication No. 2021 / 088433 [Patent Document 2] U.S. Patent No. 1,149,903 Summary of the Invention [Problem to be solved by the invention]

[0010] However, to maximize operational performance, any power variations must be perfectly compensated. One source of power variations stems from the highly polarized nature of the diode lasers employed in many laser spectrometers. Any change in polarization state, combined with polarization-dependent losses (PDL) in various components such as lenses, beam splitters, fibers, and detectors, results in off-track power variations, which degrade the accuracy of the measured extinction spectrum.

[0011] Polarization-maintaining (PM) fibers, such as single-mode polarization-maintaining (PANDA) fibers, are also sometimes used between the tunable laser spectrometer and the probe and / or sample cell and / or sample holder in contact with the sample. Such PM fibers provide the polarization stability needed to combat PDL. However, the use of polarization-maintaining fibers does not completely solve the problem. The unwanted polarization state usually contains some power, and this optical mode can beat with the power of the desired polarization mode, resulting in power fluctuations. This can lead to the combined effects of fiber polarization beating and PANDA ripple. Overall, when two waves with different linear polarization states propagate through a birefringent PM fiber, their phases evolve differently. The differential phase delay is proportional to the fiber length. [Means for solving the problem]

[0012] Generally speaking, in one aspect, the invention features a monitoring system including a tunable laser spectrometer that generates and transmits a swept wavelength signal through an optical fiber; a reference light detection means that detects the swept wavelength signal after transmission through the optical fiber from the tunable laser spectrometer; and a sample light detection means that detects the swept wavelength signal after transmission through a sample detection region.

[0013] In an embodiment, a polarizing means is provided to improve the polarization state of the swept wavelength signal before the reference light detecting means.

[0014] The optical fiber may also include polarization-maintaining fiber, one example being single-mode polarization-maintaining fiber.

[0015] In some embodiments, the sample interface includes at least one beveled light transmission port and light detection port, such as provided by an input waveguide rod and an output waveguide rod.

[0016] In one example, the tunable laser spectrometer sweeps wavelengths within a spectral band that includes 2.3 micrometers and / or 6.5 micrometers.

[0017] The system may be implemented as a probe that is inserted into the bioreactor, or may include a sample interface that receives a droplet of sample removed from the bioreactor.

[0018] Typically, a control unit is provided which monitors the response of the sample light detection means and the reference light detection means to resolve the absorption spectrum of the sample in the sample detection region, and preferably compensates for noise associated with ripple from the optical fiber and / or temperature detected by one or more thermistors provided in the reference light detection means and the sample light detection means.

[0019] Typically, there is no optical fiber between the reference detector and the sample light detector, which allows the reference detector to be used to compensate for the sensitivity of the system to polarization states.

[0020] Generally speaking, in another aspect, the invention features a monitoring system including a tunable laser spectrometer that generates a swept wavelength signal; a reference light detection means that detects the swept wavelength signal from the tunable laser spectrometer; a polarization means that improves the polarization state of the swept wavelength signal before the reference light detection means; and a sample light detection means that detects the swept wavelength signal after passing through a sample detection region.

[0021] Generally speaking, in yet another aspect, the invention features a probe for a bioreactor, the probe including: a port forming a sample detection region within the bioreactor; a reference light detection means for detecting an optical signal received from a polarization means prior to the sample detection region; and a sample light detection means for detecting the optical signal after transmission through the sample detection region.

[0022] In general, in yet another aspect, the invention features a method for online or offline monitoring of a bioreactor, the method including generating a swept wavelength signal, delivering the swept wavelength signal to a sample detection region of a sample in the bioreactor, polarizing the swept wavelength signal, detecting the polarized swept wavelength signal before transmission through the sample detection region, detecting the swept wavelength signal after transmission through the sample detection region, and resolving an absorbance spectrum of the sample in the sample detection region using the swept wavelength signal before and after transmission through the sample detection region.

[0023] Techniques such as those described herein also improve the quality of the analysis. For example, the embodiments described herein improve, and in some cases even maximize, the signal-to-noise ratio. This is achieved by launching a light beam from a fiber and / or free-space link through a sample gap and directing the transmitted light to a light detection means. Instead of using a fiber optic cable back to a photodiode that may be outside the reactor, the approaches described herein allow the detection means cable to run the length of the probe, relying on the signal-to-noise ratio (SNR) of electrical cables, which are typically superior to fiber optics.

[0024] These and other features of the present invention, including various novel details of construction and combination of parts and other advantages, are more particularly described hereinafter with reference to the accompanying drawings and as particularly pointed out in the claims. It will be understood that the specific methods and apparatus embodying the invention are merely illustrative and not limiting. The principles and features of the invention can be applied to numerous and varied embodiments without departing from the scope of the invention.

[0025] In the accompanying drawings, where reference characters refer to the same structures / components throughout the different views, the drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a schematic diagram of a monitoring system according to the present invention comprising a tunable laser spectrometer and an in-situ probe. [Figure 2] FIG. 2 is a cross-sectional view of a head portion of the probe. [Figure 3] FIG. 2 is a cross-sectional view of the tip portion of the probe. [Figure 4] FIG. 2 is a side perspective view of a cross section showing each rod of the tip portion of the probe. [Figure 5]FIG. 1 is a side view of another monitoring system according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, which depict exemplary embodiments of the invention. However, the present invention may be embodied in many other forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0028] As used herein, the terms "and / or" and "and / or," when used in conjunction with one or more items, are intended to encompass any and all combinations of those items. Furthermore, the singular and articles "a," "an," and "the" are intended to encompass the plural, unless expressly stated otherwise. Furthermore, as used herein, the terms "having," "including," "comprising," and / or "having" are intended to identify the presence of stated features and / or entities and / or steps and / or processes and / or elements and / or components, but not to exclude the presence or addition of one or more other features and / or entities and / or steps and / or processes and / or elements and / or components and / or combinations thereof. Furthermore, when an element, such as a component or subsystem, is referred to and / or shown as being connected or coupled to another element, it is understood that one may be directly connected or coupled to the other element, as well as that there may be elements therebetween.

[0029] Unless otherwise defined, the meanings of all terms used in this specification (including technical and scientific terms) are the same as those commonly understood by those skilled in the art to which this invention belongs. Furthermore, even for terms defined in commonly used dictionaries, the meanings should be interpreted in accordance with the meanings in the context of the relevant field, and unless clearly defined in this specification, they should not be interpreted in an idealized or overly formal way.

[0030] Many processes carried out in vessels such as bioreactors require or benefit from strict control of one or more parameters, such as pH, oxygen levels, nutrients, metabolites, or waste products. In numerous embodiments, the present invention relates to devices and methods for offline or online (often continuous) analysis of the contents of a bioreactor. Reactor components can be detected at various time intervals, and the data can be used to assess status and, if necessary, adjust or optimize process parameters. Examples of processes that can be monitored include cell growth protocols, biopharmaceutical manufacturing, tissue engineering, fermentation, algae cultivation, wastewater treatment, bioremediation, research and development, and the food and beverage industry.

[0031] In one embodiment, the techniques described herein are practiced using a bioreactor, a cell culture system for the three-dimensional assembly, growth, and differentiation of cells and tissues, or a bioreactor containing such a system, typically containing a combination of cells, media, nutrients, metabolites, enzymes, hormones, cytokines, etc.

[0032] The analysis may involve a spectroscopic system that determines the spectral response of each component in the sample detection region in one or more of the infrared (IR) region, including one or more of the near-infrared, mid-infrared, and far-infrared, the visible region, and the ultraviolet (UV) region, and may measure various properties of the analytes in the bioreactor, such as one or more of the absorbance, emission (including blackbody and fluorescence), elastic scattering and reflectance, impedance (e.g., refractive index, etc.), and inelastic scattering (e.g., Raman, Compton, etc.) spectra.

[0033] While certain embodiments described herein utilize ultraviolet and visible spectroscopy, most commonly infrared spectroscopy spans wavelengths from 700 nanometers (nm) to 1 millimeter (mm) across the spectrum, specifically including near-infrared (0.75-1.4 μm; NIR), short-wavelength infrared (1.4-3 μm; SWIR), mid-wavelength infrared (3-8 μm; NWIR), long-wavelength infrared (8-15 μm; LWIR), and / or far-infrared (15-1000 μm; FIR). NIR-SWIR spectroscopy examines molecular overtone and combination vibrations, covering the 750 nanometer (nm) to 2500 nm region of the electromagnetic spectrum. In one embodiment, a tunable laser sweeps wavelengths within a spectral band that includes 2.3 micrometers, more specifically sweeping over 100 nanometers around 2.3 micrometers. In one example, the laser sweeps the wavelength over 150 nanometers or over 200 nanometers, for example, from 2.2 micrometers to 2.4 micrometers. An overview of NIR spectroscopy can be found, for example, in the article "An Introduction to Near Infrared (NIR) Spectroscopy" by AMC Davies (impublications.com / content / introduction-near-infrared-nir-spectroscopy).See also Cervera, A.E., Petersen, N., Lantz, A.E., Larsen, A., and Gernaey, K.V., "Application of near-infrared spectroscopy for monitoring and control of cell culture and fermentation," Biotechnol. Prog. 25, 1561-1581 (2009), and Roggo, Y. et al., "A review of near infrared spectroscopy and chemometrics in pharmaceutical technologies," Journal of Pharmaceutical and Biomedical Analysis, Volume 44, Issue 3, 2007.

[0034] In the embodiments described herein, measurements are taken in-situ in the reactor (typically without the need to draw samples to a sample cell or flow cell) to provide online monitoring, or samples are drawn to an external (ex-situ) arrangement for offline readings.

[0035] Many embodiments of the present invention utilize at least one outer tube, e.g., stainless steel, containing at least one light detection means, e.g., a photodiode, and at least one optical component, e.g., at least one waveguide, e.g., a free space transmission line, an optical fiber, and / or at least one window and / or at least one focusing lens. These elements form a sample interface that includes a light transmission port and a light detection port that form the sample detection region. The air gap between these windows contacts the fluid within or removed from the bioreactor, and thus can be the path length of the laser light within the fluid.

[0036] Figure 1 shows a monitoring system 5 including a tunable laser spectrometer 8 and an in-situ probe 10 that provides a sample interface configured to position and / or maintain the sample interface as a sample detection region 12 within a vessel such as a bioreactor. Figure 2 shows a cross-sectional view of the head portion 11 of the in-situ probe 10, and Figure 3 shows a longitudinal cross-sectional view of the tip portion 14 of the in-situ probe 10.

[0037] The tunable laser spectrometer 8 is connected to the in-situ probe 10 by a fiber optic patch cable 110. The fiber optic patch cable 110 terminates in a fiber optic connector 112. The fiber optic connector 112 is mated to a fiber optic receptacle 114 in a housing 118 of the head unit 11.

[0038] The tunable laser spectrometer 8 is electrically connected to the head unit 11 by a spectrometer electrical wiring harness 116 .

[0039] Generally, the head portion 11 is characterized by a probe optical housing 118 that mates at its lower end with a probe outer tube 120. The probe outer tube 120 is metal, for example, 12 millimeter (mm) outer diameter (OD) stainless steel.

[0040] The metal probe outer tube 120 terminates in a sample assembly 122. The tip section 14 has a lower end tube 124 below the sample assembly.

[0041] Many bioreactors have ports in their cover plates to accept various threadable fittings. Thus, fitting 18 is provided to seal the cover plate on the bioreactor. More specifically, fitting 18 can be a PG 13.5 fitting, a threaded standard typically used on bioreactor cover plates.

[0042] Figure 2 shows the internal components of head unit 11. Fiber optic patch cable 110 terminates in APC-SM fiber connector 112, which connects to a suitable fiber receptacle or port 114. The port contains collimating lens 115, which collimates the beam emerging from fiber end face 111. The beam propagates through port 118P in probe optical housing 118.

[0043] In this embodiment, the optical fiber patch cable 110 is PM, typically PANDA fiber, which ensures that the polarization state from the tunable laser spectrometer 8 to the probe 10 is stable.

[0044] The collimated free-space beam exiting the fiber is received by a polarizing means 152 supported by a rotating mount 153. The polarizing means 152 filters or removes orthogonally polarized light. The polarizing means is secured to a bench 150 of the head unit 11 by the rotating mount 153. The rotating mount 153 allows the polarizing means 152 to rotate in a plane perpendicular to both the top surface of the bench 150 and the optical axis of the beam exiting the fiber. During calibration, the rotating mount 153 allows the polarizing means 152 to be finely adjusted in its rotation to align with the desired polarization axis of the PANDA fiber.

[0045] A partially reflective sapphire window 154, such as a wedge-shaped window, is held on a pitch-yaw mount 156. The window 154 is secured to the bench 150 by the pitch-yaw mount 156. The partially reflective sapphire window 154 reflects a portion of the beam toward a ripple reference detector 158, such as an In-GaAs detector. A focusing lens 160 couples the beam to the active area of ​​the ripple reference detector 158. The ripple detector is mounted on a head-side printed circuit board (PCB) 162. The head-side PCB 162 includes a transimpedance amplifier 163 that amplifies the electrical response of the ripple reference detector. The head-side PCB 162 also preferably includes a thermistor 161 that enables temperature compensation of the detector 158 and the transimpedance amplifier 163. The response of the ripple reference detector is then transmitted to the spectrometer 8 via an internal harness 180 and the spectrometer electrical wiring harness 116.

[0046] The pitch-yaw mount 156 allows the free-space beam reflected and transmitted through the sapphire window 154 to be adjusted so that a portion of the beam propagates through the center of the inner bore 131 of the inner tube 130 within the outer tube 120, while the remainder is incident on the active area of ​​the ripple reference detection means 158.

[0047] 3, the sample detection region 12 is located in the tip portion 14 of the probe 10 and is defined by a recess formed in the sample portion 122. The sample detection region 12 is defined by an optical transmission port 126P and an opposing optical detection port 128P. In this example, the optical transmission port 126P is defined by an input quartz or sapphire waveguide rod 126 held in the body of the sample portion 122, and the optical detection port 128P is defined by an output quartz or sapphire waveguide rod 128 also held in the body of the sample portion 122. The air gap between these two rods and each port in the air gap define the sample detection region 12 and typically determine the path length of the light traversing the sample.

[0048] The input waveguide 126 is optically coupled to a free-space path formed by the inner bore 131 of the inner tube 130. The output waveguide 128 is optically coupled to a sample light detector 132. In this example, the sample light detector 132 is a TO-46 In-GaAs detector with a dome-shaped lens. The sample light detector 132 is electrically connected to a sample detector-side PCB 134 housed in the lower tube 124. The detector-side PCB 134 contains a transimpedance amplifier 135 that amplifies the detector response and transmits it to the head-side PCB 162 via an internal wiring harness 136 and to the spectrometer 8 via the spectrometer electrical wiring harness 116. The detector-side PCB 134 also contains a thermistor 137 that detects the internal temperature of the chip and enables temperature offsetting.

[0049] The sample section 122 is fitted at its upper end to the metal probe outer tube 120, and is fitted at its lower end to the bottom tube 124. Specifically, the upper necked-down section 82 is inserted into the bottom end of the metal probe outer tube 120. The outer surface of the necked-down section 82 is joined to the inner surface of the bottom end of the metal probe outer tube 120. Similarly, the lower necked-down section 84 of the sample section 122 is inserted into the top end of the bottom tube 124.

[0050] The dimensions of the tip portion 14 and the metal probe outer tube 120 may be selected depending on the size of the reactor. In most cases, the longitudinal distance between the fixture 18 and the optical sensing region 12 of the tip portion 14 is set so that the sensing region 12 is exposed to the reactor medium being monitored, specifically a portion of the medium that represents the entire medium in the bioreactor, rather than the medium along the reactor wall, which may be unmixed, or the medium near the reactor surface. In one illustrative example, the distance between the fixture 18 and the optical detection region 12 is 1 centimeter (cm) or greater, e.g., 2 cm or greater, 3 cm or greater, 4 cm or greater, 5 cm or greater, 10 cm or greater, 15 cm or greater, 20 cm or greater, 25 cm or greater, 30 cm or greater, 35 cm or greater, 40 cm or greater, 45 cm or greater, 50 cm or greater, 55 cm or greater, 60 cm or greater, 65 cm or greater, 70 cm or greater, 75 cm or greater, 80 cm or greater, 85 cm or greater, 90 cm or greater, 95 cm or greater, or 100 cm or greater. For example, the tip portion 14 may be smaller for miniature reactor configurations or larger for some industrial-scale applications.

[0051] Generally, in operation, swept wavelength light from a tunable laser 201 is coupled into the fiber optic patch cable 110 toward the probe optical housing 118. In one embodiment, the tunable laser includes a gallium antimonide (GaSb) gain chip and sweeps wavelengths in a spectral band that includes 2.3 micrometers and over 100 nanometers. In one example, the laser sweeps wavelengths from about 2.2 micrometers to 2.4 micrometers.

[0052] In another example, the tunable laser 201 operates in the MIR and includes a quantum cascade gain chip, where the tunable laser sweeps a spectral band including 6,500 micrometers, preferably over 500 nanometers or over 1,000 nanometers, and in one example, sweeps from about 5.5 micrometers to 7.5 micrometers.

[0053] The PANDA fiber optic patch cable 110 acts to eliminate any higher-order spatial modes from the laser, minimizing ripple. However, due to fiber loss, a short length is desirable. Typically, the PANDA fiber optic patch cable 110 is between 0.5 meters and 3-4 meters in length. Additionally, a polarization means 152 addresses polarization-dependent losses in the optical components by forcing a single polarization state while eliminating any modal or random polarization fluctuations. A ripple reference detection means 158 further improves operation by addressing random power attenuation introduced by the PANDA fiber optic patch cable 110.

[0054] By polarizing the light with polarizing means 152, there is no risk of polarization dependent losses in the optical components. A portion of the polarized light is then detected by ripple reference light detection means 158. This ripple reference signal is sent back to the spectrometer 8 on spectrometer electrical wiring harness 116.

[0055] The remaining light (from the partially reflective sapphire window 154) travels in the free space path formed by the inner bore 131 of the inner tube 130. This light is coupled into the input waveguide 126 and exits through the optically transparent port 126P, where it propagates through the sample detection region 12. It exits the sample detection region 12 at the optical detection port 128P and propagates through the output waveguide 128. The light is then modified by the bioreactor medium in region 12, which absorbs some wavelengths preferentially over others, and is detected by the sample light detection means 132.

[0056] The spectrometer 8 control unit 200 monitors the response of the sample light detection means 132 and the ripple reference light detection means 158. That is, the control unit can resolve the sample absorption spectrum by monitoring the spectral scan across the scan band of the tunable laser 201 against the time response of the sample light detection means 132. Any noise associated with sources such as ripple from the optical fiber is compensated for by the response from the ripple reference light detection means. Typically, the tunable laser or tunable laser system sweeps its emission across a region or narrow band across a portion of the electromagnetic spectrum, such as the NIR and / or SWIR regions.

[0057] The control unit 200 uses the temperature detected by the thermistor 161 on the PCB 162 on the head side and the temperature detected by the thermistor 137 on the PCB 134 on the detection means side to compensate for changes in the response of the ripple reference light detection means 158 and the sample light detection means 132, as well as changes in the gain of the transimpedance amplifier on the PCB 162 on the head side and the transimpedance amplifier on the PCB 134 on the detection means side caused by temperature changes.

[0058] The probe optical housing 118 of the head 11 is configured to be detachable from the lower section 170 to which the outer tube 120 and inner tube 130 are connected, allowing the head to be removed and the remainder of the probe to be autoclaved.

[0059] FIG. 4 shows some of the structural details relating to the input and output waveguide rods 126 and 128.

[0060] Between parallel reflective surfaces, an etalon may be formed. Reflections occur at each surface due to a refractive index mismatch between the air in the free-space path of the beam within inner tube 130 and the bulk material of input waveguide 126. A refractive index mismatch typically also exists between the fluid in sample region 12 and input and output waveguides 126, 128. The beam also propagates in free space between output waveguide 128 and detection means 132. Even with anti-reflection coatings on surfaces, residual reflectivity still exists.

[0061] In a preferred embodiment, ports 126A, 126P, 128P, and 128A of input waveguide 126 and output waveguide 128 are sloped. Typically, the slope is a few tenths of a degree, with 0.125° shown. Preferably, the slope of input waveguide 126 and the slope of output waveguide 128 are rotated 90° relative to each other. In this example, ports 126A and 126P of input waveguide 126 are sloped from left to right on the page. Ports 128A and 128P of output waveguide 128 are sloped front to back on the page.

[0062] Other systems also require polarization control for optimal performance.

[0063] Figure 5 shows another monitoring system 300 with a clamshell design often used for offline monitoring. The addition of a flow cell also enables online monitoring. Details are described, for example, in U.S. Provisional Patent Application No. 63 / 423,856 (Attorney Docket No. 0376.0029PR1), filed November 9, 2022; U.S. Provisional Patent Application No. 63 / 469,502 (Attorney Docket No. 0376.00029PR2), filed May 29, 2023; and U.S. Provisional Patent Application No. 63 / 512,996 (Attorney Docket No. 0376.0031PR1), filed July 11, 2023, the entire teachings of which are incorporated herein by reference.

[0064] First portion 315 is secured or fixed to a base or support 327. In this embodiment, first portion 315 forms the lower or bottom portion of the open / close device. In this configuration, first portion 315 is suspended from support 327 and does not move or tip over. Rather, the opening and closing action is performed by second portion 319. In this embodiment, second portion 319 forms the upper, top, or lid portion. Other arrangements and orientations of these two portions are also possible.

[0065] The top portion 319, supported by supports 327, provides a clamshell or "flip-over" configuration that allows the device to be opened and closed. Various mechanisms can be used to move the top portion between the open and closed configurations. For example, the linkage 323 can be or include one or more elements known in the art, such as a jar, hinge, soft-close arrangement, rod, or lever. The top portion 319 is raised and lowered relative to the bottom portion 315 by knobs, rods, and soft-close mechanisms. In some approaches, a lip is provided that allows the portion 319 to be raised and lowered by a linkage with, for example, a hinge mechanism. In general, the linkage 323 can be constructed with elements and techniques known in the art.

[0066] In some embodiments, the two sections are directly connected by a connecting portion 323. For example, connecting portion 323 can be supported by the fixed first section and can include a mechanism that allows the second section to be attached and moved between the open and closed configurations. In other embodiments, additional components are used to support connecting portion 323 or components of connecting portion 323. For example, supports 327 can be used to position top portion 319 to a structure to ensure precise butting of the top and bottom sections in the closed configuration.

[0067] During operation, the sample interface portion 337 is exposed by opening the portion 319, and the sample, for example in the form of a droplet, is placed inside the portion 319, which is then closed like a lid, allowing the sample to be analyzed.

[0068] Light from the tunable laser in tunable laser spectrometer 8 is transmitted over optical fiber 110. Electrical signals from one or more of the photodetector means used may be collected and transmitted to tunable laser spectrometer 8 via a cable, such as one or more electrical harnesses. The tunable laser, together with the control unit, are typically part of tunable laser spectrometer 8.

[0069] The light beam generated by the tunable laser of spectrometer 8 is transmitted through optical fiber 110. The light then enters portion 315 at fiber port 114. In some embodiments, fiber patch cable 110 includes a PANDA fiber that transmits light from the tunable laser. Fiber port 114 can be configured as a collimator for light exiting the fiber optic patch cable and directed to polarizing means 152 (which can be used to filter out orthogonally polarized light). Rotation mount 153 allows polarizing means 152 to rotate in a plane perpendicular to both bench 150 and the optical axis of the beam exiting the fiber. In some embodiments, mount 153 allows fine rotational adjustment of polarizing means 152 to align it with the desired polarization axis of PANDA fiber 110.

[0070] The beam splitter includes a partially reflective sapphire window 154, such as a wedge-shaped window, held on a pitch-yaw mount 156. The window 154 is secured to the bench 150 by the pitch-yaw mount 156. The partially reflective sapphire window 154 reflects a portion of the beam, referred to herein as the "reference" beam, toward a ripple reference light detector 158, such as an In-GaAs detector. A focusing lens 160 couples the beam onto the active area of ​​the ripple reference detector 158. In addition to holding the sapphire window 154, the pitch-yaw mount 156 also adjusts the free-space beam reflected and transmitted through the window 154 so that it propagates toward the active area of ​​the ripple reference detector 158.

[0071] The ripple detection means is mounted on a head-side printed circuit board (PCB) 162. The head-side printed circuit board 162 includes a transimpedance amplifier that amplifies the electrical response of the ripple reference detection means. The head-side printed circuit board may be provided with a thermistor that enables temperature compensation of the detection means 158 and the transimpedance amplifier. The response of the ripple reference light detection means, together with the response of the thermistor, may then be transmitted as an electrical signal to the tunable laser spectrometer 8, for example, via an electrical connection in an electrical wiring harness arrangement (not shown).

[0072] In addition to the reference beam, the beam splitter generates a second beam portion, referred to herein as the "input" or "probe" beam, which propagates from the partially reflective sapphire window 154 toward the sample interface 337, specifically toward the sample detection region 12, which is formed by an optical transmission port and an opposing optical detection port. In this example, the optical transmission port is formed by a quartz or sapphire input rod 326, and the optical detection port is formed by a quartz or sapphire output rod 328. The rod-shaped waveguide arrangement described herein eliminates the need for a cuvette, a typical component of existing instrumentation.

[0073] In some embodiments, rod 326 is held by rod holder 331 and rod 328 is held by rod holder 333. Heating of one or both rod holders may occur under the control of the controller of spectrometer 8.

[0074] The input rod 326 is mounted on the sample platform 335, and the output rod 328 is part of the open / closed section 319 of the device. In the open position, a sample (or a blank for calibration purposes) can be introduced onto the rod 326 of the sample platform 335. By moving the section 319 to the closed position, the two rods are aligned. The distance between these ports determines the path of the incident (probe) beam within the sample detection region 12. A precision ball screw 375 extending from one end of the section 319 towards the sample platform 335 allows the rods to be offset to set the path to a constant length.

[0075] The path length between the ends of the rods 326, 328 that define the sample detection region 12 may be in the range of about 0.010 millimeters (mm) to about 5 mm, and in some cases up to about 10 mm, e.g., in the range of about 0.01 mm to about 0.05 mm, about 0.1 mm, about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm, e.g., in the range of about 0.05 mm to about 0.1 mm, about 0.5 mm, about 1 mm, Within the range of about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm, within the range of about 0.1 mm to about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm, within the range of about 0.5 mm to about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm mm, or up to about 10 mm, in the range of about 1 mm to about 2 mm, to about 3 mm, to about 4 mm, to about 5 mm, to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or up to about 10 mm, in the range of about 2 mm to about 3 mm, to about 4 mm, to about 5 mm, to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or up to about 10 mm, in the range of about 3 mm to about 4 mm, to about 5 mm, to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or up to about 10 mm, It can be selected from the range of about 5 mm to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or to about 10 mm, from about 5 mm to about 6 mm, to about 7 mm, to about 8 mm, to about 9 mm, or to about 10 mm, from about 6 mm to about 7 mm, to about 8 mm, to about 9 mm, or to about 10 mm, from about 7 mm to about 8 mm, to about 9 mm, or to about 10 mm, from about 8 mm to about 9 mm, or to about 10 mm, from about 9 mm to about 10 mm, etc.

[0076] An example path length is about 5 mm. Another example path length is 1 mm. For mid-infrared (MIR) systems, an example path length may be reduced to, for example, about 100 microns.

[0077] In a detailed embodiment, the diameter of rods 326, 328 is in the range of about 2 to about 8 millimeters (mm), e.g., about 4 mm. This configuration allows for surface tension to hold a droplet of about 15 microliters (mL), while allowing a beam of 0.5 to 3 mm, preferably about 1 mm, to travel through the rod and the sample in sample detection region 12. In many cases, both rods have the same diameter. In other cases, the rods have different diameters. In one example, the window in the base is enlarged to allow for cleaning.

[0078] The output rod 328 is optically coupled to a lensed sample light detector 132. The electrical signal registered by the light detector 132 is transmitted to the tunable laser spectrometer 8 via an electrical connection in a wire harness arrangement (not shown). In one example, the light detector is a TO-46 In-GaAs detector with a dome lens. The detector may be electrically connected to a sample detector printed circuit board (PCB) 134. The detector PCB may itself include a transimpedance amplifier that amplifies the detector response and transmits it to the tunable laser spectrometer 8. The detector PCB may include a thermistor to sense temperature and allow for temperature offset compensation by the controller.

[0079] In operation, swept wavelength light from the tunable laser of the spectrometer 8 is coupled into the fiber optic patch cable 110. The PANDA fiber optic patch cable operates to eliminate modes and minimize ripple. The short length of the fiber patch cable reduces or minimizes loss. Additionally, the polarization means 154 eliminates modes and random polarization fluctuations and accounts for polarization-dependent loss in optical components. The ripple reference detection means 158 further enhances operation by accounting for random power attenuation. Polarizing the light enforces a stable polarization state, eliminating the risk of polarization-dependent loss in optical components. A portion of the polarized light is detected by the ripple reference light detection means 158. This ripple reference signal is transmitted back to the tunable laser spectrometer 8, for example, via the spectrometer electrical wiring harness.

[0080] The remaining light travels towards input rod 326, couples into input rod 326 and exits through the light transmission port. It then propagates through sample detection region 12. It exits sample detection region 12 through the light detection port and propagates through output rod 328. The light is modified by the analyte in sample detection region 12 and detected by sample light detection means 132. The analyte will absorb some wavelengths preferentially over others.

[0081] In many cases, the light beam entering the gated device travels in free space (from one optical element to the next) and not just through an optical fiber.

[0082] The controller, which may be part of the tunable laser spectrometer 8, monitors the response of the sample light detector 132 and the ripple reference detector 158. That is, the controller can resolve the sample absorption spectrum by monitoring the spectral scan across the tunable laser scan band against the time response of the sample light detector 132. Any noise associated with sources such as ripple from the optical fiber is compensated for by the response from the ripple reference detector 158. Typically, the tunable laser or tunable laser system sweeps its emission across a region or narrow band across a portion of the electromagnetic spectrum, such as the NIR and / or SWIR regions.

[0083] The control unit of the spectrometer uses the temperature detected by the thermistor on the PCB on which the sample detection means 132 is provided and the temperature detected by the thermistor on the reference detection means 158 to compensate for changes in the response of the ripple reference light detection means 158 and the sample light detection means 132, as well as changes in the gain of the transimpedance amplifiers on these PCBs.

[0084] While the present invention has been particularly shown and described with reference to preferred embodiments, those skilled in the art will recognize that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A monitoring system, a tunable laser spectrometer that generates a swept wavelength signal and transmits it over an optical fiber; a reference light detection means for detecting the swept wavelength signal from the tunable laser spectrometer after transmission through the optical fiber; a sample light detection means for detecting the swept wavelength signal after passing through the sample detection region; A system comprising:

2. 10. The system of claim 1 further comprising: a polarization means for improving the polarization state of the swept wavelength signal before the reference light detection means; A system comprising:

3. 3. The system of claim 1 or 2, wherein the optical fiber comprises a polarization-maintaining fiber.

4. 4. The system of claim 3, wherein the polarization-maintaining fiber is a single-mode polarization-maintaining fiber.

5. 5. The system of claim 1, wherein the sample interface includes a light transmission port and a light detection port having at least one beveled surface.

6. 6. The system of claim 5, wherein the light transmission port and the light detection port are provided by input and output waveguide rods.

7. 7. The system of claim 1, wherein the tunable laser spectrometer sweeps wavelengths within a spectral band including 2.3 micrometers and / or 6.5 micrometers.

8. 8. The system of claim 1, wherein the tunable laser spectrometer sweeps over more than 100 nanometers.

9. 9. The system of claim 1, wherein the tunable laser spectrometer sweeps wavelengths over wavelengths on the order of 2.2 micrometers to 2.4 micrometers.

10. The system of any one of claims 1 to 9, further comprising: a probe inserted into the bioreactor; A system comprising:

11. The system of any one of claims 1 to 9, further comprising: a sample interface for receiving a droplet of sample removed from the bioreactor; A system comprising:

12. 12. The system of claim 1, further comprising: a control unit that monitors the response of the sample light detection means and the reference light detection means to resolve the absorption spectrum of the sample in the sample detection region; A system comprising:

13. 13. The system of claim 12, wherein the control unit compensates for noise associated with ripples from the optical fiber.

14. 14. The system according to claim 12, wherein the control unit performs compensation based on temperatures detected by one or more thermistors provided in the reference light detection means and the sample light detection means.

15. 15. A system according to any one of claims 1 to 14, wherein no optical fibre is provided after the reference detection means to the sample light detection means.

16. A monitoring system, a tunable laser spectrometer that generates a swept wavelength signal; a reference light detection means for detecting the swept wavelength signal from the tunable laser spectrometer; a polarization means for improving the polarization state of the swept wavelength signal before the reference light detection means; a sample light detection means for detecting the swept wavelength signal after passing through the sample detection region; A system comprising:

17. 17. The system of claim 16, further comprising:

16. A configuration according to any one of claims 2 to 15, A system comprising:

18. A probe for a bioreactor, comprising: a port forming a sample detection region within the bioreactor; a reference light detection means for detecting a light signal received from the polarizing means and prior to the sample detection region; a sample light detection means for detecting the optical signal after passing through the sample detection region; A probe comprising:

19. 1. A method for online or offline monitoring of a bioreactor, comprising: generating a swept wavelength signal; delivering the swept wavelength signal to a sample detection region of a sample in the bioreactor; polarizing the swept wavelength signal; detecting the swept wavelength signal after polarization and before transmission through the sample detection region; detecting the swept wavelength signal after transmission through the sample detection region; resolving the absorption spectrum of the sample in the sample detection region with reference to the swept wavelength signals before and after transmission through the sample detection region; A method comprising:

20. 17. The method of claim 16, carried out using a system according to any one of claims 1 to 17.

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