Improved sample holder

Abstract

The present invention relates to a sample holder for use in molecular absorption spectroscopy. The sample holder comprises a first surface having a first predetermined geometric shape and a second surface having a second predetermined geometric shape. The first surface opposes the second surface. The sample holder is configured to hold a measurement sample between the first surface and the second surface such that the distance between the first surface and the second surface defines an optical path length of the sample holder. The predetermined geometries of the first and second surfaces provide a continuously varying cross-section across the sample holder to provide a continuous range of optical path lengths.

Description

[Technical field]

[0001] The present invention relates to an improved sample holder. In particular, embodiments of the present invention relate to a sample holder for use in molecular absorption spectroscopy, a spectrophotometer including the sample holder, and a method of using the spectrophotometer to analyze a measurement sample placed in the sample holder. [Background technology]

[0002] In the field of absorption spectroscopy, optical absorption spectra of liquid substances are measured. Absorption spectra are a measurement of light attenuation as a function of light wavelength. In a simple spectrophotometer, the measurement sample is placed in a transparent container commonly known as a cuvette, sample cell, or sample holder. Light of known wavelength and intensity is incident on one side of the sample holder, and a detector measures the intensity of the light exiting the sample holder. The shape of the sample holder defines the distance the light travels through the sample. This distance is called the optical pathlength of the sample holder. Generally, the light transmitted through the measurement sample follows a known relationship such that the properties of the measurement sample can be determined based on the absorption spectrum. In other words, if the absorption spectrum of a given substance is known, its presence and concentration in the measurement sample can be determined.

[0003] In many cases, compounds of interest in solution are highly concentrated. For example, certain biological samples, such as proteins, DNA, or RNA, are often present at concentrations outside the linear range of the spectrophotometer when absorbance is measured. Thus, dilution of the sample is often required to measure absorbance values ​​that are within the linear range of the instrument. Traditionally, concentrated samples are diluted before analysis to obtain suitable absorbance (often in the range of 0.2 absorbance units to 1.2 absorbance units) at optical path lengths of about 1 mm to 10 mm. In some instances, the need to dilute samples multiple times can introduce human error and inaccuracies in measurements and downstream applications. Furthermore, the need to perform additional dilution steps makes measurements time- and labor-intensive. Therefore, it is desirable to take existing samples without knowing the expected concentration and measure the absorbance of these samples without dilution.

[0004] Traditionally, the optical path length associated with a spectrophotometer's sample holder is fixed. In recent years, spectrometers and sample handling techniques have been developed that allow sample measurements to be performed at two or more optical path lengths to achieve absorbance values ​​within a measurable range.

[0005] For example, the CTech® SoloVPE™ system allows for measurement of sample absorbance over variable path lengths by providing a fiber optic probe that is vertically movable within a sample holder. The probe provides light to measure a sample within the sample holder. A detector is positioned to receive light emanating from the probe that is transmitted through the sample. Movement of the probe through the sample within the sample holder effectively provides a variable path length for analysis.

[0006] However, slowly moving the probe through the sample relative to the detector to achieve accurate and viable measurements can be a time-consuming process. Additionally, the CTech® SoloVPE® system moves the probe to take measurements one at a time for each path length and calculates the sample properties based on regression analysis. In many cases, it can be difficult to achieve an optimal signal-to-noise ratio for the measurements.

[0007] Probe movement may also cause instability in the intensity of the optical radiation emitted from the probe, affecting the accuracy of measurements for samples with low absorbance. In addition, the probe is associated with a drive assembly that includes a motor for affecting the linear movement of the probe. The ability of the system to provide accurate and repeatable measurements is dependent on the accuracy and reproducibility in the operation of the drive assembly. Inevitably, the movement of mechanical components in the drive assembly creates inaccuracies due to, for example, hysteresis, thermal expansion, friction, clearance movement, and various motor-related errors. The drive assembly is also subject to wear and tear. Thus, frequent inspection and maintenance is required to maintain an acceptable level of accuracy. Furthermore, for low concentration samples, relatively long path lengths (e.g., up to 15 mm) must be used, requiring relatively large sample volumes. This may be a disadvantage when samples are in very short supply and / or very expensive (i.e., very scarce and / or very expensive).

[0008] In another example, the NanoDrop® spectrophotometer provides a sample holder that holds a measurement sample between two opposing surfaces. The two opposing surfaces can move relative to one another to effectively provide a change in optical path length. However, relying on moving parts to provide a change in path length has inherent limitations in measurement accuracy and repeatability. In a further example, Unchained Labs' Lunatic® spectrophotometer provides a sample cell with two path lengths to carry the sample. The sample cell provides a discrete change in cell thickness in the measurement direction, thereby providing two different discrete path lengths. In both examples, only a limited number of discrete path lengths are provided. Furthermore, it can be difficult to achieve acceptable accuracy in a particular measurement, especially at short path lengths (e.g., often in the 50 μm to 100 μm range). For a 50 μm path length, an error of less than 500 nm is required to achieve 1% accuracy, which can be very difficult to achieve. Summary of the Invention [Problem to be solved by the invention]

[0009] Embodiments of the present invention may provide a sample holder, spectrophotometer, and method of operation that overcomes or ameliorates one or more of the above-mentioned shortcomings or problems, or at least provides a useful choice for consumers.

[0010] The reference in this specification to a patent document or any other matter identified as prior art should not be construed as an admission that the document or other matter was known or that the information contained therein was part of the common general knowledge at the priority date of any of the claims. [Means for solving the problem]

[0011] According to one aspect of the invention there is provided a sample holder for use in molecular absorption spectroscopy, comprising: a first surface having a first predetermined geometric shape; a second surface having a second predetermined geometric shape, the first surface being opposed to the second surface; and Equipped with the sample holder is configured to hold a measurement sample between a first surface and a second surface such that a distance between the first surface and the second surface defines an optical path length of the sample holder; A sample holder is provided where the predetermined geometry of the first and second surfaces provides a continuously variable cross-section across the sample holder to provide a continuous range of optical path lengths.

[0012] The sample holder can be configured to hold any suitable measurement sample having any suitable consistency. For example, the measurement sample can have any viscosity. Typically, the sample is a liquid sample.

[0013] The first predetermined geometric shape can be the same as the second predetermined geometric shape. Alternatively, the first predetermined geometric shape can be different from the second predetermined geometric shape.

[0014] The predefined geometry of the first and second surfaces provides a continuously varying cross-section across the sample holder which advantageously provides a continuous range of optical path lengths without the need for moving parts in contact with the measurement sample, thereby avoiding inaccuracies associated with such moving parts.

[0015] The continuously varying cross-section can comprise a first measurement zone where a minimum distance between the first and second surfaces defines a minimum optical path length and a second measurement zone where a maximum distance between the first and second surfaces defines a maximum optical path length. The continuously varying cross-section can provide a continuous range of optical path lengths between the first and second measurement zones.

[0016] In some embodiments, the first surface can contact the second surface to provide a minimum optical path length of zero in the first measurement zone. In other embodiments, the first surface cannot contact the second surface and the minimum optical path length can be close to zero. Thus, the sample holder can advantageously provide a continuous range of optical path lengths from zero to any desired maximum path length. In practice, positioning the first and second surfaces of the sample holder to have a point of contact or near a point of contact as described herein provides precise positioning of the surfaces relative to one another and precise control of the non-zero path length.

[0017] The first and second surfaces can have any suitable geometric shape to provide the desired continuously varying cross-section of the sample holder. For example, one or both of the first and second surfaces can be curved. The curvature of one or more surfaces can follow a regular or irregular predetermined geometric shape. Typically, the first surface can be curved. The second surface can be flat or substantially flat.

[0018] In some embodiments, one or both of the first predetermined geometric shape and the second predetermined geometric shape are: A sphere and A parabolic cylinder and A circular paraboloid; An elliptical paraboloid; Any smooth aspheric surface It is defined by one of the following:

[0019] In sample holders according to embodiments of the present invention, a continuous range of optical path lengths can be provided that provide optical path length variations on the order of about 1 μm to 3 mm, which advantageously allows high resolution and accurate sample measurements to be performed at high speed.

[0020] The sample holder can thus provide a continuous range of spatially varying optical path lengths from 0 μm to any maximum path length with path length variations on the order of about 1 μm without any moving parts in contact with the measurement sample. The sample holder can thus provide a measurable path length in at least one region of the sample holder for a sample of any unknown concentration, or any concentration within a wide range of acceptable concentrations.

[0021] Furthermore, because the sample holder enables measurement of all path lengths within a continuous range of path lengths, the need for an operator to manually select a range of measurable path lengths is avoided, thereby improving work efficiency and reducing human error.

[0022] In practice, the sample holder may include a plano-convex lens having curved sides that provide the first surface, which may be movable between an open position in which the first surface is separated from the second surface and a closed position in which the first surface is in contact with and positioned opposite the second surface.

[0023] Typically, in the closed position, the position of the first surface is fixed relative to the second surface and a variable distance between the first and second surfaces is created by their respective predetermined geometric shapes.

[0024] The sample holder can be made from any suitable material or combination of different suitable materials. In some embodiments, the sample holder can be disposable. In these embodiments, the sample holder can be made from a plastic material. In other embodiments, the sample holder can be reusable and made from a glass material.

[0025] In some embodiments, the sample holder can be a flow cell having an inlet and a vent that allows injection of a measurement sample into the sample holder via the inlet. In these embodiments, the first and second surfaces of the sample holder can be flexible, thereby allowing cleaning of the first and second surfaces, for example, via flushing the sample holder.

[0026] Advantageously, sample holders according to embodiments of the present invention enable rapid and accurate quantification of concentrations (e.g., typically protein concentrations) over a wide dynamic range (e.g., over four orders of magnitude) using only very low sample volumes (e.g., a few microliters) without the need for any moving parts in contact with the sample.

[0027] Because the sample holder of the present invention is capable of providing path length variations on the order of 1 μm over a continuous range of path lengths starting from zero or near zero, the sample holder also allows low volume samples to be accurately measured.

[0028] According to another aspect of the present invention, there is provided a spectrophotometer having a sample holder as described herein.

[0029] The spectrophotometer can have any suitable configuration, and the sample holder can be positioned in any suitable location within the spectrophotometer.

[0030] In some embodiments, the spectrophotometer can operate in the ultraviolet-visible (UV-VIS) range, in some embodiments in the near-infrared (NIR) range or in the infrared (IR) range.

[0031] The spectrophotometer may further include a detector to detect the absorbance of the measurement sample at each of a continuous range of optical path lengths. Any suitable detector may be used. In some embodiments, the detector is a: Single point detector, 1D array detector, or 2D array detector It can be any one of the following:

[0032] The spectrophotometer may further include a light source. Any suitable light source may be used. In some embodiments, the light source is: A broadband light source; LEDs and Laser and It may include any one of the following:

[0033] In some embodiments, the light source provides light simultaneously across the entire measurement sample. In these embodiments, the light source can be fixed. In a spectrophotometer with a stationary source, no moving parts are required across the instrument. This further provides the benefits associated with higher measurement speed while maintaining high accuracy in the measurement. Additionally, no maintenance of moving parts is required, thereby reducing downstream costs.

[0034] The spectrophotometer can have any suitable configuration, and the sample holder can be positioned in any suitable location relative to the light source and detector within the spectrophotometer.

[0035] The spectrophotometer may further include a mask positioned between the light source and the sample holder, such that light passing through the mask projects a predetermined shadow pattern for detection by the detector, and the spectrophotometer is configured to calibrate effects caused by refraction at one or both of the first and second surfaces of the sample holder based on the detected shadow pattern.

[0036] In some embodiments, the spectrophotometer can further include one or more optical elements. The one or more optical elements can be located in any suitable location relative to the sample holder, the light source, and the detector. The one or more optical elements can be any one or more of a light dispersing element, a light focusing element, or the like. For example, the optical element can include one or more mirrors, lenses, diffraction gratings, prisms, crystals, fibers, waveguides, or any combination thereof.

[0037] In some embodiments, the light source can provide a light beam that is movable relative to the measurement sample to scan the measurement sample such that a transmission value is detectable sequentially for each path length within the range of path lengths. In one embodiment, the light beam moves from one end of the sample holder to an opposite end of the sample holder. In other embodiments, the light beam can move in a circular motion.

[0038] The spectrophotometer may further include a temperature controller for controlling the temperature of the measurement sample.

[0039] According to a further aspect of the invention, there is provided a computer-implemented method of analyzing a measurement sample disposed in a sample holder as described herein using a spectrophotometer, the spectrophotometer including a light source that emits light through the measurement sample and a detector that detects the intensity of light transmitted through the measurement sample, the method comprising: obtaining a detected transmittance value corresponding to each optical path length of the sample holder, the detected transmittance value being based on one or more light intensity measurements from a detector; calculating an estimated transmission value corresponding to each optical path length of the sample holder; determining an attenuation coefficient of the measurement sample that minimizes the error between the detected transmittance value and the estimated transmittance value; Includes.

[0040] The step of calculating the estimated transmittance value comprises: calculating an estimated attenuation coefficient of the measurement sample based on the corresponding detected transmittance values; calculating an estimated transmittance value based on the estimated attenuation coefficient; may include.

[0041] Determining the attenuation coefficient of the measurement sample may be an iterative process.

[0042] The method may further include determining a value for each optical path length as a function of the refractive index of the measurement sample.

[0043] According to yet another aspect of the invention, there is provided a computer-implemented method for analyzing a measurement sample disposed in a sample holder as described herein using a spectrophotometer including a light source that emits light through the measurement sample, a detector that detects the light intensity transmitted through the measurement sample, and a higher spatial frequency modulation pattern associated with the light source such that high spatial frequency features can be detected by the detector. The method includes: providing a predetermined modulation model that predicts optical intensity values ​​at the detector corresponding to each refractive index within a predefined range of refractive indices and corresponding to each optical path length; obtaining a detected transmittance value corresponding to each optical path length, the detected transmittance value being based on one or more light intensity measurements from a detector; calculating an estimated transmittance value of the measurement sample corresponding to each optical path length and each refractive index based on a predetermined modulation model; determining an attenuation coefficient of the measurement sample that minimizes the error between the detected transmittance value and the estimated transmittance value; may include.

[0044] According to another aspect of the invention, there is provided a computer-implemented method for analyzing a measurement sample disposed in a sample holder as described herein using a spectrophotometer including a light source that emits light through the measurement sample, a detector that detects an illumination signal representative of the light intensity transmitted through the measurement sample, and a higher spatial frequency modulation pattern associated with the light source such that high spatial frequency features can be detected in the detector illumination signal. The method includes: filtering the illumination signal to obtain first filtered illumination data in which high spatial frequency features are extracted from the illumination signal and second filtered illumination data in which high spatial frequency features are removed from the illumination signal; calculating a refractive index value based on the first filtered illumination data; obtaining a detected transmittance value corresponding to each optical path length, the detected transmittance value being based on the second filtered illumination data; calculating an estimated transmittance value for the measurement sample corresponding to each optical path length based on the calculated refractive index; determining an attenuation coefficient of the measurement sample that minimizes the error between the detected transmittance value and the estimated transmittance value; may include.

[0045] According to yet another aspect of the present invention, one or more tangible, non-transitory computer-readable media having computer-executable instructions for performing the computer-implemented methods described herein are provided.

[0046] According to a further aspect of the present invention there is provided a method of manufacturing a sample holder as described herein, the method comprising the steps of: forming a first portion of a sample holder to provide a first surface; forming a second portion of the sample holder to provide a second surface; Attaching the first part to the second part such that the first part is movable relative to the second part between an open position allowing placement or removal of a measured sample and a closed position in which the first surface is in a fixed position relative to the second surface and provides a continuously varying cross-section across the sample holder; may include.

[0047] According to a further aspect of the present invention there is provided a method of manufacturing a sample holder as described herein, the method comprising the steps of: forming a first portion of a sample holder to provide a first surface; forming a second portion of the sample holder to provide a second surface; attaching the first portion to the second portion such that the first surface is fixed relative to the second surface to provide a continuously varying cross-section across the sample holder; forming an opening to allow injection of a measurement sample into the cavity between the first portion and the second portion; Includes.

[0048] In order that the invention may be more readily understood and put into practice, one or more preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0049] [Figure 1A] FIG. 2 is a schematic diagram showing a partial cross-section of a sample holder according to an embodiment of the present invention. [Figure 1B] FIG. 2 is a schematic diagram showing a partial cross-section of a sample holder according to another embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram of a spectrophotometer according to one embodiment of the present invention. [Diagram 3] FIG. 2 is a schematic diagram of a spectrophotometer according to another embodiment of the present invention. [Figure 4] FIG. 4 is a schematic diagram of a spectrophotometer according to a further embodiment of the present invention; [Diagram 5]FIG. 1 is a flow diagram illustrating a method of analyzing a sample using a spectrophotometer according to an embodiment of the present invention. [Figure 6A] 1B is an XY plot showing pre-calculated sample path lengths between a first surface and a second surface of the sample holder shown in FIG. 1A. [Figure 6B] 1B is an XY plot showing relative sample transmittance using the sample holder of FIG. 1A for samples having different absorbances. The relative sample transmittance is plotted versus multiple pixel locations across a one-dimensional array detector. [Figure 7A] 1B is an XYZ plot showing relative sample transmittance using the sample holder of FIG. 1A. The relative sample transmittance is plotted versus multiple pixel locations across a two-dimensional array detector. [Figure 7B] 7A-7C are XYZ diagrams showing relative sample transmittance using the sample holder of FIG. 1A. The sample corresponding to FIG. 7B has higher absorbance compared to the sample of FIG. 7A. The relative sample transmittance is plotted for multiple pixel locations across a two-dimensional array detector. [Figure 8] 1 is an XY plot showing measured and predicted transmittance values ​​after parameter optimization of attenuation coefficient and relative light source intensity. [Figure 9] FIG. 1 is a flow diagram illustrating a method of analyzing a sample using a spectrophotometer according to an embodiment of the present invention. [Figure 10A] 3 is a line graph showing the intensity variation on an array detector when the source illumination is modulated by a mask that produces regularly spaced thin shadows according to the spectrophotometer configuration shown in FIG. 2. [Figure 10B] 1 is a line graph showing the expected displacement of high spatial frequency features on an array detector due to changes in refractive index. [Figure 11A] 1 is a line graph showing the output from low pass filtering to remove high spatial frequency features from the original detector signal. [Figure 11B] 1 is a line graph showing the output from low pass filtering to remove high spatial frequency features from the original detector signal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] 1A shows a partial cross-sectional view of a sample holder 100 for use in molecular absorption spectroscopy according to one embodiment of the present invention. The sample holder 100 has a first portion 112 providing a first curved surface 102 having a first predetermined geometric shape and a second portion 114 providing a second substantially flat surface 104 having a second predetermined geometric shape. Although not specifically shown in the drawings, the first predetermined geometric shape of the first curved surface 102 may be any one of a sphere, a parabolic cylinder, a circular paraboloid, and an elliptical paraboloid.

[0051] One advantage of the symmetrical nature of these geometries is that any phenomena such as air bubbles in the sample 201 or insufficient filling of the cavity between the first surface 102 and the second surface 104 can be easily detected so that appropriate measures can be taken to correct any errors. Furthermore, in embodiments where the first curved surface 102 is spherical, any variation in the angle between the first portion 102 and the second portion 104 of the sample holder 100 does not affect operation due to the symmetrical nature of the spherical geometry.

[0052] The sample holder 100 is configured to hold a measurement sample 201 (see also FIG. 2 ) between the first surface 102 and the second surface 104 such that a distance d ( FIG. 1A ) between the first surface 102 and the second surface 104 defines a plurality of different optical path lengths 106 of the sample holder 100. In particular, the predetermined geometry of the first surface 102 and the second surface 104 provides a continuously varying cross-section across the sample holder 100 so as to provide a continuous range of different optical path lengths 106.

[0053] 1A, the continuously varying cross-section of the sample holder 100 includes a first measurement zone 108 in which a minimum distance between the first surface 102 and the second surface 104 defines a minimum optical path length 106. The minimum optical path length 106 can be zero, in which case the first surface 102 contacts the second surface 104 in the first measurement zone 108. Alternatively, the minimum optical path length 106 is a small value close to zero, in which case the first surface 102 does not contact the second surface 104 in the first measurement zone 108. The continuously varying cross-section of the sample holder 100 further includes a second measurement zone 110 in which a maximum distance between the first surface 102 and the second surface defines a maximum optical path length 106. A continuous range of different optical path lengths 106 is provided ranging from a minimum path length 106 in a first measurement zone 108 to a maximum path length 106 in a second measurement zone 110 .

[0054] The sample holder 100 is symmetrical about a central plane (not shown) that intersects the first measurement zone 108, such that a first set of continuous ranges of optical path lengths 106 are provided on one side of the central plane, and a second set of continuous ranges of optical path lengths 106 that match the first set are provided on the other side of the central plane. In embodiments in which the first curved surface 102 has a regular geometric shape (such as a sphere, a parabolic cylinder, a circular paraboloid, or an elliptical paraboloid), an infinite set of continuous ranges of matching optical path lengths can be provided. Typically, the continuous ranges of optical path lengths within each set can provide optical path length variations on the order of about 1 μm to 3 mm. The ability to provide small path length variations in the continuous ranges of path lengths allows suitable measurement ranges to be determined for a wide range of different sample concentrations, and highly accurate measurements can be performed within the suitable measurement ranges.

[0055] 1A, it is understood that the first portion 112 can be movably attached or fixedly attached to the second portion 114. Alternatively, the first portion 112 can be separate from the second portion 114. The sample holder 100 can be made from any suitable material, including one or more plastic and / or glass materials (i.e., a plastic material or a glass material or both).

[0056] Advantageously, it is possible to produce a continuous range of optical path lengths 106 with very high accuracy, since the accuracy of the optical path length depends only on the accuracy of the geometry of first surface 102 and second surface 104. Using current optical manufacturing techniques, high accuracy can be achieved for a given surface geometry.

[0057] In use, light 113 from light source 208 (see FIG. 2) is incident on one side of sample holder 100. The light beam 113 is transmitted through sample holder 100 and measurement sample 201 and is incident on detector 202. As will be explained in more detail below with reference to FIGS. 2-4, the transmitted light detected by detector 202 at different wavelengths can be used to construct an absorption spectrum of measurement sample 201.

[0058] Typically, near the center (or first measurement zone) 108 of the sample holder 100, where the path length is close to zero, the light beam 108 is barely attenuated. At the edge (or second measurement zone 110), where the path length is greatest, the light beam 108 is more strongly attenuated (see FIG. 6B).

[0059] In practice, the light 110 transmitted through the sample holder 100 and the measurement sample 201 is subject to refraction 116. In some embodiments, it is necessary to take into account the effect of refraction of the light 110 by determining a value of the refractive index when analyzing the sample 201. In these embodiments, the refractive index of the sample 201 influences the intensity pattern or absorption spectrum detected by the detector 202. As shown in FIG. 2, a mask 210 can be provided to project a known shadow pattern on the surface of the detector 202. Changes in this shadow pattern can be detected to calculate the refractive index. This can be used to correct the absorption spectrum or as an additional analysis output, or both. Calculations regarding the refractive index are explained in more detail below with reference to FIGS. 9 to 11B.

[0060] FIG. 1B shows a partial cross-sectional view of a sample holder 120 according to another embodiment of the invention. The sample holder 120 has a curved first surface 122 and a substantially flat second surface 124. The first surface 122 is any smooth aspheric surface. Like numbers in FIG. 1B refer to like features previously described with reference to FIG. 1A. The predetermined geometry of the first surface 122 and the second surface 124 provides a continuously varying cross-section across the sample holder 120 to provide a continuous range of different optical path lengths 106.

[0061] FIG. 2 shows a spectrophotometer 200 according to one embodiment of the present invention. In general, the spectrophotometer is configured to operate in the ultraviolet-visible (UV-Vis) range. The spectrophotometer 200 includes a sample holder 100 according to the partial cross-section shown in FIG. 1A. In the spectrophotometer 200, the sample holder 100 includes a first portion 112 and a plano-convex lens 204 providing a first curved surface on its underside. The sample holder 100 also includes a substantially flat window 206 providing a second portion and a second substantially flat surface on the upper side of the window 206. The plano-convex lens 204 touches or nearly touches the window 206 at a central portion of the lens 204. The measurement sample 201 is placed between the lens 204 and the window 206.

[0062] In the spectrophotometer 200, light 113 from the light source 208 is projected onto a mask 210 to generate a higher spatial frequency modulation pattern in the source illumination. The mask 210 generates a shadow pattern on the detector 202 to facilitate calculation of the refractive index. Changes in the shadow pattern are detectable, and the refractive index can be estimated based on the measured changes in the shadow pattern. The absorptivity of the sample is the imaginary part of the refractive index. As described in more detail below with reference to Figures 9-11B, the real part of the measured refractive index can be used to correct for the absorptivity of the sample.

[0063] In the particular configuration shown in Figure 2, focusing elements 212 and 213 image mask 210 through sample holder 100 and measurement sample 201 onto detector 202. Detector 202 detects light 113 transmitted through sample holder 100 and measurement sample 201. The transmittance detected by detector 202 is processed, for example by a processor, to determine the absorption spectrum and other characteristics of measurement sample 201. Although not shown in Figure 2, it is understood that the processor can be any suitable processor built into the hardware of spectrophotometer 200 or provided separately from spectrophotometer 200.

[0064] Additionally, in other specific embodiments, different configurations are possible for spectrometer 200 without departing from the scope of the present invention. For example, in some embodiments, a wavelength selective element may be provided in the optical path before sample holder 100. In some embodiments, a monochromator may be provided in the optical path upstream of sample 201. In some embodiments, a polychromator may be provided in the optical path downstream of sample 201.

[0065] Furthermore, the position of lens 204 relative to detector 202 is not important, since the center of the symmetrical pattern of the absorption spectrum can be easily determined as a reference. In fact, sample holder 100 can be positioned in any suitable location relative to light source 208 and detector 202 within spectrophotometer 200.

[0066] Typically, the detector 202 is a two-dimensional (2D) array detector. In practice, the detector 202 can include a charge coupled device (CCD) array detector, a complementary metal oxide semiconductor (CMOS) sensor array detector, or a microbolometer array detector. In some embodiments, a one-dimensional (1D) array detector can be used.

[0067] In some embodiments, the light source 208 can provide white light, and wavelength selectivity can be provided by a polychromator after the measurement sample 201. This configuration can be used to create an output of transmittance versus wavelength and path length, for example, on a 2D array detector where the wavelength is dispersed in one dimension and the path length variations are spread across the other dimension.

[0068] In some embodiments, LEDs (260 nm, 280 nm, 320 nm) can be used for the light source 208. In these embodiments, no additional wavelength selectivity is required to perform the sample analysis.

[0069] In some embodiments, a pre-dispersion element can be provided in the optical path before the measurement sample 201. In some embodiments, a post-dispersion element can be provided in the optical path after the sample 201.

[0070] Typically, for a highly absorbing sample 201, the outer parts of the absorption image typically have very little light and a poor signal-to-noise ratio. In some embodiments, an optimized weighting function can be applied in the calculation such that parts of the absorption spectrum image where there is enough light to give a good signal-to-noise ratio are included and poor regions are excluded.

[0071] Furthermore, the detector 202 does not need to operate at very low light levels, since for any sample 201 in the sample holder 100, the associated absorption spectrum image will always include regions with relatively high light levels.

[0072] 2, the light source 208 is fixed and provides light simultaneously across the entire measurement sample 201. With spectrophotometer 200, no moving parts are required throughout the instrument.

[0073] In an alternative embodiment as shown in FIG. 3, the light source 302 of the spectrophotometer 300 provides a light beam 304 that is movable relative to the measurement sample 201 to scan the measurement sample 201 from one end of the sample holder 100 to the opposite end of the sample holder 100. With this configuration, the detector 202 sequentially detects the transmittance values ​​for each path length within the range of path lengths as the light source 302 moves from one end of the sample holder 100 to the other. The spectrophotometer 300 shown in FIG. 3 can be used to operate in the near-infrared (NIR) range or the infrared (IR) range. By using the movable light source 302 as shown in FIG. 3, the light beam 304 can be set substantially perpendicular to the surface 122 and the sample 201, thereby reducing the effect of the refractive index.

[0074] In general, infrared measurements present challenges that typically do not arise in the UV-VIS spectral range. Of particular importance is the high attenuation of some wavelength ranges by water. For example, at 1650 cm -1 The protein amide I band at 1653 cm -1The absorbance band of the analyte is significantly overlapped with the water absorbance band centered at . This means that to be able to detect low concentrations of protein, the spectrophotometer 300 must be able to resolve very small changes in absorbance due to the analyte in the presence of large absorbance due to the solvent (water). The repeatability of the geometry of the first surface 102 and the second surface 104 in the sample holder 100 allows for accurate subtraction of the reference measurement independent of the spacing between the surfaces 102, 104.

[0075] In another embodiment, to achieve measurements where the path length varies with time, the light source 302 is stationary and the sample holder 100 is movable to allow the light beam 304 to move from one end of the sample holder 100 to the other. In the above embodiments, a single point detector can be used to detect the transmission of light through the sample 201. In one example, the single point detector can be a mercury cadmium telluride (MCT) detector. In some embodiments, the light source 302 can be radially aligned with the single point detector.

[0076] In practice, the movement of the sample holder 100 may be relatively small, for example on the order of a few millimeters. In some embodiments, the movement of the sample holder 100 may be achieved using a flexure mechanism. In some embodiments, the detector 202 may be movable relative to the sample 201.

[0077] In a further alternative embodiment, to operate in the IR or NIR range, the spectrophotometer can be configured to allow a comparison between two samples. In particular, the absorption of a solvent-only sample can be compared to a sample containing solvent and analyte. The spectrometer can include two identical sample holders to hold the two samples, or a single sample holder with two cavities to hold the two samples. The comparative analysis can be performed simultaneously using a double beam configuration, in which a light beam is projected simultaneously onto both samples. Alternatively, a single sample holder can be provided to hold the two samples sequentially.

[0078] In these embodiments, the spectrophotometer may include a temperature controller that controls the temperature of the measurement sample. In particular, the temperature controller may be used to ensure that the temperature of the sample is substantially constant during the measurement. Changes in temperature can often cause changes in the absorbance of the solvent. Therefore, errors related to temperature changes may be minimized by using a temperature controller to maintain a substantially constant temperature in the measurement sample. In particular, a temperature controller may be provided to maintain a substantially constant temperature spatially across the sample when measurements of the two comparative samples are performed simultaneously. Alternatively, a temperature controller may be provided to maintain a substantially constant temperature temporally for the sample when measurements of the two comparative samples are performed sequentially.

[0079] In practice, a quantum cascade laser (QCL) can be used as the light source 302. A QCL is a powerful source of monochromatic IR radiation. A QCL is a relatively compact light source that can be efficiently coupled into an optical system. The relatively high power allows relatively efficient results to be achieved. Furthermore, the detector requirements are less critical, thus reducing potential costs and complexity. For example, instead of a cryogenic temperature detector, a room temperature or thermoelectrically cooled detector can be used. In some applications, only a few wavelengths are of interest. The use of a QCL source is particularly suitable for such applications.

[0080] In some embodiments, the detector 202 may be a microbolometer array detector. In other embodiments, the detector 202 may be a 1D or 2D mercury cadmium telluride detector.

[0081] FIG. 4 shows a spectrophotometer 400 according to a further embodiment of the present invention. In the spectrophotometer 400, a sample holder 402 includes a plano-convex lens 204 providing a curved first surface on its underside and a mirrored sample slide 404 providing a substantially flat second surface on its upper side. The sample holder 402 is adapted to hold a measurement sample 201 between the first and second surfaces. In the spectrophotometer 400, a light beam from a light source 406 is redirected onto the sample holder 402 and the sample 201 via a focusing mirror 408. The mirrored sample slide 404 reflects the light back through the sample 201 and the plano-convex lens 204 onto an imaging mirror 410, which focuses the reflected light onto a detector 412 positioned on the same side of the sample 201 as the light source 406. The effective optical path length detected in the embodiment shown in FIG. 4 is approximately doubled because the light passes through the measurement sample 201 twice. Optionally, a mask 403 can be provided to create a shadow pattern on the detector 412 to facilitate calculations for the refractive index, as will be explained in more detail below.

[0082] A method for calculating the attenuation coefficient of an analyte in a measurement sample 201 using a sample holder and a spectrophotometer according to an embodiment of the present invention will now be described below with reference to FIGS. 5-11B. Typically, the method described below is a computer-implemented method that runs automatically on a computer processor, provided together with or separately from the spectrophotometer. The computer processor may include a software application installed thereon to perform one or more of the steps of the computer-implemented method. In an alternative embodiment, the software application may be a cloud-based application accessible via a network such as the Internet. In some embodiments, the software application may be accessible remotely via a local network.

[0083] Each pixel measurement from a 1D or 2D array detector (or single point detector) measures light intensity through an optical path. Typically, each pixel measurement corresponds to a path length of the sample holder 100. In practice, the optical path includes transmission and / or reflection losses from optical components (i.e., transmission from or reflection losses from the optical components, or both), transmission losses at interfaces between components, detection losses due to the quantum efficiency of the detector, and absorbance in the measurement sample 201.

[0084] The following calculation methods refer to pixel measurements from an array detector, however it will be understood that an alternative configuration having a moveable light source, such as that shown in FIG. 3, can use equivalent calculations.

[0085] The calculation method 500 shown in the flow diagram of FIG. 5 assumes that there is little variation in the sample refractive index between different samples or that the geometry of the sample holder 100 is designed to keep image motion due to refraction negligible. In some embodiments, this can be achieved by providing a sample holder 100 in which the first curved surface 102 has a regular geometry with a relatively large radius of curvature. In these scenarios, the optical path length in the sample 201 corresponding to each pixel can be pre-calculated based on the predetermined geometry of the first surface 102 and the second surface 104 of the sample holder 100. In one example, the path length can be pre-calculated using a commercially available optical ray tracing software program. In one embodiment, a look-up table 503 of sample path lengths for each detector pixel can be pre-calculated for a particular sample holder design and stored in the memory of the computer processor. Alternatively, the computer processor can calculate the sample path length in real time based on the predetermined geometry of the sample holder 100.

[0086] Once the optical path length is known for each pixel, the following calculation method can be used regardless of whether the detector array is one-dimensional or two-dimensional, or whether any positioning of multiple detectors is used. This calculation method can also be applied to sample holders with different optical geometries that have different distributions of sample path lengths.

[0087] With respect to the exemplary diagrams provided herein in Figures 5-11B, the sample path length is for a sample holder having a parabolic cylinder with a first surface and a substantially flat second surface when used with a 1D array detector, or for a sample holder having a spherical first surface and a substantially flat second surface when used with a 2D array detector.

[0088] In step 502 of method 500, a reference or baseline sample is placed in the sample holder 100. Each pixel of the detector 202 receives light from the light source 208 with some attenuation due to the total light path and detector sensitivity. The path attenuation remains substantially constant over time, but the intensity of the light source may not be constant. The signal received at each pixel of the detector 202 is referred to herein as a reference signal S ref The reference signal is measured by the detector 202 and recorded by the computer processor. The measured and recorded reference signal S for each detector pixel is ref can be expressed as follows using equation (1): S ref [p]=I ref ×T[p] (1) Where: S ref [p] is the reference signal received by detector pixel p, I ref is the reference signal S ref [p] is the light source intensity when it was measured and recorded, T[p] is the transmittance of the entire optical path from the light source 208 to the detector pixel p (including the reference sample path).

[0089] In step 504, a measurement sample q is placed in the sample holder 100. The light reaching each detector pixel is attenuated by the absorbance of sample q. The signal received at each pixel of the detector 202 is referred to herein as the sample signal S q The measured and recorded sample signal S for each detector pixel is called q can be expressed as follows using equation (2):

number

[0090] In step 506, the variable T[p] (transmittance of the entire optical path) is cancelled out by the ratio of equations (1) and (2), and equation (3) can be derived as follows:

number

[0091] The detected signal at each pixel p provides a new set of values ​​for equation (3), so in step 506, the relative intensity of the light source (K q ) and the relative attenuation coefficient of the sample (A q ) can be calculated.

[0092] For example, the graph in FIG. 6A shows a range of sample path lengths corresponding to pixels of a linear array detector. The graph in FIG. 6B shows the expected relative sample transmission for each pixel of the linear array detector for a number of different samples with different absorbances. For any detector pixel that corresponds to a zero or near-zero sample path length (i.e., within the first measurement zone 108 of the sample holder where the two optical surfaces are in contact), an anomalous transmission is expected. For these optical paths, Fresnel losses from the optical surfaces to the sample vanish, resulting in a discontinuity in the expected transmission. Therefore, detector pixels that correspond to zero or near-zero sample path lengths can be excluded from the measurement.

[0093] The graphs in Figures 7A and 7B show the relative sample transmittance for a 2D array detector in which the sample holder 100 has a spherical first surface 102 and a flat second surface 104. The sample absorbance corresponding to the transmittance values ​​in Figure 7B is higher than the transmittance values ​​in Figure 7A.

[0094] In one embodiment, the processor converts the 2D data points shown in FIGS. 7A and 7B into multiple 1D arrays of transmittance versus path length (e.g., as shown in FIG. 6B) before applying equations (1)-(3) above to perform calculation method 500.

[0095] In step 508, the processor calculates the relative intensity (K q ) and the relative attenuation coefficient of the sample (A q ) and refine these values ​​using an optimization algorithm to minimize the error between the estimated transmittance value for each pixel and the corresponding detected transmittance value of the sample's attenuation coefficient (A q ) is determined. In one embodiment, an iterative optimization process based on the following FitError function (4) can be used: A q ≧0;K q ≧0, FitError(A q,K q ) (4)

[0096] The FitError function calculates a set of predicted transmittance values ​​and generates a value that represents how much those transmittance values ​​differ from the measured transmittance values. One option for minimizing the error is by minimizing the sum of the squared differences, for example:

number

[0097] 8 is a plot of measured transmission, marked by data points "+", and fitted transmission, marked by data points "·", after application of the optimization algorithm in step 508. The iterative optimization process described herein is not limited to regular symmetric optical surfaces, and therefore advantageously avoids over-constraining the optical design while providing flexibility for the system.

[0098] In some scenarios, the effects of the refractive index cannot be ignored when it comes to distortion of the signal received at the detector 202. Typically, in these scenarios, the path length corresponding to each detector pixel changes due to refraction.

[0099] If the geometry of the first surface 102 of the sample holder 100 is a parabolic cylinder or a sphere, and the second surface 104 is substantially flat, a change in the refractive index will result in a change in the transmission pattern similar to a change in the attenuation coefficient of the sample. This ambiguity in the measurement can be resolved in several different ways, for example: (a) Use uniform source illumination and select the geometry of the first surface 102 and the second surface 104 of the sample holder 100 such that when either the refractive index or the attenuation coefficient changes, the transmission pattern changes in a readily distinguishable way. (b) Including a higher spatial frequency modulation pattern in the source illumination (e.g., using a patterned filter 210 in the optical path that focuses a shadow pattern onto the detector 202 as shown in FIG. 2 ) so that changes in the sample attenuation coefficient cause absorption-based changes in the transmission pattern, but changes in the refractive index distort the position of the spatial modulation.

[0100] In one embodiment, solution (a) uses a geometry for the first surface 102 of the sample holder 100, for example a parabolic surface, that has different surface slopes in two different dimensions. z=a×x 2 +b×y 2 Here, a≠b.

[0101] In this embodiment, an optical ray tracing program is used to calculate the sample path length corresponding to each detector pixel, where the path length depends on the refractive index of the sample. By using multiple ray traces with different assumed refractive indices, a model of the path length as a function of refractive index can be created for each detector pixel. A look-up table of path length as a function of refractive index, or a set of predefined functions defining the relationship between path length and refractive index for a particular sample holder surface geometry, can be stored in memory 510 (see FIG. 5).

[0102] For solution (a) above, each path length is a function of the sample refractive index n and the detector pixels, i.e., L[p,n]. The general steps of method 500 as described above with reference to FIG. 5 can be followed. However, method 500 takes into account the refractive index as an additional parameter to be optimized. At each iteration of the optimization, an estimated transmission can be determined based on the following equation (5):

number

[0103] In case (b) above, high spatial frequency features are intentionally introduced into the source illumination, for example as shown in Figure 2. Distortion of the detector signal based on changes in refractive index affects the location of the high spatial frequency features detected by detector 202. To address the combined effects of absorbance and refractive index, two exemplary methods are described below with reference to Figures 9-11B.

[0104] 9 is a flow chart illustrating a method 900 for determining the attenuation coefficient of a measurement sample 201 using spatial filtering. According to the method 900, the source illumination provides a rapidly changing spatial pattern that can be resolved by the pixels of the detector 202. In particular, changes in the refractive index cause the detector signal pattern to be stretched or distorted. The amount of pattern stretching at various parts of the detector depends on the optical geometry of the surfaces 102, 104 of the sample holder 100, but is independent of the attenuation coefficient of the sample 201.

[0105] By providing a smooth variation in sample path length, the low spatial frequency pattern of illumination on the detector 202 allows for estimation of the sample's attenuation coefficient. As discussed above, distortions due to refractive index differences introduce errors into that estimation. Independent estimation of the refractive index by method 900 allows for the correction of these errors.

[0106] In step 902, spatial filtering is applied to the original detector signal to separate high spatial frequency features from low spatial frequency features. Figure 10A shows the original detector signal including high spatial frequency features received from the array detector 202 when the source illumination is spatially modulated by an optical mask 210 that creates regularly spaced thin shadows at the detector 202. As shown in Figure 10A, when the sample absorbance changes and the refractive index remains constant, the high spatial frequency features align with the detector signal. Figure 10B shows the lateral displacement of the high spatial frequency features when the sample absorbance remains constant and the refractive index changes.

[0107] In step 904, the refractive index of the sample 201 can be estimated based on the extracted high spatial frequency components of the detector signal. In one embodiment, peak identification techniques can be used to estimate the spacing between high spatial frequency features. In one embodiment, multiple ray tracings with different refractive indices are used to create a model or look-up table that is used to determine the relationship between feature spacing and the corresponding refractive index. In these embodiments, a refractive index n that best predicts the observed feature spacing can be determined.

[0108] In step 902, the detector signal is also filtered to extract low spatial frequencies, providing a set of transmittance values ​​for each detector pixel that attenuates or avoids high frequency illumination features. T q =LPF(S q ) / LPF(S ref ) Where: LPF is a low-pass spatial filter (dimensions are selected to suit 1D or 2D detectors), S ref is the array of pixel signals received from the reference sample, S q is the array of pixel signals received from sample q, T q is the pixel transmittance T for all pixels p q [p] is an array of

[0109] Figure 11A shows the output of a combination of median and Savitzky-Golay filtering that removes high spatial frequency features, and Figure 11B shows the output of a filter that excludes any pixels likely to be affected by light shadows for a range of expected sample refractive indices.

[0110] In step 906, the refractive index n previously estimated in step 904 can be substituted into L[p,n], and parameter optimization can be performed to obtain the parameter K in equation (6) below: q (relative intensity of the light source) and A q (the relative attenuation coefficient of sample q) can be estimated.

number

[0111] In step 908, an optimization algorithm is applied to minimize the fitting error in an objective function such as: FitError = Sum((T q [p]-T est [p]) 2 ) In addition, the sample's attenuation coefficient A, which is corrected for distortion from the sample's refractive index by optimization, q can be used to obtain an estimate of

[0112] According to another exemplary method, a modulation model can be created to determine how the relative intensity of each detector pixel varies with refractive index. The modulation model can include a look-up table that provides the expected relative signal distribution across the pixels of the detector for any possible combination of sample attenuation coefficient and refractive index. Typically, if the expected relative illumination pattern can be predicted for any sample attenuation coefficient and refractive index, then one can measure the relative illumination pattern produced by the sample and then determine the attenuation coefficient and refractive index that predicts a pattern consistent with that observed.

[0113] The modulation module can be used to optimize parameters (including the refractive index n) to obtain an estimated transmittance that matches the observed transmittance according to equation (7) below.

number

[0114] [interpretation] The specification, including the claims, is intended to be interpreted as follows.

[0115] The embodiments or examples described herein are intended to illustrate the present invention without limiting the scope of the invention. The present invention can be implemented with various modifications and additions that would be easily thought up by a person skilled in the art. It is therefore understood that the scope of the present invention is not limited to the exact configuration and operation described or shown, but is limited only by the scope of the appended claims.

[0116] The mere disclosure of a method step or product element within this specification should not be construed as essential to the invention claimed herein unless expressly stated as such or expressly recited in the claims.

[0117] The terms in the claims have the broadest meaning that would be given to them by a person of ordinary skill in the art as of the reference date.

[0118] The terms "a" and "an" mean "one or more," unless expressly specified otherwise.

[0119] Neither the title nor the Abstract of this application should be construed in any way as limiting as to the scope of the invention claimed.

[0120] The preamble of a claim may recite a purpose, benefit, or contemplated use of the claimed invention, but may not limit the claimed invention to having only that purpose, benefit, or contemplated use.

[0121] In this specification, including the claims, the term "comprise" and variations of terms such as "comprises" or "comprising" are used to mean "including but not limited to," unless expressly specified otherwise or the context or usage requires an exclusive interpretation of the term.

[0122] The disclosures of any documents referenced herein are incorporated by reference into this patent application as part of this disclosure, but merely for documentary description and enablement, and should in no way be used to limit, define, or otherwise interpret any terms of this application, which necessarily provide an ascertainable meaning even without such incorporation by reference. Any incorporation by reference does not in itself support or approve any statements, opinions, or arguments contained in any incorporated document.

Claims

1. A first surface having a first predetermined geometric shape, A second surface having a second predetermined geometric shape, wherein the first surface faces the second surface A sample holder for use in molecular absorption spectrometry, comprising: The sample holder is configured to hold a measurement sample between the first surface and the second surface such that the distance between the first surface and the second surface defines the optical path length of the sample holder. The predetermined geometric shapes of the first surface and the second surface provide a continuously varying cross-section across the sample holder to provide a continuous range of optical path lengths. Sample holder.

2. The continuously varying cross-section is A first measurement zone where the minimum distance between the first surface and the second surface defines the minimum optical path length, And A second measurement zone where the maximum distance between the first surface and the second surface defines the maximum optical path length, Comprising, The continuously varying cross-section provides a continuous range of optical path lengths between the first measurement zone and the second measurement zone. The sample holder according to claim 1.

3. In the first measurement zone, the first surface contacts the second surface to provide a minimum optical path length of zero. The sample holder according to claim 2.

4. The continuous range of optical path lengths provides a variation in optical path length of about 1 μm to 3 mm. The sample holder according to claim 1.

5. The first surface is curved. The sample holder according to claim 1.

6. One or both of the first predetermined geometric shape and the second predetermined geometric shape is A sphere, A parabolic cylinder, A circular paraboloid, An elliptical paraboloid, Any smooth aspherical surface Defined by any one of. The sample holder according to claim 5.

7. The second surface is substantially flat. The sample holder according to claim 5.

8. The sample holder is Including a plano-convex lens having a curved side surface providing the first surface, The plano-convex lens is movable between an open position where the first surface is separated from the second surface and a closed position where the first surface contacts the second surface and is disposed opposite the second surface. The sample holder according to claim 1.

9. The position of the first surface is fixed with respect to the second surface, the sample holder according to claim 1.

10. The sample holder is made of one or more plastic materials, the sample holder according to claim 1.

11. The sample holder is made of one or more glass materials, the sample holder according to claim 1.

12. A spectrophotometer having the sample holder according to any one of claims 1 to 11.

13. The spectrophotometer operates in the UV-VIS range, the spectrophotometer according to claim 12.

14. The spectrophotometer operates in the NIR range or the IR range, the spectrophotometer according to claim 12.

15. Further comprising a detector for detecting the absorbance of the measurement sample at each optical path length in a continuous range of the optical path length, a single point detector, a one-dimensional array detector, a two-dimensional array detector and any one of the detectors, the spectrophotometer according to claim 12.

16. Further comprising a light source, the light source is, a broadband light source, an LED, a laser and any one of them, the spectrophotometer according to claim 12.

17. The light source provides light simultaneously over the entire measurement sample, the spectrophotometer according to claim 16.

18. The light source provides a light beam, the light beam is movable with respect to the measurement sample, and the measurement sample is scanned so that transmittance values can be sequentially detected for each optical path length within the range of the optical path length, the spectrophotometer according to claim 16.

19. A mask positioned between the light source and the sample holder, the mask being configured such that light passing through the mask projects a predetermined shadow pattern for detection by the detector, and the spectrophotometer is configured to calibrate an effect caused by refraction at one or both of the first surface and the second surface of the sample holder based on the detected shadow pattern, the spectrophotometer according to claim 16.

20. Further comprising a temperature controller for controlling the temperature of the measurement sample, the spectrophotometer according to claim 12.

21. A computer-implemented method for analyzing a measurement sample placed in a sample holder according to any one of claims 1 to 11 using a spectrophotometer, wherein the spectrophotometer has a light source that emits light through the measurement sample and a detector that detects the light intensity transmitted through the measurement sample, Obtaining a detected transmittance value corresponding to each optical path length of the sample holder, wherein the detected transmittance value is based on one or more measured light intensity values from the detector, the step of obtaining; Calculating an estimated transmittance value corresponding to each optical path length of the sample holder; Determining an attenuation coefficient of the measurement sample that minimizes an error between the detected transmittance value and the estimated transmittance value A method including.

22. The step of calculating the estimated transmittance value includes Calculating an estimated attenuation coefficient of the measurement sample based on the corresponding detected transmittance value; Calculating an estimated transmittance value based on the estimated attenuation coefficient The method according to claim 21, including.

23. The step of determining the attenuation coefficient of the measurement sample is an iterative process, the method according to claim 21.

24. The method according to claim 21, further including the step of determining a value of each optical path length according to the refractive index of the measurement sample.

25. A computer-implemented method for analyzing a measurement sample placed in a sample holder according to any one of claims 1 to 11 using a spectrophotometer, wherein the spectrophotometer has a light source that emits light through the measurement sample, a detector that detects the light intensity transmitted through the measurement sample, and a higher spatial frequency modulation pattern associated with the light source such that high spatial frequency features can be detected by the detector. Providing a predetermined modulation model that predicts a light intensity value of the detector corresponding to each refractive index within a predefined refractive index range and corresponding to each optical path length; Obtaining a detected transmittance value corresponding to each optical path length, wherein the detected transmittance value is based on one or more measured light intensity values from the detector, the step of obtaining; Calculating an estimated transmittance value of the measurement sample corresponding to each optical path length and each refractive index based on the predetermined modulation model; Determining an attenuation coefficient of the measurement sample that minimizes an error between the detected transmittance value and the estimated transmittance value A method comprising the steps of: **Claim 26** A computer-implemented method of analyzing a measurement sample disposed within a sample holder according to any one of claims 1 to 11 using a spectrophotometer, the spectrophotometer comprising: a light source that emits light through the measurement sample; a detector that detects an illumination signal representing the light intensity transmitted through the measurement sample; and a higher spatial frequency modulation pattern associated with the light source such that a high spatial frequency feature can be detected within the illumination signal of the detector. Filtering the illumination signal to obtain first filtered illumination data from which the high spatial frequency feature is extracted and second filtered illumination data from which the high spatial frequency feature is removed from the illumination signal. Calculating a refractive index value based on the first filtered illumination data. Obtaining a detected transmittance value corresponding to each optical path length, the detected transmittance value being based on the second filtered illumination data. Calculating an estimated transmittance value of the measurement sample corresponding to each optical path length based on the calculated refractive index. Determining an attenuation coefficient of the measurement sample that minimizes an error between the detected transmittance value and the estimated transmittance value A method comprising the steps of: **Claim 27** One or more tangible non-transitory computer-readable media having computer-executable instructions for performing the computer-implemented method of claim 21. **Claim 28** One or more tangible non-transitory computer-readable media having computer-executable instructions for performing the computer-implemented method of claim 25. **Claim 29** One or more tangible non-transitory computer-readable media having computer-executable instructions for performing the computer-implemented method of claim 26. **Claim 30** A method of manufacturing a sample holder according to any one of claims 1 to 11, Forming a first portion of the sample holder to provide the first surface. Forming a second portion of the sample holder to provide the second surface. The first portion is movable relative to the second portion between an open position that enables placement or removal of the measured sample and a closed position in which the first surface is in a fixed position relative to the second surface and provides the continuously varying cross-section across the sample holder, attaching the first portion to the second portion A method comprising. **Claim 31** A method of manufacturing a sample holder according to any one of claims 1 to 11, forming a first portion of the sample holder to provide the first surface; forming a second portion of the sample holder to provide the second surface; attaching the first portion to the second portion such that the first surface is fixed relative to the second surface and provides the continuously varying cross-section across the sample holder; forming an opening that enables injection of the measurement sample into a cavity between the first portion and the second portion A method comprising.

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