Improved light transmission sample holder and multi-wavelength analysis

Abstract

This disclosure provides an apparatus, device, and method for improving optical analysis of a sample thin layer between two plates, particularly for optical analysis of multiple waves.

Description

[0001] Cross-citation of related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 864,491, filed June 20, 2019, which is incorporated herein in its entirety for all purposes. Technical Field

[0003] This invention relates to an apparatus and method for improving the light transmission analysis of a thin sample layer sandwiched between two plates. Background Technology

[0004] This disclosure generally relates to the field of optical transmission analysis of samples. More specifically, this disclosure relates to a method and apparatus for optical transmission analysis of samples such as hemoglobin.

[0005] Light absorption in thin-layer samples is one method for determining the light absorption of biological and chemical samples. One way to measure light absorption is to measure the intensity of incident and transmitted light directly entering and leaving the sample, respectively.

[0006] However, in many practical situations, it is difficult to directly measure these light intensities for various reasons. One reason is that thin-layer samples typically require a sample holder for measurement, and the transmitted light being measured is light that passes through both the sample and the sample holder. Therefore, a method is needed that can separate the light absorption of the sample holder from the light absorption of the sample.

[0007] Another reason is that the incident and transmitted light are on opposite sides of the sample, making it difficult to use a single detector for both types of light. Therefore, a single photodetector is required for absorption measurements.

[0008] In existing methods for measuring the light transmittance of thin samples, a sample holder comprising two plates has been used to clamp the sample in a thin layer between the two plates, and the light transmittance through a bubble within the sample layer (which can occur under certain conditions) is used as a reference signal to separate the light absorption of the sample holder from that of the sample. This method also allows for light absorption measurements using a single photodetector. In this method, it is assumed that (i) the light transmittance through the bubble region is the same as the light transmittance through a zero-thickness sample, and (ii) the light absorption of the sample holder is the same in the bubble region (where the reference signal is measured) and the sample region (where a single sample is measured). However, in reality, both assumptions can be incorrect. Bubbles can be generated at locations significantly away from the sample signal location, resulting in a significant difference in sample holder absorption between the two locations. Bubbles may be too small, causing significant light scattering and a significantly different reference signal from that of a sample with zero thickness. Furthermore, the generation of bubbles is random in both occurrence (may or may not occur) and location (e.g., random locations).

[0009] Therefore, the object of this invention is to provide an apparatus and method for generating reference light, simplifying light transmission measurement, and simplifying sample handling. This invention can overcome or reduce the disadvantages of existing apparatuses or systems. Summary of the Invention

[0010] In some embodiments, this disclosure provides apparatus and methods for measuring different wavelengths using a monochrome camera (e.g., RGB) for object sensing and imaging.

[0011] In some embodiments, this disclosure provides apparatus and methods for measuring or determining certain properties of, for example, biological or chemical samples by interrogating them with light.

[0012] In some embodiments, this disclosure provides apparatus and methods for optical transmission analysis of samples, for example, located in a sample holder comprising two plates.

[0013] In some embodiments, the device (e.g., having a Bayer filter preceding the sample chamber) comprises, in the order listed:

[0014] A single white light source;

[0015] Dual-band or triple-band bandpass filters are used, for example, to selectively filter white light into quantitative color before interrogating a sample;

[0016] A cavity for receiving samples, such as a sample holder having a slot for receiving a plate (e.g., a QMAX card) with a sample;

[0017] Imaging lenses (e.g., a single lens or multiple lenses. The function of an imaging lens is to allow for enhanced imaging on the sensor);

[0018] RGB Bayer filter, used to filter light emitted from the sample being questioned;

[0019] A sensor for receiving filtered light from an RGB Bayer filter; and

[0020] Optionally, a processor for image processing of the light signal information recorded by the sensor.

[0021] RGB Bayer filters and sensors or sensor arrays are available on the market in combination, either as a single or integrated component with a combination of filters and sensors.

[0022] In the embodiments: the sensor may be, for example, at least one pixel of a camera; the sensor may be, for example, part of a smartphone camera; the RGB Bayer filter and the sensor are combined into a single component.

[0023] In some embodiments, the method includes:

[0024] A dual-band or triple-band bandpass filter is illuminated with light from a single white light source to produce a mixed filtered light with a narrow bandwidth of dual or triple wavelengths, for example, the narrow bandwidth may be 50 to 100 nm.

[0025] Question the sample with filtered light;

[0026] Optionally, the light emitted from the sample being questioned may be adjusted or directed through an imaging lens, such as at least one lens or multiple lenses;

[0027] Light emitted from the imaging lens is passed through an RGB Bayer filter;

[0028] Sensors are used to sense and record light emitted from an RGB Bayer filter; and

[0029] Image processing and analysis of sensed and recorded light.

[0030] In some embodiments, the sensed and recorded light may, for example, be further processed, i.e., the method further includes, for example, performing image processing on the sensed and recorded light to remove or eliminate color overlap (e.g., subtracting overlapping color signals); and

[0031] Data for analyzing specific analytes (such as hemoglobin and similar analytes).

[0032] The following embodiments are shown in Figure N5, where the RGB filter is located after or following the sample.

[0033] In some embodiments, the device (e.g., having a Bayer filter after the sample chamber) (i.e., for the sample and sample holder) comprises, in the order listed:

[0034] A single white light source;

[0035] A cavity for receiving samples, such as a sample holder;

[0036] Dual-band or triple-band bandpass filters are used to selectively filter light emitted from the sample being questioned into component colors;

[0037] Imaging lenses, such as a single lens or multiple lenses (the function of an imaging lens is to allow imaging on a sensor);

[0038] An RGB Bayer filter for filtering light emitted from the imaging lens; and

[0039] A sensor for receiving filtered light from an RGB Bayer filter; and

[0040] Optionally, a processor is used to perform image processing on the light sensed by the sensor.

[0041] In some embodiments, the method includes:

[0042] Interrogating the sample with light from a single white light source; and

[0043] The light emitted from the sample being questioned is filtered using a dual-band or triple-band bandpass filter to produce a mixture of light with two or three wavelengths that has a narrow band or wide bandwidth.

[0044] Optionally, an imaging lens can be used to adjust a mixture of two-wavelength or three-wavelength light with a narrow bandwidth;

[0045] Light emitted from the imaging lens passes through the RGB Bayer filter;

[0046] Sensors are used to sense and record light emitted from RGB Bayer filters; and...

[0047] Optionally, a processor for image processing of the light signal information recorded by the sensor.

[0048] The present invention relates to apparatus and methods for improving the optical analysis of a thin sample layer sandwiched between two plates, particularly for generating a reference signal that improves optical analysis, and for applications such as determining hemoglobin in blood.

[0049] The properties of a sample (e.g., biological or chemical properties) can be determined by the sample's optical density (OD) and the ratio of the intensity of transmitted light through a thin sample layer to the intensity of incident light (i.e., Beer-Lambert's law). However, thin-layer samples typically require a sample holder for measurement, and the light being measured also passes through the sample holder. It is necessary to separate the sample's optical transmission signal and light absorption (e.g., optical density) from the total transmitted light, which includes light transmission through the sample and through the sample holder.

[0050] One object of the present invention is to provide apparatus and methods for certain embodiments of a sample holder, and to improve the use of light transmission measurements.

[0051] References

[0052] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated by reference. Attached Figure Description

[0053] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. In some of the drawings, the figures are drawn to scale. In figures showing experimental data points, the lines connecting the data points are only used to guide the view of the data and have no other significance. For clarity, some elements are enlarged when shown in the figures. It should be noted that the drawings are not intended to show elements at strict scale. The dimensions of the elements should be depicted in accordance with the description provided herein and incorporated herein by reference. The drawings are not intended to limit the scope of the invention in any way.

[0054] Figure 1A A device that uses a color camera to determine the intensity of each wavelength in light with two different wavelengths.

[0055] Figure 1B A device for measuring the light absorption of a sample at each of two different wavelengths.

[0056] Figure 1c shows the optical setup for measuring hemoglobin in a QMAX card with a dual bandpass filter configuration.

[0057] Figure N4 is a schematic diagram of an apparatus and method for measuring dual-wavelength transmitted light signals in the green, blue, and red regions using an RGB color camera sensor through a single sensor image or a single camera capture.

[0058] Figure N5 is a schematic diagram of an apparatus and method for measuring transmitted light signals at three wavelengths in the green, blue, and red regions using an RGB color camera sensor through a single sensor image or a single camera capture.

[0059] Figure N6 An example of a sample card for on-the-spot colorimetric determination of sample liquid is shown, comprising a top plate, a bottom plate, a diffuse layer laminated on the back of the bottom plate, and the sample liquid between the top plate and the bottom plate.

[0060] Figure N7 A test apparatus for measuring the colorimetric signal of a sample liquid in a sample card with a laminated diffuse layer is shown. The apparatus includes, for example, a light source, a colorimetric sample card with a laminated diffuse layer, and a sensor.

[0061] Figure X1 The optical setup for measuring hemoglobin in a QMAX card with a dual bandpass filter configuration is shown. The filter before the LED can be (a) a single wavelength setting (550 nm), (b) two separate wavelength settings (550 nm + 650 nm), and (c) a dual BP wavelength (525 nm + 680 nm). The bandwidth of the filter is between 20 nm and 60 nm.

[0062] Figure X2The HgB performance on the iMOST platform with the HemoCue 301 commercial HgB analyzer is shown using (a) one wavelength setting (550 nm), (b) two separate wavelength settings (550 nm + 650 nm), and (c) dual BP wavelengths (525 nm + 680 nm).

[0063] Figure X3 shows the HgB performance on the iMOST platform relative to the HemoCue 301 commercial HgB analyzer, using a total of 60 samples (K2EDTA venous blood collected from patients at a local hospital) collected over 5 days. The error |Δ| for each sample was calculated. The mean difference was 2.3%, with a correlation R = 98.5%, a slope of 0.975, and a bias of 0.2%. 59 (98%) of the 60 samples were within the allowable total error ±7%.

[0064] Figure X4 shows fluorescence photographs of two Q-cards: (a) before and (b) after blood addition. One Q-card was printed using an inkjet dot printing method in water with a droplet size of 10 nL, a period of 600 μm, and reagents. The other Q-card used a guided flow printing method with a volume of 8 μL and reagents in alcohol exceeding 90%. Clearly, the guided flow printing method provides a uniform coating without droplet boundaries before and after blood addition.

[0065] Figure X5 A flowchart illustrating the workflow of HgB absorption measurement image processing is shown.

[0066] Figure X6 illustrates the components of HgB image processing, including: horizontal column inspection, vertical column inspection, and intensity ratio calculation.

[0067] Figure X7 A flowchart of the lateral dimension correction (LDC) calculation is shown.

[0068] Figure 1M shows a cross-sectional view of one embodiment of a sample holder known as an OAC (e.g., an optical analysis card) for analyzing analytes (e.g., hemoglobin in a blood sample) using light transmission, comprising: a first plate, a second plate, and a light-guiding spacer (LGS); wherein the LGS has a columnar shape, sandwiched between the two plates, each end of the column being in direct contact with one of the plates to form an LGS plate contact area, and is configured to allow light to be transmitted from the first plate through the LGS to the second plate without passing through the sample.

[0069] Figure 1J shows a cross-sectional view of an embodiment of a sample holder known as an OAC (e.g., an optical analysis card) for analyzing analytes (e.g., hemoglobin in a blood sample) using light transmission, comprising: a first plate, a second plate, and a light-guiding spacer (LGS), wherein the two plates are movable relative to each other and the column has a flat top.

[0070] Figure 1N is an illustration of an embodiment of CROF (Compression-Regulated Open Flow). Figure (a) shows a first plate and a second plate, wherein the first plate has a spacer. Figure (b) illustrates sample deposition on the first plate (shown) or the second plate (not shown), or both (not shown), in an open configuration. Figure (c) illustrates (i) using two plates to disperse the sample (the sample flows between the plates) and reduce the sample thickness, and (ii) using spacers and plates to regulate the sample thickness in a closed configuration. The inner surface of each plate has one or more binding sites and / or storage sites (not shown).

[0071] Figure 2A shows a cross-sectional view of an embodiment of a sample holder having a first plate, a second plate, and an LGS, the position of the sample in the holder, the sampling area and the reference area, and the incident light and transmitted light in the sample area and the reference area, respectively.

[0072] Figure 2B A perspective view of one embodiment is shown, illustrating a sample holder having a first plate, a second plate, and an LGS, the sample in the holder, and the positions of the sampling area and the reference area.

[0073] Figure 3 A top view of an embodiment is shown, illustrating a sample holder having a first plate, a second plate, and an LGS, the location of the LGS, a sampling area, a reference area, and exemplary locations of the edges of the reference area and the sample area. Note that the edges are selected during imaging processing.

[0074] Figure 4 The molar extinction coefficients of oxyhemoglobin [HbO2] and deoxyhemoglobin [Hb] are shown at wavelengths from 200 nm to 1000 nm.

[0075] Figure 5 The optical setup for measuring hemoglobin in a QMAX card is shown.

[0076] Figure 6 An exemplary hemoglobin measurement from a QMAX card obtained by an iPhone is shown.

[0077] Figure 7 An exemplary QMAX hemoglobin measurement compared to the gold standard (Abbott Emerald blood cell counter) is shown.

[0078] Figure 8 shows an exemplary hemoglobin measurement from a QMAX card obtained by an iPhone.

[0079] Figure 9 An exemplary method for selecting the sampling region and the reference region is shown. Detailed Implementation

[0080] The following detailed description illustrates certain embodiments of this disclosure by way of example and not limitation. Section headings and any subheadings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. The content under a section heading or subheading is not limited to the section heading or subheading, but applies to the entire description of this disclosure.

[0081] References to any publication are made because their publication date predates the application date and should not be construed as an admission that the claims of this invention are not entitled to precede these publications by virtue of a prior invention. Furthermore, the provided publication date may differ from the actual publication date, which can be independently verified.

[0082] definition

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may also be used to practice or test the teachings herein, some exemplary methods and materials are described here.

[0084] "Interrogation," "interrogating," and "interrogated" refer to using light or illumination to address or contact a sample or specimen to obtain information about the sample or specimen arising from the interaction between the sample or specimen and light. The light generated by the interrogation or interaction between the initial light or illumination and the sample or specimen can be analyzed to reveal information about the sample or specimen. For example, when a sample such as fresh blood may contain hemoglobin or oxyhemoglobin, analyzing light can reveal the presence of hemoglobin, oxyhemoglobin, or both, and if present, the relative amounts or individual concentrations of hemoglobin and oxyhemoglobin in the sample.

[0085] The term "average error" or similar terms like "average difference" typically refers to the average of all errors in a set. In this article, "error" refers to the uncertainty in a measurement, or the difference between a measured value and the true / correct value. The more formal term for error is measurement error, also known as observation error.

[0086] "OTSA" or similar terms refer to light transmission sample analysis, which measures the optical density (OD) of a thin sample layer by light transmission.

[0087] The terms “imager,” “sensor,” “camera,” and similar terms are used interchangeably.

[0088] The terms “include”, “includes”, or similar terms mean including but not limited to, that is, inclusive rather than exclusive.

[0089] The term "about," as used to describe, for example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, viscosity, and similar values ​​and ranges thereof of a component in a composition describing embodiments of the present disclosure, or the size and similar values ​​and ranges thereof of a component, refers to variations in numerical quantities that may occur, for example: by typical measurement and processing procedures used to prepare materials, compositions, composites, concentrates, components, articles, or formulations; by unforeseen errors in these procedures; by differences in the manufacture, origin, or purity of the starting materials or components used to carry out the methods; and similar considerations. The term "about" also includes amounts that differ due to aging of a composition or formulation having a particular initial concentration or mixture, and amounts that differ due to mixing or processing of a composition or formulation having a particular initial concentration or mixture.

[0090] "Optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the possibility that the event or situation occurs and the possibility that it does not occur.

[0091] Unless otherwise stated, the indefinite article “a” or “one” and its corresponding definite article “the” as used herein refer to at least one, or one or more.

[0092] Abbreviations well known to those skilled in the art may be used (e.g., “h” or “hrs” for hours or more, “g” or “gm” for grams, “mL” for milliliters, “rt” for room temperature, “nm” for nanometers, and similar abbreviations).

[0093] The term "light-guiding spacer" or "LGS" can refer to a column that, during light transmission measurement of a sample, brings one end of the column into direct contact with a first plate and the other end into direct contact with a second plate, wherein in some embodiments, the first and second plates sandwich the sample between the two plates. In some embodiments, the optical index and the dimensions of the column are predetermined and known. In some embodiments, the LGS is made of the same material as one or both of the plates. In some embodiments, the LGS is bonded, molded, imprinted, or otherwise attached to one or both plates.

[0094] The term "sample with no significant amount" can refer to a sample amount that is not significant for light transmission measurements when measured in an area containing two plates and a sample.

[0095] The term "LGS plate contact area" can refer to the area at each end of the LGS (which has a columnar shape) that is in direct contact with one of the plates. In some embodiments, the LGS and a plate are made of a single piece of material, and the LGS plate contact area at the end of the LGS used to attach to the plate is the cross-section of the LGS. In some embodiments, the LDG and two plates are made of a single piece of material, then the LGS plate contact area at both ends of the LGS is the cross-section of the LGS.

[0096] The term "transverse section of LGS" can refer to the cross section of LGS that is parallel to the plates when LGS is sandwiched between two plates.

[0097] The term "LGS contact area or LGS cross section larger than the wavelength of light" can refer to an LGS contact area or LGS cross section larger than the wavelength of light, or an area of ​​a disk with a diameter equal to the wavelength of light.

[0098] The term "OTSA" refers to optical transmission sample analysis, which measures the optical density of a thin sample layer by transmitting light.

[0099] The terms “SR region” or “a pair of SR regions” are interchangeable and can refer to a sampling region and a corresponding reference region, wherein the OD of the thin sample layer is determined by taking the ratio of the intensity of light transmitted through the sample region to that transmitted through the reference region.

[0100] The term "reference area" in an OAC device can refer to the area of ​​the device through which wavelengths and polarizations of light pass via a first plate, a photoconductive spacer, and a second plate, wherein the photoconductive spacer is in direct contact with the first and second plates. Alternatively, the term "reference area" in an OAC device can refer to the area of ​​the device in which the photoconductive spacer is sandwiched between two plates and in direct contact with each plate, wherein, in the reference area, probe light sequentially passes through the first plate, the photoconductive spacer, and the second plate without passing through the sample (Figures 1 and 2).

[0101] The term "sampling area" of an OAC device can refer to a device area in which light of sample wavelength and polarization passing through a reference area passes through a first plate, a sample between the two plates, and a second plate without passing through a light guide spacer.

[0102] The term "sampling area" of an OAC device can refer to the area between two plates in which the sample does not encounter an LGS; that is, in the sampling area, the probe light passes through the first plate, the sample between the two plates, and the second plate in sequence without encountering an LGS.

[0103] The term "exclusion distance for reference" refers to the minimum distance between the edge of the optical guide spacer and the edge of the reference area.

[0104] The term "exclusion distance for sampling" refers to the minimum distance between the edge of the optical guide spacer and the edge of the sampling area.

[0105] The terms "distance between sampling area and reference area" and "exclusion distance between sampling and reference" in OAC devices are interchangeable; they refer to the shortest interval between the boundary of the reference area and the boundary of the sampling area. That is, the exclusion distance between sampling and reference is the sum of the exclusion distance of sampling and the exclusion distance of reference.

[0106] The terms "imager" and "camera" are used interchangeably.

[0107] The term "embossing" refers to the process of integrally fixing spacers and plates by embossing (e.g., treading) a sheet of material to form spacers on the surface of the plate. The material can be a single layer or multiple layers.

[0108] The term "etched" refers to the process of fixing spacers and plates together by etching a sheet of material to form spacers on the surface of the plate. The material can be a single layer or multiple layers.

[0109] The term "fusion to" refers to the integral fixation of spacers and plates by attaching them together, with the original materials of the spacers and plates fused together and a clear material boundary existing between the two materials after fusion.

[0110] The term "bonded to" means that the spacer and the plate are fixed together as a whole by adhesive bonding.

[0111] The term "attached to" means that spacers and plates are connected together.

[0112] The terms “CROF card (or card),” “COF card,” “QMAX card,” “Q card,” “CROF device,” “COF device,” “QMAX device,” “CROF plate,” “COF plate,” and “QMAX plate” are interchangeable, except that in some embodiments, the COF card does not include a spacer; and these terms refer to a device comprising a first plate and a second plate, the first plate and the second plate being movable relative to each other into different configurations (including open configurations and closed configurations), and the device comprising a spacer for adjusting the spacing between the plates (except in some embodiments of COF). The term “X plate” may refer to one of the two plates in a CROF card to which the spacer is fixed. Further description of the COF card, CROF card, and X plate is described in Provisional Application Serial No. 62 / 456065, filed February 7, 2017, all of which are incorporated herein by reference in their entirety for all purposes.

[0113] In the QMAX process, the term "open construction" for two plates refers to a construction in which the two plates are either partially or completely separated, and the spacing between the plates is not adjusted by spacers.

[0114] In the QMAX process, the term "closed configuration" for two plates refers to a configuration in which the plates face each other, the spacer and the relevant volume of the sample are between the plates, the relevant spacing between the plates and thus the thickness of the relevant volume of the sample are adjusted by the plates and the spacer, wherein the relevant volume is at least a portion of the total volume of the sample.

[0115] In the QMAX process, the term "sample thickness adjusted by plate and spacer" means that, given the plate, sample, spacer, and plate compression method, the thickness of at least one port of the sample under the closed configuration of the plate can be predetermined based on the properties of the spacer and plate.

[0116] In QMAX cards, the term "inner surface" or "sample surface" of a plate refers to the surface of the plate that contacts the sample, while the other surface of the plate (that does not contact the sample) is called the "outer surface".

[0117] Unless otherwise specified, the terms "height" or "thickness" for objects in QMAX processes may refer to the dimension of an object in a direction perpendicular to the surface of the plate. For example, spacer height is the dimension of a spacer in a direction perpendicular to the surface of the plate, and spacer height and spacer thickness refer to the same thing.

[0118] Unless otherwise specified, the term "area" in QMAX processes can refer to an area of ​​an object that is parallel to the surface of the plate. For example, a spacer area is an area of ​​spacers that is parallel to the surface of the plate.

[0119] The term QMAX card can refer to a device that performs QMAX (e.g., CROF) processes on a sample and has or does not have a hinge connecting the two plates.

[0120] The terms "QMAX card with hinge" and "QMAX card" are interchangeable.

[0121] The terms “angle self-maintenance” or “rotation angle self-maintenance” refer to the property of a hinge that essentially maintains the angle between two plates after an external force that moved the plates from an initial angle to that angle is removed from the plates.

[0122] The terms "spacer with a predetermined height" and "spacer with a predetermined spacing distance" mean that the values ​​of the spacer height and spacing distance are known prior to the QMAX process. If the values ​​of the spacer height and spacing distance are not known prior to the QMAX process, then the values ​​of the spacer height and spacing distance are not predetermined. For example, in the case where beads are sprayed onto a plate as spacers, where the beads land at random locations on the plate, the spacing distance is not predetermined. Another example of a spacer not being predetermined is when the spacer moves during the QMAX process.

[0123] In the QMAX process, the term "spacer fixed to its corresponding plate" means that the spacer is attached to the plate at a certain position and remains attached to that position during the QMAX process (i.e., the spacer's position on the corresponding plate does not change). An example of "spacer fixed to its corresponding plate" is that the spacer is made integrally from a single piece of material of the plate, and the spacer's position relative to the plate surface does not change during the QMAX process. An example of "spacer not fixed to its corresponding plate" is that the spacer is bonded to the plate with adhesive, but during the use of the plate, during the QMAX process, the adhesive cannot hold the spacer in its original position on the plate surface, and the spacer moves away from its original position on the plate surface.

[0124] Multi-wavelength intensity and sample absorption were measured using a monochrome camera.

[0125] In some embodiments, the disclosed apparatus and methods may use a single white light source and a dual-band or tri-band bandpass filter to convert or purify a single white light into a mixture of different colors of light. For example, the bandpass filter may convert or purify a single white light before interrogating a sample (see Figures N1 to N4), after interrogating a sample (see Figure N5), or before and after interrogating a sample (not shown).

[0126] Two or three wavelengths will be sufficiently separated, for example, one wavelength in the red region and another in the green region. After the light is finally collected by the R, G, and B channels of a sensor (such as a digital camera or CCD array), the signal of each channel consists almost exclusively of a single wavelength of light with a narrow bandwidth. By capturing (i.e., making or recording) a single sensor image (a single “shot”), the emitted signal at two and at most three narrow bandwidths can be obtained using Bayer filters.

[0127] In some embodiments, the disclosed apparatus and methods may use a single white light source and a dual-band or triple-band bandpass filter. In some embodiments, the disclosed apparatus and methods first cause or pass white light through a dual-band or triple-band bandpass filter for purification, i.e., separating the white light into two or three color light components, such as red and green light from a dual-band bandpass filter, or blue-red-green light from a triple-band bandpass filter. After purification, the light is a mixture of light separated into different colors. The dual or triple wavelengths can be sufficiently separated, for example, one wavelength in the red region and another wavelength in the green region. After the light is finally collected by the R, G, and B channels of a sensor (e.g., a digital camera, CCD array, and similar imager), the signal of each channel consists almost exclusively of light of a single wavelength with a narrow bandwidth. If the signal of a channel consists of light of more than a single wavelength or a narrow bandwidth wavelength range, it may include wavelengths or overlaps from adjacent colors, such as blue light slightly overlapping with a green wavelength, red light slightly overlapping with a green wavelength, or green light slightly overlapping with one or both of the blue and red wavelengths. By capturing (i.e., creating or recording) a single sensor image (i.e., a “single shot” or a single exposure; or a composite exposure), a Bayer filter can be used to obtain the transmitted signal at two wavelengths or at most or no more than three wavelengths in a narrow bandwidth.

[0128] In some embodiments, this disclosure provides a method for manufacturing the above-described device, comprising, for example, assembling the components described and shown into, for example... Figure 5 The structure shown.

[0129] In some embodiments, this disclosure provides a method for using the above-described apparatus to analyze a sample (such as a blood sample) against an analyte (such as hemoglobin).

[0130] In some embodiments, the disclosed apparatus and method sequentially: interrogate a sample in an imaging field using white emitted light from a single light source; process synthetic light from the imaging field; generate digital information for the sample from the synthetic light; and generate a response file for the sample from the digital information. The generated response file can be used for analysis, such as the size, enumeration, characterization, and classification of sample content.

[0131] The processing steps may include, for example, an optical detection step, followed by an electronic processing step.

[0132] The applicant's aforementioned application, entitled "Improved Light Transmission Sample Holder and Analysis, Particularly Hemoglobin," mentions devices and methods for measuring analytes such as hemoglobin. This application mentions a device or apparatus with a specific sample holder, referred to as an OAC (Optical Analysis Card). This application also mentions a measurement method in which the optical density of a material is determined by interrogating the sample in the holder, for example, with a single light source (such as an LED), and taking the ratio of the intensities of two transmitted lights: one being light transmitted through a sampling area of ​​the sample holder, and the other being light transmitted through a reference area of ​​the sample holder, wherein the optical density (OD) of the sample is measured without directly measuring the incident light.

[0133] Example. The two color channels are the red and green channels of a color camera.

[0134] A method for distinguishing the light absorption of a sample for each of two different wavelengths using a monochrome camera, the method comprising:

[0135] A color camera is provided, comprising three sets of detection elements, each set detecting a different color, wherein the three different colors are red, green and blue;

[0136] Provides a non-transient computer-readable medium for storing the algorithm, the algorithm

[0137] The sample simultaneously transmits light of the first wavelength and light of the second wavelength.

[0138] The total transmitted light is detected using a camera; the total transmitted light is the sum of the first wavelength and the second wavelength of light that are transmitted through the sample and collected by the camera.

[0139] The total transmitted light and the algorithm are used to determine the transmitted light at the first wavelength and the transmitted light at the second wavelength through the sample;

[0140] The algorithm described includes formulas.

[0141]

[0142] in and The transmitted light intensities at the first and second wavelengths to be determined; I R and I G It is the sum of the total transmitted light intensity measured in the camera's green and red channels;

[0143] in It is the channel crosstalk matrix.

[0144] in It is the percentage of light entering the camera's red channel when only the first wavelength of light enters the camera; It is the percentage of light entering the camera's red channel when only the second wavelength of light enters the camera; where It is the percentage of light entering the camera's green channel when only the first wavelength of light enters the camera; It is the percentage of light entering the camera's green channel when only the second wavelength of light enters the camera;

[0145] Calculate the sample region and column reference region The ratio of the two values ​​is calculated based on the sample absorption at the first wavelength, and the values ​​are used to calculate the ratio between the sample region and the column reference region. The ratio of the two values ​​is the sample absorption at the second wavelength.

[0146] Principles and some examples

[0147] In some embodiments, this disclosure provides an apparatus, device, and method for improving the light transmission analysis of a thin sample layer, such as that located between two plates, specifically for generating a sample signal that can improve optical analysis, and for determining analytes (e.g., hemoglobin in a blood sample) in a sample.

[0148] In the light transmission experiment through the sample layer, the absorption coefficient α of the thin sample layer was measured. s Certain biological or chemical properties of a sample can be determined using Beer-Lambert's law. The light absorption coefficient α of a thin sample layer... s With the incident light intensity (i.e., the light incident on the sample) I i and transmitted light intensity (i.e., light passing through the sample) I t related:

[0149]

[0150] Where L s This is the length (i.e., thickness) of the sample layer, and OD is the optical density through the sample layer. The light absorption coefficient α of the thin sample layer... s This can be related to the characteristics of the sample. Therefore, using the Beer-Lambert law, the characteristics of the sample can be determined by measuring the OD of the sample layer.

[0151] However, in practice, it is difficult to directly measure the intensity of incident light (i.e., light directly incident on the sample layer) and transmitted light (i.e., light directly transmitted through the sample layer). Typically, what is measured in experiments is the total light transmittance through the sample and sample holder. This is because thin-layer samples usually require a sample holder for measurement, and the light being measured also passes through the sample holder. Therefore, it is necessary to separate / determine the OD of the sample from the total light transmittance.

[0152] In some embodiments, a specific sample holder called an OAC (i.e., optical analysis card) is provided, and the optical density of the material is determined by taking the ratio of the intensities of two transmitted lights: one is the light transmitted through the sampling area of ​​the sample holder, and the other is the light transmitted through the reference area of ​​the sample holder, wherein the OD of the sample is determined without directly measuring the incident light.

[0153] The specific and preferred values ​​and ranges of components, ingredients, additives, dimensions, conditions, times, etc., disclosed herein are for illustrative purposes only; they do not exclude other defined values ​​or other values ​​within the defined range. The apparatus, devices, and methods disclosed herein may include any value or any combination of said values, specific values, more specific values, and preferred values, including explicit or implicit intermediate values ​​and ranges.

[0154] Bayer Color Filter Array (CFA) CMOS Sensor

[0155] Bayer filter mosaics are color filter arrays (CFAs) used to arrange RGB color filters on a square grid, such as that of a light sensor. Their specific filter arrangement is used in most monolithic digital image sensors used in digital cameras, camcorders, and scanners to produce color images. The filter pattern is 50% green, 25% red, and 25% blue.

[0156] Bryce Bayer (US Patent No. 3,971,065) refers to the green filter photosensitive point as the luminance-sensitive element and the red and blue points as the chromaticity-sensitive elements. Bayer uses twice as many green elements as red or blue to simulate the physiology of the human eye. The Bayer pattern data from the sensor is called RAW image data.

[0157] Optical bandpass filters are used to selectively transmit a portion of the spectrum of incident radiation while suppressing or blocking all or substantially all other wavelengths. The original optical bandpass filter is a prism capable of splitting or dispersing white light into a spectrum of colors. Analyzing white light by dispersing it with a prism is an example of spectroscopy. Various bandpass filters and fluorescence bandpass filters are commercially available (see, for example, Edmund Optics, Inc., edmundoptics.com), such as conventional coated 400–699 nm bandpass interferometer filters, available at UV, visible, and IR center wavelengths, offering bandwidths of 10–80 nm, and ideal for biomedical applications and instrument integration.

[0158] To reconstruct a full-color RGB image from data collected by a color filter array (such as "RAW" image data), some form of interpolation is used to fill in the blanks. This mathematical process is called demosaicing and can be performed in different ways. A simple method is to interpolate the color values ​​of pixels of the same color in the neighborhood. For example, a pixel with a green filter provides an accurate measurement of its green component. The red and blue components of that pixel are obtained from its neighboring pixels. For a green pixel, two red neighboring pixels can be interpolated to produce a red value. Similarly, two blue pixels can be interpolated to produce a blue value.

[0159] RAW refers to any of several local data formats that contain complete data captured by a sensor. Typically, these local data formats are proprietary, and each company uses its own. Because a RAW file is a single set of brightness values ​​for each pixel on the sensor, it does not contain actual per-pixel color information. Color is derived by comparing neighboring pixels filtered for one of three colors with a Bayer mask.

[0160] In some embodiments, alternatives to Bayer color filter arrays may be considered, and may include, for example:

[0161] Panasonic's low-light, filterless sensor technology, namely OPF (Organic Photoconductive Film) technology, achieves color separation through diffraction using "miniature dichroists" and potentially offers higher sensitivity, wider dynamic range, improved global shutter speed, and variable sensitivity; and

[0162] The article, titled "(BRIGHT IDEA FOR LOWLIGHT PHOTOGRAPHY)" (https: / / unews.utah.edu / bright-idea-for-lowlight-photography / ) (published October 27, 2015, accessed June 18, 2019), mentions a camera color filter for digital cameras that allows three times more light than conventional filters, resulting in cleaner, more accurate images in low light. This new filter can be used in any type of digital camera, but it is particularly useful for smartphone cameras. The filter is approximately micrometers thick (100 times thinner than a human hair). The filter comprises a glass wafer with precisely designed micro-ridges etched on one side. These ridges bend light in a certain way as it passes through, creating a series of color patterns or codes. Software then reads these codes to determine what colors they represent.

[0163] In some embodiments, an apparatus and method are provided for measuring transmitted light signals at dual wavelengths in the red and green regions using an RGB color camera sensor via a single sensor image or a single camera capture. White light from a white light source first passes through a dual-band bandpass filter to make the white light a mixture of two wavelengths with narrow bandwidths, for example, green and red light, respectively. After the bandpass filter, the resulting purified light passes through a sample (i.e., an interrogation sample) and an imaging lens, and finally reaches an RGB color filter located in front of the camera sensor or similar sensor. The R filter or R channel of the RGB color filter almost exclusively collects the narrow-bandwidth red light in the mixed light, and the G filter or R channel of the RGB color filter almost exclusively collects the narrow-bandwidth green light in the mixed light. The center wavelength of the dual-band bandpass filter in the green region can be any wavelength, for example, from 500 to 550 nm, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value. The center wavelength of the dual-band bandpass filter in the red region can be any wavelength from 600 to 700 nm, for example, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value.

[0164] In some embodiments, an apparatus and method are provided for measuring emitted signals at dual wavelengths in the red and blue regions using an RGB color camera sensor via a single sensor image or a single camera capture. White light from a white light source first passes through a dual-band bandpass filter to make the white light a mixture of two wavelengths with narrow bandwidths, such as blue and red light, respectively. After the bandpass filter, the resulting purified light passes through a sample (i.e., an interrogation sample) and an imaging lens, and finally reaches an RGB color filter located in front of the camera sensor or similar sensor. The R filter or R channel of the RGB color filter almost exclusively collects the narrow-bandwidth red light in the mixed light, and the B filter or B channel of the RGB color filter almost exclusively collects the narrow-bandwidth blue light in the mixed light. The center wavelength of the dual-band bandpass filter in the blue region can be any wavelength, for example, from 400 to 450 nm, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value. The center wavelength of the dual-band bandpass filter in the red region can be any wavelength from 600 to 700 nm, for example, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value.

[0165] In some embodiments, an apparatus and method are provided for measuring emitted signals at dual wavelengths in the green and blue regions using an RGB color camera sensor via a single sensor image or a single camera capture. White light from a white light source first passes through a dual-band bandpass filter to make the white light a mixture of two wavelengths with narrow bandwidths, such as green and blue light, respectively. After the bandpass filter, the resulting purified light passes through a sample (i.e., an interrogation sample) and an imaging lens, and finally reaches an RGB color filter located in front of the camera sensor or similar sensor. The G filter or G channel of the RGB color filter almost exclusively collects the narrow-bandwidth green light in the mixed light, and the B filter or B channel of the RGB color filter almost exclusively collects the narrow-bandwidth blue light in the mixed light. The center wavelength of the dual-band bandpass filter in the blue region can be any wavelength, for example, from 400 to 450 nm, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value. The center wavelength of the dual-band bandpass filter in the green region can be any wavelength from 500 to 550 nm, for example, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value.

[0166] In some embodiments, an apparatus and method are provided for measuring emitted signals at three wavelengths in the green, blue, and red regions using an RGB color camera sensor via a single sensor image or a single camera capture. White light from a white light source first passes through a three-band bandpass filter to make the white light a mixture of three wavelengths of light with narrow bandwidths, for example, green, blue, and red, respectively. After the bandpass filter, the resulting purified light passes through a sample (i.e., an interrogation sample) and an imaging lens, and finally reaches an RGB color filter located in front of the camera sensor or similar sensor. The G filter or G channel of the RGB color filter almost exclusively collects the narrow bandwidth green light in the mixed light, the B filter or B channel of the RGB color filter almost exclusively collects the narrow bandwidth blue light in the mixed light, and the R filter or R channel almost exclusively collects the narrow bandwidth red light in the mixed light. The center wavelength of the three-band bandpass filter in the blue region can be any wavelength, for example, from 400 nm to 450 nm, and the bandwidth can be 1 nm, 5 nm, 20 nm, 50 nm, or any value between them. The center wavelength of the three-band bandpass filter in the green region can be any wavelength from, for example, 500 nm to 550 nm, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value. The center wavelength of the three-band bandpass filter in the red region can be any wavelength from, for example, 600 nm to 700 nm, and the bandwidth can be, for example, 1 nm, 5 nm, 20 nm, 50 nm, or any intermediate value.

[0167] In some embodiments, an apparatus and method are provided for measuring transmitted light signals at three wavelengths in the green, blue, and red regions using an RGB color camera sensor through a single sensor image or a single camera capture. The location of the dual-band or triple-band bandpass filter can be, for example, between two lenses or between imaging lens groups.

[0168] In one embodiment, this disclosure provides:

[0169] An apparatus comprising, in the order listed:

[0170] A single white light source;

[0171] Dual-band or triple-band bandpass filters are used to filter white light into quantity and color before interrogating the sample;

[0172] A cavity for receiving samples;

[0173] An optional imaging lens is used to enhance the imaging on the sensor;

[0174] An RGB Bayer filter, used to filter light emitted from the sample being questioned; and

[0175] A sensor used to receive filtered light from an RGB Bayer filter.

[0176] A method comprising:

[0177] A dual-band or tri-band bandpass filter is illuminated with light from a single white light source to produce filtered light that is a mixture of two or three wavelengths of light with narrow bandwidths, each of which is 50 nm to 100 nm.

[0178] Question the sample with filtered light;

[0179] Optionally, the light emitted from the sample being questioned can be adjusted or directed through the imaging lens;

[0180] Light emitted from the imaging lens is passed through an RGB Bayer filter;

[0181] Sensors are used to sense and record light emitted from an RGB Bayer filter; and

[0182] Image processing senses and records light to remove or eliminate color overlap; and

[0183] Analyze data for a specific analyte.

[0184] An apparatus comprising, in the order listed:

[0185] A single white light source;

[0186] A cavity for receiving samples;

[0187] Dual-band or triple-band bandpass filters are used to selectively filter light emitted from the sample being questioned into component colors;

[0188] Imaging lens;

[0189] An RGB Bayer filter for filtering light emitted from the imaging lens; and

[0190] A sensor used to receive filtered light from an RGB Bayer filter.

[0191] A method comprising:

[0192] Interrogating the sample with light from a single white light source; and

[0193] The light emitted from the sample being questioned is filtered using a dual-band or triple-band bandpass filter to produce a mixture of light with two or three wavelengths that has a narrow band or wide bandwidth.

[0194] Optionally, an imaging lens can be used to adjust a mixture of two-wavelength or three-wavelength light with narrow bands;

[0195] Light emitted from the imaging lens passes through the RGB Bayer filter;

[0196] Sensors are used to sense and record light emitted from an RGB Bayer filter; and

[0197] Image processing senses and records light to remove or eliminate color overlap; and

[0198] Analyze data for a specific analyte.

[0199] Example. An optical setup for measuring hemoglobin in a QMAX card with a dual bandpass filter setup.

[0200] Figure X1 The optical setup for measuring hemoglobin in a QMAX card with a dual bandpass filter configuration is shown. The filter before the LED can be, for example: (A) a single wavelength setting (550 nm); (b) two separate wavelength settings (550 nm + 650 nm); and (c) a dual BP wavelength (525 nm + 680 nm). The bandwidth of the filter is between 20 nm and 60 nm.

[0201] In the experiment, the QMAX device consisted of two plates. The first plate was a flat PMMA substrate, 1 mm thick and measuring 30 mm × 24 mm. The second plate was a 175 μm thick PMMA film with a 24 mm × 22 mm micropillar array on it. This column array had a column size of 30 μm × 40 μm, a column-to-column distance of 80 μm, and a column height of either 10 μm or 30 μm. A reagent containing acridine orange dye and a zwittergent was uniformly coated onto the second plate (X-plate).

[0202] The sample was fresh whole blood (7 uL, 30 μm column height), which was dropped into the first plate and pressed down by the second plate.

[0203] Figure X2 The HgB performance on the iMOST platform relative to the HemoCue 301 commercial HgB analyzer is shown using: (A) a single wavelength setting (550 nm), (b) two separate wavelength settings (550 nm + 650 nm), and (c) dual BP wavelengths (525 nm + 680 nm).

[0204] A total of 38 samples were measured, including 25K2EDTA venous blood collected from patients at a local hospital (Hunterdon Medical Center, NJ, US) and 13 fresh finger-prick blood samples. The error |Δ| for each sample was calculated. The mean errors were (a) 3.2%, (b) 2.6%, and (c) 2.5%. Compared with the other two results, the dual-bandpass method had the lowest error and provided superior results, i.e., greater accuracy.

[0205] Furthermore, the correlation factor between iMOST and the HemoCue 301 commercial HgB analyzer was 95.3% for (a) single-wavelength measurements, 96.1% for (b) two separate wavelength measurements, and 96.6% for (c) dual-bandpass wavelength measurements. Similarly, dual-bandpass wavelength measurements showed the best correlation between iMOST and the reference method.

[0206] In another measurement using the dual-bandpass filter iMOST setup, Figure X3 shows the HgB performance of the iMOST platform relative to the HemoCue 301 commercial HgB analyzer, using a total of 60 samples (K2EDTA venous blood collected from patients at a local hospital) collected over 5 days. The difference |Δ| for each sample was calculated. The mean difference was 2.3%, with a correlation R = 98.5%, a slope of 0.975, and a bias of 0.2%. Notably, 59 out of the 60 samples (i.e., 98%) were within the allowable total error of ±7%.

[0207] Microstructured flow-guided printing for uniform reagent coating

[0208] A novel reagent printing method has been used to produce new Qmax cards. These new cards can be used to measure, for example, HgB, WBC, and other analytes.

[0209] In previous disclosures, inkjet droplet printing was used. For example, small droplets in the range of 1 nL to 100 nL were dropped onto an X-plate at a period of 100 μm to 1000 μm and then dried. This printing method typically creates droplet boundaries on the plate and is non-uniform.

[0210] In some embodiments, this disclosure provides a guided flow printing method with novel reagents. The reagents are hydrophilic and are used to make the plate hydrophilic and to give the plate a contact angle of, for example, less than 45 degrees. In the case where the plate has microstructures (e.g., pillars), the hydrophilic reagent is uniformly guided by the microstructures to form a coating on the plate, and a uniform coating is obtained without the aforementioned unsightly droplet boundaries.

[0211] In the disclosed guided flow printing method, the solvent in the reagent is selected to have a small contact angle with the plate. The solvent may include, for example, alcohols, IPA, acetone, aromatic compounds, benzene, toluene, methanol, esters and ethers, ketones, acetone, amines, nitrated and halogenated hydrocarbons, or similar solvents, and mixtures thereof.

[0212] In the disclosed guided flow printing method, the plate is treated to have a small contact angle with the reagent. Treatment may include, for example, coating a hydrophilic layer, coating the plate with a surfactant, plasma treatment, ozone treatment, generating hydrophilic chemical bonds, or combinations thereof.

[0213] In one example of the guided-flow coating method, the X-plate in the Q-card uses a reagent formulation containing acridine orange dye at a concentration of 0.4 mg / mL to 1.0 mg / mL and a zwitterionic detergent at a concentration of 0.5 mg / mL to 1.5 mg / mL in an aqueous alcohol solution of over 90%. 5 μL to 8 μL of the reagent is dropped onto the X-plate in a column array with a thickness of 175 μm, a column height of 30 μm, a period of 100 μm, and a column size of 30 × 40 μm. The reagent is evenly spread on the X-plate into squares approximately 15 mm to 25 mm in size and then dried.

[0214] Figure X4 shows fluorescence photographs of two Q-cards: (a) before blood addition and (b) after blood addition. One Q-card was printed in water using an inkjet dot printing method with a droplet size of 10 nL, a period of 600 μm, and reagents. The other Q-card used a guided flow printing method with an 8 μL volume, and the reagents were dissolved in alcohol of more than 90%. Clearly, the guided flow printing method provided a uniform surface coating on the card without droplet boundaries, both before and after blood addition.

[0215] Other transmission measurement applications

[0216] Other applications of this guided flow printing method may include, for example:

[0217] (1) Applications based on transmission and / or absorption, such as HgB and HCT measurements in CBC;

[0218] (2) Applications based on transmission and / or absorption, such as OD (optical density) measurement for any biological or chemical sample;

[0219] (3) Applications based on transmission and / or absorption, such as molecular density measurements for any biological or chemical sample;

[0220] (4) Applications based on transmission and / or absorption, such as cell or tissue density measurements for any biological or chemical sample;

[0221] (5) Applications based on transmission and / or absorption, such as bead and particle density measurements for any biological or chemical sample;

[0222] (6) Applications based on transmission and / or absorption, such as turbidity measurement for any biological or chemical sample;

[0223] (7) Colorimetric applications, such as colorimetric enzyme reactions, for example, in glucose measurement, cholesterol measurement and triglyceride measurement;

[0224] (8) All applications can use transmission or absorption in sample analysis.

[0225] The apparatus of any of the preceding device claims may have one or more of the following functions:

[0226] Compositional analysis, such as fiber identification and mixture analysis;

[0227] Color fastness tests in washing, laundry, bleaching, etc.;

[0228] Analysis of wet treatment processes such as scrubbing and bleaching in laboratory samples;

[0229] Sample defect analysis;

[0230] General chemical tests include carbonization, dissolution, peeling and re-dyeing, textile absorbency, bleaching loss, drying shrinkage, etc.

[0231] Parameter testing includes density, nitrogen content, foaming tendency, emulsion stability, etc.

[0232] Analysis of water, effluent and sludge, including parameters such as pH, density, conductivity, odor, turbidity, total dissolved solids, total hardness, acidity, and total chlorine;

[0233] Ecological parameter testing, including tests for free formaldehyde, copper, cobalt, lead, mercury, polyvinyl chloride, APEO / NPEO, and other parameters;

[0234] The apparatus of any of the preceding device claims may have one or more of the following functions and purposes:

[0235] 1) Determine the interactions between the sample and other known substances;

[0236] 2) Determine the composition of the sample;

[0237] 3) Provide standard data for other scientific, medical, and quality assurance functions;

[0238] 4) Confirm the suitability for the end use;

[0239] 5) To provide a basis for technical exchanges;

[0240] 6) Provide technical means for comparing several options;

[0241] 7) Provide evidence for legal proceedings;

[0242] 8) Determine or verify whether the quality standards, regulations, or contractual requirements are met.

[0243] I. Colorimetric sample cards with laminated diffused layers

[0244] In measurements involving the detection of optical signals, such as colorimetry, a small container holds a liquid sample, and a beam of light is passed through the sample to measure the light absorption spectrum or the color of the sample. When the sample is very dilute, the light or color becomes faint and difficult to measure.

[0245] In a specific test scenario of interest, the sensor used to collect the light signal can be located above and very close to the sample. The light source can be placed below the sample (i.e., on the opposite side of the sample's plane), and the sensor measures, for example, the optical density after the light has passed through the sample.

[0246] To enhance the color signal generated from a sample, light should travel a longer distance within the sample to be absorbed more. In some embodiments, this disclosure provides a colorimetric sample card (e.g., a surface-modified Qmax card) device for holding thin samples, which can enhance the color signal by increasing the light path through the sample.

[0247] The sample card device includes a top plate, a bottom plate, and a thin sample layer located between the top plate and the bottom plate, as well as a diffuse layer located on the sample surface that is first illuminated by interrogation light. The diffuse layer may be, for example, a translucent layer attached to or integrated with the first illuminated surface of the sample card, such as the diffuse layer being laminated on the back of the bottom plate.

[0248] In an embodiment, this disclosure provides a testing apparatus for measuring the colorimetric signal of a sample liquid in a sample card device having a laminated diffuse layer, the apparatus comprising:

[0249] light source;

[0250] Cavities or holders for sample cards (i.e., workpieces, such as improved Qmax colorimetric sample cards with a laminated diffuse layer on the surface of the sample card, which is the first irradiated surface closest to the light source); and

[0251] sensor.

[0252] Light from the back of the sample card is incident on a light source and first strikes a diffuser layer. The diffuser layer deflects the light beam and increases the light path through the liquid layer of the sample, thereby increasing the exposure of the colorimetric sample. A sensor is placed above the sample card (i.e., on the opposite side of the sample) to collect the transmitted light signal.

[0253] Figure N6 An example of a sample card for on-the-spot colorimetric determination of sample liquid is shown, comprising a top plate, a bottom plate, a diffuse layer laminated on the back of the bottom plate, and the sample liquid between the top plate and the bottom plate.

[0254] Figure N7 A test apparatus for measuring the colorimetric signal of a sample liquid in a sample card with a laminated diffuse layer is shown. The apparatus includes, for example, a light source, a colorimetric sample card with a laminated diffuse layer, and a sensor. The light source illuminates light from the back of the sample card and first illuminates the diffuse layer. The diffuse layer deflects the light beam and increases the optical path of the light traveling in the sample liquid layer to increase the exposure of the colorimetric sample. The sensor is placed above the sample card to collect the transmitted light signal.

[0255] In some embodiments, the diffuse layer may be, for example, a white diffuse film laminated on the back of a substrate, comprising: a film made of a white plastic material; a transparent film material having a textured surface structure; or a combination thereof.

[0256] In some embodiments, lamination can be achieved, for example, by thermal bonding, adhesives, or other methods.

[0257] In some embodiments, the diffuse layer may be, for example, a white coating applied to the back of the substrate.

[0258] In some embodiments, the diffuse layer may be formed, for example, by surface treatment on the back side of a substrate having a textured structure.

[0259] II. Lateral Dimension Correction (LDC) Algorithm

[0260] Columns are used for lateral dimension correction (LDC), such as Figure X7 As shown, an HgB image is divided into non-overlapping regions.

[0261] For each region, the LDC algorithm is run as follows:

[0262] 1. Operation column detection;

[0263] 2. Check the column detection score (if the check fails, reject the image sample);

[0264] 3. After the image passes the column detection score check, run the horizontal column check;

[0265] 4. Run a vertical column check;

[0266] 5. Check the intercolumn distance (if the check fails, reject the image sample);

[0267] 6. After the image is checked for inter-cylinder distance, the algorithm generates cylindrical loops; and

[0268] 7. Calculate the area under the center of the column and derive the LDC.

[0269] Sample holder

[0270] According to the present invention, as shown in Figures 1 and 2, one embodiment of a sample holder (i.e., optical analysis card) called OAC for analyzing analytes (e.g., hemoglobin in a blood sample) using light transmission comprises:

[0271] The components include a first plate, a second plate, a light guide spacer (LGS), a sampling area, and a reference area, wherein:

[0272] (i) The first plate and the second plate are configured to sandwich a sample for light transmission analysis between the plates in a thin layer, and each plate has a sample contact area on its inner surface that contacts the sample.

[0273] (ii) The light-guiding spacer (LGS) has a cylindrical shape, sandwiched between two plates, wherein each end of the column is in direct contact with one of the plates forming the LGS-plate contact area, and is configured to allow light to pass from the first plate through the LGS to the second plate without passing through the sample.

[0274] (iii) The sampling region is the area where the light sequentially passes through the first plate, the sample, and the second plate, wherein the sampling region does not have the LGS; and

[0275] (iv) The reference area is the region in which light passes through the first plate, the light-guiding spacer and the second plate in sequence but does not pass through the sample;

[0276] The LGS contact area and the lateral cross-section of the LGS are larger than the wavelength of light.

[0277] The optical guide spacer is surrounded by or close to the sample; and

[0278] The sample in the sampling area has a thickness of 500 μm or less.

[0279] At least a portion of the plate in the reference area and the sampling area is transparent.

[0280] According to the present invention, as shown in Figures 1 and 2, a sample holder called OAC (i.e., optical analysis card) has at least a "sampling area" and a "reference area", and the light absorption coefficient of the sample layer is determined by obtaining the ratio of light transmitted through the sampling area to light transmitted through the reference area.

[0281] In some embodiments, the sample holder (also referred to as a device) further includes a plurality of light-guiding spacers having substantially uniform height, and wherein at least one of the light-guiding spacers is located within the sample contact area.

[0282] In some embodiments, the first and second boards are connected to LGS ( Figure 1A ) Fixed. In some embodiments, such as Figure 1B As shown, the first and second plates can be moved relative to each other into different configurations, including an open configuration and a closed configuration. In the open configuration, the plates are partially separated and a sample is deposited. In the closed configuration, the first and second plates are in contact with the flat ends of the LGS, respectively.

[0283] In some embodiments, the first and second plates in the sample area and the reference have a uniform thickness and are transparent.

[0284] The material of the plate is plastic, glass, or other materials described in this disclosure.

[0285] In some embodiments, other spacers are used to adjust the spacing between the first plate and the second plate, and thus adjust the sample thickness.

[0286] Sample OD measurement method.

[0287] In some embodiments, the properties of a sample can be determined by measuring the OD of a thin layer of the sample, wherein the OD is determined by the ratio of light transmitted through a sampling region of the OAC to light transmitted through a reference region of the OAC.

[0288] In some embodiments, images of the sample in the sample holder are captured by a camera and analyzed.

[0289] In some embodiments, the wavelength of the light is in the range of 500nm to 1200nm, 200nm to 3000nm, 3000nm to 30,000nm, or 100nm to 200nm.

[0290] A) Light absorption through the sample, determined by light transmission in the sampling and reference regions.

[0291] For light with incident intensity of 0, using Beer-Lambert's law, the transmitted light intensity I through the sample... s It is given by the following formula:

[0292]

[0293] Where, ε s ε is the extinction coefficient of the sample (e.g., hemoglobin), c is the average concentration of the sample (e.g., hemoglobin), and L is the length of the optical path through the sample. (ε in cm) -1 / M is the unit, c is the unit of M, L is the unit of cm), and OD s It can refer to the optical density of the sample.

[0294] For light with incident intensity of 0, applying Beer-Lambert's law, passing through a certain length L... r The light intensity I transmitted through the optical guide spacer r It is given by the following formula:

[0295]

[0296] Where α r is the absorption coefficient of the photoconductor spacer, and L is the length of the optical path through the sample, and OD s It can refer to the optical density passing through the optical guide spacer as a reference.

[0297] Subtracting the second equation from the first equation yields:

[0298]

[0299] According to the present invention, the above equation shows that, without measuring the incident light (assuming that the incident light in the two regions is substantially the same), the absorption coefficient of the sample layer can be determined by taking the ratio of the transmitted light passing through the sampling region to the transmitted light passing through the reference region.

[0300] Use spacers to form a uniform thin sample layer.

[0301] Figure 1 is an illustration of a CROF (Compression-Regulated Open Flow) embodiment. Figure (a) shows a first plate and a second plate, wherein the first plate has a spacer. Figure (b) illustrates sample deposition on the first plate (shown), the second plate (not shown), or both (not shown) in an open configuration. Figure (c) illustrates (i) using two plates to disperse the sample (the sample flows between the plates) and reduce the sample thickness, and (ii) using spacers and plates in a closed configuration to regulate the sample thickness. The inner surface of each plate has one or more binding sites and / or storage sites (not shown).

[0302] B) Two types of hemoglobin

[0303] There are two types of hemoglobin in the blood. Oxyhemoglobin [HbO2] is the form of hemoglobin that binds oxygen, while deoxyhemoglobin [Hb] is the form of hemoglobin that does not bind oxygen. Typically, oxyhemoglobin [HbO2] accounts for about 75% in veins and about 90% in arteries.

[0304] Total hemoglobin concentration = [HbO2] + [Hb].

[0305] Two types of hemoglobin can have different extinction coefficients (i.e., light absorption) at different wavelengths, such as... Figure 4 As shown. Therefore, by measuring the light absorption of blood in different wavelength ranges, the concentrations of [HbO2] and [Hb] in the blood can be determined separately.

[0306] C) Optical transmission sample analysis is performed by comparing the light transmission from the sampling area and the reference area.

[0307] According to the present invention, light absorption (and optical density (“OD”) through a thin sample layer is measured by comparing light transmission from a sample region and a reference region.

[0308] In some cases, the comparison is made on the ratio of light transmission from the sample region to the reference region.

[0309] D) Improved optical transmission sample analysis

[0310] In many practical measurement scenarios, there are numerous drawbacks that can significantly reduce the accuracy of OD measurements. For example, the sample in the sample holder and / or the sample holder itself may have uneven thickness. Defects may exist in the sample or sample holder, such as bubbles, dust, or other defects that may have different light transmission than those passing through a perfect (i.e., ideal) sample. Light intensity may be uneven throughout the measurement area.

[0311] This invention offers several ways to reduce errors in optical transmission sample analysis (OTSA) caused by defects. According to the invention, to improve the accuracy of OD measurements, the following features, apparatuses, and methods are used individually or in combination (i.e., in Section 1.4 and its subsections).

[0312] I. Reduction of light scattering at LGS sidewalls and / or LGS-plate interface

[0313] According to one embodiment of the OD measurement method of the present invention, which measures the light intensity of the sample region and the reference region and then takes the ratio of the two intensities, the measurement accuracy will be significantly reduced if the light passing through the reference region has strong scattering from (a) the LGS sidewall or (b) the LGS, or if the light from the sample region has significant scattering from the nearby LGS sidewall.

[0314] To reduce the influence of light scattered from the LGS sidewalls on the light from the reference region, the edge of the reference region used for OD determination should be a certain distance away from the LGS sidewalls. Since the reference region cannot be smaller than the wavelength of light to avoid significant light diffraction, the cross-section of the LGS should be at least larger than the wavelength of light to reduce the influence of light scattered from the LGS sidewalls on the light from the reference region.

[0315] In some embodiments, the edge of the reference area used for OD determination is a certain distance from the LGS sidewall.

[0316] In some embodiments, the cross-section of the LGS should be larger than the wavelength of light, and the edge of the reference area used for OD determination should be a certain distance from the sidewall of the LGS.

[0317] Similar to light from the reference region, in order to reduce the impact of light scattering on light from the sampling region, the edge of the sampling region should be a certain distance from the LGS sidewall.

[0318] In some embodiments, the edge of the sampling area used for OD determination is a certain distance from the LGS sidewall.

[0319] In some embodiments, the edge of the reference region used for OD determination is a certain distance from the LGS sidewall, and the edge of the sampling region used for OD determination is a certain distance from the LGS sidewall.

[0320] In some embodiments, the cross-section of the LGS should be larger than the wavelength of light, the edge of the reference area used for OD determination is a certain distance from the LGS sidewall, and the edge of the sampling area used for OD determination is a certain distance from the LGS sidewall.

[0321] II. The areas of the reference region and the sampling region, and the distance between them.

[0322] When determining the OD of a sample by taking the ratio of the light intensity passing through the sample region to that passing through the reference region, it is assumed that the incident light in each region has the same intensity, or that the thicknesses of the first and second plates and the sample are the same or known in both the sampling and reference regions. However, in many practical optical systems, these assumptions do not hold, which introduces uncertainty (i.e., error) in the determination of OD. For example, in practice, the incident light intensity in sample light transmission measurements is not uniform, especially with a large illuminated area; and the thicknesses of the first and second plates and the sample are different or known in both the sampling and reference regions, and each can have significant variations.

[0323] According to the present invention, one way to reduce error is to limit the area of ​​the sampling region and the reference region used to determine the OD of the sample, or to optimize the distance between the sampling region and the reference region (i.e., the sampling exclusion distance) to avoid significant light scattering caused by the LGS sidewalls, or both.

[0324] In some embodiments, the area of ​​the sampling region and the distance between the sampling region and the reference region are a combination of the above two segments.

[0325] III. Multiple sampling regions and reference regions

[0326] Using a pair of sample and reference regions can lead to large errors. This is for several reasons: (i) since the spatial variations in the thickness of the first plate, the second plate, and the sample are random, a single pair of sample and reference regions may not represent most of the sample; and (ii) since the number and location of optical defects are also random, these defects may occur in the sampling and / or reference regions, making the sample and reference region pair unusable in OTSA.

[0327] To address these issues, according to the present invention, multiple pairs of SR regions are used.

[0328] In some embodiments, the OAC comprises multiple pairs of SR regions, wherein the distance between the centers of two adjacent SR regions is substantially periodic or aperiodic.

[0329] According to the present invention, reagents for facilitating testing are deposited on the inner surface of the OAC plate, said reagents including but not limited to staining reagents, surfactants, antibodies, proteins, and nucleic acids.

[0330] Optical guide pillars (e.g., spacers)

[0331] In some embodiments, the optical guide spacers are substantially periodic and predetermined in terms of spacing.

[0332] Light guide post height

[0333] In some embodiments, the height of the light guide spacer is 1 μm, 2 μm, 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1,000 μm, 2,000 μm, 5,000 μm, 10,000 μm, or within any two values.

[0334] Optical guide post period

[0335] In some embodiments, the spacers (also referred to as LGS) are arranged in a periodic array, which are 1 μm, 2 μm, 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 300 μm, 500 μm, 1,000 μm, 2,000 μm, 5,000 μm, 10,000 μm, or within any two values.

[0336] In some embodiments, preferred spacers (also known as LGS) are arranged in a periodic array, which are 1 μm, 2 μm, 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 300 μm, or in any range between two values.

[0337] Light guide post spacing

[0338] In some embodiments, the spacer (also referred to as LGS) has a spacing distance of 1 μm, 2 μm, 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 300 μm, 500 μm, 1,000 μm, 2,000 μm, 5,000 μm, 10,000 μm, or within any two values.

[0339] In some embodiments, preferred spacers (also referred to as LGS) have a spacing distance of 1 μm, 2 μm, 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 300 μm, or within any two values.

[0340] Geometry of Light Guide Spacer (LGS)

[0341] In some embodiments, the LGS has a columnar shape with substantially flat ends. In some embodiments, one or both ends of the LGS are made from a single piece by bonding, fusion, or other methods of attaching the LGS to one or both ends of the plate.

[0342] In some embodiments, the shape of the transverse cross section of LGS includes, but is not limited to, a circle, a rectangle, a square, a triangle, a polygon, a letter, a number, or a combination thereof.

[0343] In some embodiments, the average lateral cross-section of each light guide spacer (LGS) is 1 μm² (square micrometers), 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², 100,000 μm², 200,000 μm², 500,000 μm², 1 mm², 2 mm², 5 mm², 10 mm², 50 mm², or within any two values.

[0344] In some preferred embodiments, the average lateral cross-section of each light guide spacer is 1 μm² (square micrometers), 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², 100,000 μm², 200,000 μm², or within any two values.

[0345] In some preferred embodiments, the average lateral cross-section of each optical spacer is 1 μm², 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², or within any two values.

[0346] In some preferred embodiments, the average lateral cross-section of each light guide spacer is 1 μm², 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², or within any two values.

[0347] In some embodiments, the average transverse cross section of each light guide spacer is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times larger than the wavelength of light passing through the reference region, or within any two values.

[0348] In some preferred embodiments, the average lateral cross section of each light guide spacer is 1, 2, 3, 5, 10, 20, 50, 100, 200, or 500 times larger than the wavelength of light passing through the reference region, or within any two values.

[0349] Reference area geometry

[0350] shape

[0351] In some embodiments, the reference region is smaller than the minimum lateral cross-sectional dimension of the light guide post. One advantage is that it avoids or reduces light scattering from the light guide sidewalls that could affect the reference signal.

[0352] In some embodiments, the minimum distance between the edge of the light guide spacer and the edge of the reference area (i.e., the exclusion distance for reference) is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, or within any two values.

[0353] In some preferred embodiments, the minimum distance between the edge of the light guide spacer and the edge of the reference area is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 100 μm, 200 μm, or within any two values.

[0354] In some preferred embodiments, the minimum distance between the edge of the light guide spacer and the edge of the reference area is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, or within any two values.

[0355] In some preferred embodiments, the distance between the edge of the sampling area and the reference area is 1, 2, 3, 5, 10, or 20 times greater than the wavelength of the measurement wavelength (i.e., the light passing through the reference area).

[0356] In some preferred embodiments, the distance between the edge of the sampling area and the reference area is 20, 30, 50, or 100 times greater than the wavelength of the measurement wavelength.

[0357] In some preferred embodiments, the minimum distance between the edge of the light guide spacer and the edge of the reference region is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times greater than the wavelength passing through the reference region, or within any two values.

[0358] In some preferred embodiments, the minimum distance between the edge of the light guide spacer and the edge of the reference area is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times greater than the wavelength passing through the sampling area, or within any two values.

[0359] The ratio of the reference area to the area of ​​the optical guide spacer is 3 / 10, 2 / 5, 1 / 2, 3 / 5, 7 / 10, 4 / 5, or within any two values.

[0360] Sampling region geometry

[0361] In some embodiments, the edge of the sampling region is a certain distance from the edge of the light guide post (i.e., the exclusion distance used for sampling). One advantage is to avoid or reduce light scattering from the light guide sidewalls that could affect the reference signal.

[0362] In some embodiments, the exclusion distance used for sampling is 1 μm (micrometer), 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, or within any two values.

[0363] In some preferred embodiments, the exclusion distance used for sampling is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 100 μm, 200 μm, or within any two values.

[0364] In some preferred embodiments, the exclusion distance used for sampling is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, or within any two values.

[0365] In some preferred embodiments, the exclusion distance used for sampling is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, or within any two values.

[0366] In some preferred embodiments, the area of ​​the sampling region is 3 / 5, 7 / 10, 4 / 5, 9 / 10, 1, 11 / 10, 6 / 5, 13 / 10, 7 / 5, 3 / 2 of the periodic interval distance, or within any two values.

[0367] In some preferred embodiments, the distance between the edge of the sampling area and the edge of the light guide spacer is 1 / 5, 3 / 10, 2 / 5, 1 / 2, 3 / 5, 7 / 10, 4 / 5, 9 / 10, or 1 of the light guide spacer area, or within any two values.

[0368] In some preferred embodiments, the distance between the edge of the sampling region and the edge of the optical guide spacer is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times greater than the wavelength passing through the reference region, or within any two values.

[0369] In some preferred embodiments, the distance between the edge of the sampling region and the edge of the optical guide spacer is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times greater than the wavelength passing through the sampling region, or within any two values.

[0370] In some embodiments, the exclusion distance from the column wall is 7.5 μm to 10 μm.

[0371] In some embodiments, the exclusion distance from the column wall is 1 / 4 of the area of ​​the optical guide spacer.

[0372] In some embodiments, the sampling region boundary has a size of 120μm × 110μm;

[0373] The sampling area has an edge size of 60μm × 45μm;

[0374] The optical guide spacer or post has dimensions of 40μm × 30μm;

[0375] The reference area has dimensions of 20μm × 15μm.

[0376] In some embodiments, the area of ​​the reference region is half the size of the optical spacer region, the distance between the sampling region and the edge of the optical spacer is half the size of the optical spacer region, and the area of ​​the sampling region is equal to the periodic interval distance.

[0377] The distance between the sampling area and the reference area (i.e., the exclusion distance between the sample and the reference).

[0378] In some embodiments, the distance between the edge of the sampling area and the reference area is 1 μm (micrometer), 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, or within any two values.

[0379] In some preferred embodiments, the distance between the edge of the sampling area and the reference area is 30 μm to 50 μm, 20 μm to 60 μm, 10 μm to 70 μm, 5 μm to 75 μm, or within any two values.

[0380] In some preferred embodiments, the distance between the edge of the sampling region and the reference region is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times greater than the wavelength passing through the reference region, or within any two values.

[0381] In some embodiments, the distance between the edge of the sampling area and the reference area is 2 / 5, 1 / 2, 3 / 5, 7 / 10, 4 / 5, 9 / 10, 1, 11 / 10, 6 / 5, 13 / 10, 7 / 5, 3 / 2, 8 / 5, 17 / 10 of the optical guide spacer area, or within any two values.

[0382] In some embodiments, the distance between the edge of the sampling region and the reference region is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times greater than the wavelength of the light passing through the reference region, or within any two values.

[0383] In some embodiments, the distance between the edge of the sampling region and the reference region is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times greater than the wavelength passing through the sampling region, or within any two values.

[0384] Some examples of Q card parameters

[0385] In some embodiments, the spacer height, the spacing between plates, and / or the sample thickness is approximately 30 μm. The spacer height, the spacing between plates, and / or the sample thickness is 20 μm to 40 μm. The spacer is a rectangular shape with rounded corners. The spacer is circular. The lateral dimension of the spacer is approximately 30 μm × 40 μm. The lateral dimension of the spacer is 10 μm to 40 μm. The diameter of the rounded corners of the spacer is 10 μm. The spacers are arranged in a rectangular dot array. The spacing between the spacers is approximately 80 μm. The spacing between the spacers is 70 μm to 150 μm. One plate of the Q-card is 27 mm long and 22 mm wide. One plate of the Q-card is 32 mm long and 24 mm wide. The area of ​​one plate is approximately 600 mm². 2 The area of ​​the other board is approximately 750 mm². 2 The thickness of a single Q-card is approximately 175 μm. The thickness of a single Q-card is approximately 1 mm. The notch area on a QMAX card ranges from 10 to 30 mm². 2 Within the specified range. The cut is semi-circular, with a diameter of 3 to 6 mm. The notch has a width of 3 mm and a length of 6 mm. The width of the hinge joint is approximately 6 mm. The length of the hinge joint is approximately 20 mm. The thickness of the hinge is approximately 70 μm.

[0386] Reagents are coated into an array via droplet printing. Reagents are also coated via spraying. Acridine orange or other staining reagents are applied to a first plate, a second plate, or both. Zwitterionic detergents or other detergents are applied to a first plate, a second plate, or both. Acridine orange is applied at 5-20 ng / mm. 2 The area concentration of the coating is applied to the plate, and the zwitterionic detergent is applied at a concentration of 10-30 ng / mm². 2 The area concentration is coated on the plate.

[0387] The first and second plates are made of poly(methyl methacrylate). Landing marks for the blood droplets are located on the outer surface of either the first or second plate. The landing marks are small dots or small crosses. The landing marks are outside the field of view of the image. The landing marks are located near the center of the card. In some embodiments, at least one of the plates is transparent.

[0388] Example of adapter filter parameters

[0389] In one embodiment, the dual bandpass wavelength filter has at least one center wavelength of 500 nm to 550 nm.

[0390] In one embodiment, the dual bandpass wavelength filter has at least one center wavelength of 600 nm to 700 nm.

[0391] In one embodiment, the dual bandpass wavelength filter has a bandwidth of 10 nm to 50 nm.

[0392] In one embodiment, the dual bandpass wavelength filter has a bandwidth of 50 nm to 100 nm.

[0393] In one embodiment, the dual bandpass wavelength filter has a bandwidth of 50 nm to 100 nm.

[0394] The apparatus, device, or method of any of the preceding claims, further comprising a reflector that reflects light emitted from the passive illuminator toward the diffuser.

[0395] In one embodiment, a wavelength-dependent attenuator is added before the dual bandpass filter to balance the light intensity from the two bands.

[0396] Examples of optical guide spacers, sampling areas, and reference areas

[0397] In some embodiments, the exclusion distance from the pillar wall is 7.5 μm to 10 μm. In some embodiments, the exclusion distance from the pillar wall is 1 / 4 of the area of ​​the light guide spacer. In some embodiments, the sampling area boundary has a size of 120 μm by 110 μm; the sampling area edge has a size of 60 μm × 45 μm; the light guide spacer or pillar has a size of 40 μm × 30 μm; and the reference area has a size of 20 μm × 15 μm.

[0398] In some embodiments, the area of ​​the reference region is half the size of the optical spacer region, the distance between the sampling region and the edge of the optical spacer is half the size of the optical spacer region, and the area of ​​the sampling region is equal to the periodic interval distance.

[0399] Example of color de-interference matrix and HgB calculation algorithm

[0400] A set of dc-RAW parameters (-W -r 1 1 1 1 -g 1 1 -o 0 -c -6 -T) is used before the noise reduction algorithm.

[0401] -W: Disable automatic brightness

[0402] -r 1 1 1 1: Sets the channel gain of RGBG to 1.

[0403] -g 1 1: Disable gamma correction

[0404] -c: Send the output to stdout.

[0405] -6: 16-bit output

[0406] -T: TIFF output

[0407] -o 0: Do not adjust the color space conversion; use RAW RGB.

[0408] A color de-interference matrix (also known as a "channel crosstalk matrix") is applied to derive new red and green channels to reduce color interference, as follows:

[0409]

[0410] Where a1, a2, b1, and b2 are the coefficients of the color de-interference matrix. In one example, a1, a2, b1, and b2 are in the range of 0 to 1.0.

[0411] The measurements and calibrations of a1, a2, b1, and b2 include experiments using two separate filters with precisely matched dual bandpass filters. Two images using the two separate filters are used as R... 新 and G 新 Reference standards.

[0412] The intensity ratios of the red and green channels, rred and rgreen, are used for HgB value calculation. The formula used for HgB value calculation is based on a linear combination of the logarithms of these two ratios:

[0413] HgB=Aln(r 红 )+Bln(r 绿 )+C

[0414] For example, regression analysis is performed on HgB blood samples with known HgB values ​​to predetermine three coefficients, A, B, and C. For instance, in determining A, B, and C using 50 whole blood samples (5 g / dL–25 g / dL) measured with a commercial HgB analyzer as Hemocue, regression is used to fit the values ​​of A, B, and C to match the commercial HgB analyzer. A might be in the range of 20 to 30, B in the range of -20 to -30, and C in the range of 0 to 1.

[0415] Light transmission measurement / calculation using multiple light guide structures (LGS).

[0416] A method for measuring transmission and / or light absorption, comprising:

[0417] (a) A QMAX simple holder having a plurality of optical spacers having substantially uniform height, wherein more than one of these optical spacers is located within the sample contact area.

[0418] (b) Image the sample region;

[0419] (c) Using the image in (b), measure the light transmission through a reference region of the LGS, with the sample surrounding the same LGS, and then calculate the light absorption of the region surrounding the LGS. Repeat this measurement for another LGS.

[0420] (d) Average the light absorption in the sample with the light absorption in each LGS region.

[0421] LGS can take different configurations as described in this article, such as periodic LGS.

[0422] Hybrid optical guide structure (M-LGS).

[0423] M-LGS is a light-guiding structure in which the height of the structure is less than the height of the spacer, such that a portion of the spacer is filled by the sample (light passes through both the portion in the sample and the portion in the M-LGS). This is useful in calculating light absorption for different sample thicknesses.

[0424] Predicting HgB values ​​using a wavelength-based machine learning approach.

[0425] One approach to accurately predict HgB values ​​using only one wavelength is to use (1) machine learning to learn the scattering component from existing data, and / or (2) machine learning to learn the pure absorption component from existing data.

[0426] The challenge of predicting HgB using only a single wavelength measurement from 500 nm to 550 nm is that unlysed whole blood at this wavelength has both absorbing and scattering components. Absorption is used to predict the true value of HgB in whole blood, while scattering is an interference.

[0427] In the disclosure of this patent, a single wavelength or dual bandpass wavelength is used to measure the scattering component using another wavelength of 600nm to 800nm, and it is dispersed from a wavelength of 500nm to 550nm.

[0428] One way to predict the scattering and / or absorption components is to use machine learning to learn.

[0429] Annotated images are fed into a machine learning (ML) training module, where a model trainer trains an ML model based on the training data (annotated sample images). The input data is iteratively fed into the model trainer multiple times until a specific stopping criterion is met. The output of the ML training module is an ML model—a computational model built from data according to the training process in machine learning, which gives the computer the ability to independently perform certain tasks (e.g., detecting and classifying objects).

[0430] This application uses deep learning generative adversarial networks (GANs).

[0431] In a machine learning method for predicting the pure absorption component from a wavelength, training is performed in the following manner:

[0432] (1) A pair of experimental images using the same blood annotation Q card, one of whole blood without lysis (with scattering) and the other of whole blood with the same lysis (without scattering components from cells).

[0433] (2) The network was trained to accurately predict whole blood without cleaving it into cleaved whole blood.

[0434] (3) During training, pillars can be used as a constant reference area to set the baseline of the model.

[0435] In a machine learning method for predicting the pure scattering component from a single wavelength, training is performed in this manner:

[0436] (1) Experimental images of a pair of Q cards were annotated with the same blood, one of unlysed whole blood (with scattering) measured at 500-550 nm and the other of unlysed whole blood (with scattering) measured at 600-800 nm.

[0437] (2) The network is trained so that it can accurately predict measurements from 500-550 nm to 600-800 nm in whole blood without pyrolysis.

[0438] (3) During training, pillars can be used as a constant reference area to set the baseline of the model.

[0439] A deep learning-based method is proposed to convert single-channel hemoglobin images into equivalent fragmented images, which has minimal scattering effects for colorimetric-based hemoglobin calculations.

[0440] A method for automatically simulating large-scale fragmented hemoglobin training data based on blood samples with basic true hemoglobin values. This training data is used to train a deep model that converts single-channel hemoglobin images into fragmented images.

[0441] The pyrolysis reagent coating of the iMOST-HgB device

[0442] In some embodiments, a surfactant is coated onto a plate and dissolved in blood to achieve uniform distribution of red blood cells in the device, wherein the coating may be applied to a first plate or a second plate or both.

[0443] In some embodiments, a surfactant is coated onto a plate and dissolved in blood to lyse red blood cells in the device, wherein the coating may be on a first plate or a second plate, or both.

[0444] In some embodiments, the surfactants coated on the device include, but are not limited to, zwitterionic detergents, ASB-14, ASB-16, CHAPS, cationic surfactants NN-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (IIa), IIb, IIc, IId, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), and myristyl alcohol polyol. Sodium ether sulfate, docusate, perfluorooctane sulfonic acid, alkyl-aryl ether phosphates, alkyl ether phosphates, CTAB, hexadecylpyridine chloride (CPC), benzalkonium chloride (BAC), benzyl chloride (BZT), dimethyl octadecyl ammonium chloride, octadecyl dimethyl ammonium bromide (DODAB), cocamidopropyl hydroxysulfonate, cocamidopropyl betaine, narrow-range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonoxynol, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glyceryl monostearate, glyceryl monolaurate, sorbitol monolaurate, sorbitol monostearate, sorbitol tristearate, decyl glucoside, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0445] In some embodiments, the agents coated on the device that induce erythrocyte lysis include, but are not limited to, Pluronic F-127, Cremophor EL, Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium dodecyl sulfate, Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, tamoxifen, saponins, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, antimalarial drugs (quinine compounds), arsenic, dapsone, metals (chromium / chromates, platinum salts, nickel compounds, copper, lead, cisplatin), nitrites, nitrofurantoin, penicillin, phenapyridine (marofen), rho immunoglobulin, ribavirin, sulfonamides, and sulfones.

[0446] In some embodiments, the anticoagulant coated in the device includes, but is not limited to, EDTA such as dipotassium ethylenediaminetetraacetate (K2EDTA), tripotassium ethylenediaminetetraacetate (K3EDTA), coumarin (vitamin K antagonist), warfarin (coumarin), acetocoumarin, phenylpropanoidin, atromanin, phenylindanone, heparin, fondaparin and edaparin, dabigatran, rivaroxaban, apixaban, edoxaban, betrixiban, NOAC, hirudin, lepirudin, bivalirudin, argatroban, dabigatran, batroxobin, schizofibrase, vitamin E, sodium citrate, glucose citrate, oxalate (e.g., fluoxetine), deltaparin, disiludin, and enoxaparin.

[0447] In some embodiments, to achieve uniform distribution of red blood cells in the device, a zwitterionic detergent is coated onto the plate, preferably at an area concentration of 3 ng / mm². 2 5ng / mm 2 8ng / mm 2 12ng / mm 2 15ng / mm 2 25ng / mm 2 35ng / mm 2 50ng / mm 2 80ng / mm 2 100ng / mm 2 Or within the range of any two values.

[0448] In some embodiments, in order to lyse red blood cells in the device, a zwitterionic detergent is coated onto a plate, preferably at an area concentration of 100 ng / mm². 2 120ng / mm 2 150ng / mm 2 180ng / mm 2 200ng / mm 2 300ng / mm 2 400ng / mm 2 500ng / mm 2 800ng / mm 2 1000ng / mm 2 Or within the range of any two values.

[0449] In some embodiments, in order to achieve uniform distribution of red blood cells in the device, zwitterions are coated on a plate, preferably with a final blood concentration of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 1.0 mg / mL, 2 mg / mL, or within any two values.

[0450] In some embodiments, in order to lyse red blood cells in the device, zwitterions are coated on a plate, with a preferred final blood concentration of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, 50 mg / mL, or within any two values.

[0451] Measurement range of the iMOST-HgB device

[0452] In some embodiments, the hemoglobin measurement range of the device is between 0 g / dL and 40 g / dL.

[0453] In some embodiments, the preferred hemoglobin measurement range of the device is between 0 g / dL and 30 g / dL.

[0454] In some embodiments, the preferred hemoglobin measurement range of the device is between 5 g / dL and 26 g / dL.

[0455] Scattered particle removal and compensation

[0456] In some cases, there are defects that can significantly reduce the accuracy of absorption and / or hemoglobin measurements.

[0457] For example, interfering particles may be present in the sample or sample holder, including but not limited to particles that increase turbidity, light-scattering particles, bubbles, dust, or other particles with a light transmittance different from that of an ideal (i.e., ideal sample). This invention offers several ways to reduce errors in Optical Transmission Sample Analysis (OTSA) caused by these defects.

[0458] In some embodiments, multiple pairs of SR regions are used. For each pair of SR regions, the OD of the sample is determined by taking the ratio of the light intensity passing through the sample region to that passing through the reference region. For a given pair of SR regions, a quality measurement of the SR regions is calculated; if the quality measurement value is low, the pair of SR regions is excluded from the merging algorithm in the next stage. The merging algorithm merges the ODs of the samples across all pairs of SR regions. Various merging algorithms can be used, including but not limited to median, mean, maximum, minimum, k-mean, etc.

[0459] In some embodiments, defective regions are removed or excluded from the image prior to light intensity analysis.

[0460] In some embodiments, defective regions with boundaries are removed or excluded from the image prior to light intensity analysis, wherein the boundary size is between 1 μm and 50 μm; wherein the preferred boundary size is between 5 μm and 20 μm.

[0461] Measurement with / without scanning

[0462] In some embodiments, a location on the measuring device is used for analysis, particularly for hemoglobin.

[0463] QMAX system

[0464] A) QMAX card

[0465] Details of the QMAX card are described in detail in various publications, including International Application No. PCT / US2016 / 046437 (Essenlix Case No. ESSN-028WO), which is incorporated herein by reference for all purposes.

[0466] B) Machine Learning

[0467] Detailed information about the network is described in various publications, including International Application (IA) No. PCT / US2018 / 017504 filed February 8, 2018 and No. PCT / US2018 / 057877 filed October 26, 2018, which are each incorporated herein by reference for all purposes.

[0468] One aspect of this invention provides a framework for machine learning and deep learning for analyte detection and localization. Machine learning algorithms are algorithms capable of learning from data. A more rigorous definition of machine learning is "a computer program is said to learn from experience E regarding a class of tasks T and a performance measurement P if its performance at a task T improves with experience E as measured by P." It explores the study and construction of algorithms capable of learning and predicting from data – algorithms that overcome static program instructions by building models based on sample inputs and making data-driven predictions or decisions.

[0469] Deep learning is a specific type of machine learning based on a set of algorithms that attempt to model high-level abstractions in data. In a simple case, there might be two sets of neurons: neurons that receive input signals and neurons that send output signals. When an input layer receives input, it passes a modified version of the input to the next layer. In deep networks, there are many layers between the input and output (and these layers are not made up of neurons, but it's helpful to think of it this way), allowing the algorithm to use multiple processing layers consisting of multiple linear and nonlinear transformations.

[0470] One aspect of the present invention is to provide two methods for analyte detection and localization. The first method is a deep learning method, and the second method is a combination of deep learning and computer vision methods.

[0471] (i) Deep learning approach. In the first approach, the disclosed object detection and localization workflow consists of two phases: training and prediction. We describe the training and prediction phases in the following paragraphs.

[0472] (a) Training phase

[0473] During the training phase, annotated training data is fed into the convolutional neural network (CNN). A CNN is a specialized neural network used to process data, featuring grid-like, feedforward, and hierarchical network topologies. Examples of data include time-series data and image data; time-series data can be considered as a 1D grid sampled at regular time intervals, and image data as a 2D grid of pixels. Convolutional networks have achieved success in practical applications. The name "convolutional neural network" indicates that the network employs a mathematical operation called convolution. Convolution is a specialized linear operation. A CNN is simply a neural network that uses convolution instead of general matrix multiplication in at least one of its layers.

[0474] The machine learning model receives one or more images of a sample containing analytes acquired by an imager on a sample-holding QMAX device as training data. The training data is annotated with analytes to be determined, where the annotations indicate whether the analytes are in the training data and their locations in the images. Annotations can be in the form of tight bounding boxes that fully contain the analytes or the center location of the analytes. In the latter case, the center location is further transformed into a Gaussian kernel in a circle or dot plot covering the analyte.

[0475] When training large amounts of data, training machine learning models faces two challenges: annotation (usually done by humans) is time-consuming, and training is computationally expensive. To overcome these challenges, the training data can be divided into small patches, which are then annotated and trained, or parts thereof. The term "machine learning" can refer to algorithms, systems, and devices in the field of artificial intelligence that typically use statistical techniques to train artificial neural networks from data without explicit programming.

[0476] Annotated images are fed into a machine learning (ML) training module, where a model trainer trains an ML model based on the training data (annotated sample images). The input data is iteratively fed into the model trainer multiple times until a specific stopping criterion is met. The output of the ML training module is an ML model—a computational model built from data according to the training process in machine learning, which gives the computer the ability to independently perform certain tasks (e.g., detecting and classifying objects).

[0477] During the prediction (or inference) phase, a trained machine learning model is applied by a computer. Examples of machine learning models include ResNet, DenseNet, etc., which are also referred to as "deep learning models" due to the depth of connected layers in their network structure. In some implementations, the Caffe library with fully convolutional networks (FCNs) is used for model training and prediction, and other convolutional neural network architectures and libraries, such as TensorFlow, can also be used.

[0478] The training phase produces a model that will be used in the prediction phase. This model can be reused in the prediction phase to measure the input. Therefore, the computational unit only needs access to the generated model. It does not need access to the training data, nor does it need to run the training phase again on the computational unit.

[0479] (b) Forecasting Phase

[0480] During the prediction / inference phase, the detection component is applied to the input image, which is then fed into a prediction (inference) module preloaded with the trained model generated from the training phase. The output of the prediction phase can be a bounding box containing the detected analytes and their center locations, a dot plot indicating the location of each analyte, or a heatmap containing information about the detected analytes.

[0481] When the output of the prediction phase is a list of bounding boxes, the number of analytes in the image of the sample used for measurement is characterized by the number of detected bounding boxes. When the output of the prediction phase is a dot plot, the number of analytes in the image of the sample used for measurement is characterized by the integral of the dot plot. When the output of the prediction phase is a heatmap, the localization component is used to identify locations, and the number of detected analytes is characterized by entries in the heatmap.

[0482] One implementation of the localization algorithm involves sorting the heatmap values ​​into a one-dimensional ordered list from highest to lowest. The pixel with the highest value is then selected and removed from the list along with its neighbors. This process is repeated to select the pixel with the highest value from the list until all pixels have been removed.

[0483] In the detection component using heatmaps, the input image, along with the model generated from the training phase, is fed into a convolutional neural network, and the output of the detection phase is a pixel-level prediction in the form of a heatmap. The heatmap can have the same size as the input image, or it can be a scaled-down version of the input image, and it serves as input to the localization component. We disclose an algorithm for locating the center of an object based on the heatmap. The main idea is to iteratively detect local peaks based on the heatmap. After a peak is located, we compute a local region surrounding the peak but with a smaller value. We remove this region from the heatmap and find the next peak from the remaining pixels. This process is repeated until all pixels are removed from the heatmap.

[0484] In some embodiments, the present invention provides a localization algorithm to sort heatmap values ​​from highest to lowest into a one-dimensional ordered list. The pixel with the highest value is then selected and removed from the list along with its neighbors. This process is repeated to select pixels with the highest values ​​from the list until all pixels have been removed from the list.

[0485]

[0486] After sorting, the heatmap is a one-dimensional ordered list where heatmap values ​​are sorted from highest to lowest. Each heatmap value is associated with its corresponding pixel coordinates. The first item in the heatmap is the item with the highest value, which is the output of the pop (heatmap) function. A disk is created, with the pixel coordinates of the disk containing the highest heatmap value at its center. Then, all heatmap values ​​whose pixel coordinates fall within the disk are removed from the heatmap. The algorithm repeatedly pops the highest value in the current heatmap, removing the disks surrounding it, until the item is removed from the heatmap.

[0487] In an ordered list heatmap, each item knows its predecessor and successor. When removing an item from an ordered list, we make the following changes:

[0488] • Assume the item to be removed is x r Its preceding term is x p The second term is x f .

[0489] For the preceding term x p This redefines its successor as removing the successor from the project. Therefore, x p The second term is now x f .

[0490] • For removing item x r Undefine its predecessor and successor, and remove it from the ordered list.

[0491] • For the latter term x f This redefines the project by removing the preceding item from the project. Therefore, x f The antecedent is now x p .

[0492] The localization algorithm ends after all items are removed from the ordered list. The number of elements in the set locus will be the count of the analytes, and the location information will be the pixel coordinates of each s in the set locus.

[0493] Another embodiment searches for local peaks that are not necessarily the local peak with the highest heatmap value. To detect each local peak, we start from a random starting point and search for local maxima. After a peak is found, we calculate the local region surrounding the peak but with a smaller value. We remove this region from the heatmap and find the next peak from the remaining pixels. We repeat this process until all pixels are removed from the heatmap.

[0494]

[0495] This is a breadth-first search algorithm starting from s, with a modified access point condition: only when heatmap[p] > 0 and heatmap[p] <= heatmap[q] is the neighbor p of the current position q added for coverage. Therefore, each pixel in the coverage has a non-descending path to the local peak s.

[0496]

[0497] (ii) Hybrid approach using deep learning and computer vision methods. In this second approach, detection and localization are performed using computer vision algorithms, while classification is performed using deep learning algorithms. The computer vision algorithms detect and localize potential candidates for the analyte, and the deep learning algorithms classify each potential candidate as a true analyte or a false analyte. The locations of all true analytes (along with the total count of true analytes) are recorded as the output.

[0498] (a) Detection. Computer vision algorithms detect potential candidates based on the characteristics of the analyzed object, including but not limited to intensity, color, size, shape, and distribution. Preprocessing schemes can improve detection. Preprocessing schemes include contrast enhancement, histogram adjustment, color enhancement, noise reduction, smoothing, and defocusing. The preprocessed input image is fed into the detector. The detector informs the detector of the presence of potential candidates for the analyzed object and provides an estimate of their location. Detection can be based on the structure of the analyzed object (e.g., edge detection, line detection, circle detection, etc.), connectivity (e.g., blob detection, connected components, contour detection, etc.), intensity, color, shape, etc., using schemes such as adaptive thresholding.

[0499] (b) Localization. Following detection, computer vision algorithms localize each possible candidate of the analyte by providing its boundary or a tight bounding box containing it. This can be achieved using object segmentation algorithms such as adaptive thresholding, background subtraction, flood filling, mean shift, watershed, etc. Typically, localization is combined with detection to produce the detection results and the location of each possible candidate of the analyte.

[0500] (c) Classification. Deep learning algorithms, such as convolutional neural networks, have enabled the initial stage of visual classification. We employ deep learning algorithms to classify each possible candidate of the object to be analyzed. Various convolutional neural networks can be used for object classification, such as VGGNet, ResNet, MobileNet, and DenseNet.

[0501] Given each possible candidate for an analyte, the deep learning algorithm computes high-level features through layers of neurons via convolutional and nonlinear filters to extract features that distinguish the analyte from the non-analytes. A fully convolutional layer combines these high-level features into the classification result, telling it whether it is a true analyte or the probability of it being a true analyte.

[0502] Example

[0503] A) Example 1

[0504] OAC is a QMAX device with two plates.

[0505] The first sheet is a rectangular PMMA sheet having a flat surface and a thickness of, for example, 0.8 to 1.1 mm, 0.5 to 1.5 mm, or 0.3 to 2 mm; a length of, for example, 28 to 32 mm, 25 to 35 mm, or 20 to 50 mm; and a width of, for example, 20 to 28 mm, 15 to 34 mm, or 10 to 40 mm.

[0506] The second plate is a rectangular PMMA film having a flat surface and an array of micropillars (i.e., a column array) imprinted on the flat surface. The PMMA film has a thickness of, for example, 0.8 to 1.1 mm, 0.5 to 1.5 mm, or 0.3 to 2 mm; a length of, for example, 28 to 32 mm, 25 to 35 mm, or 20 to 50 mm; and a width of, for example, 20 to 28 mm, 15 to 34 mm, or 10 to 40 mm. In some embodiments, when the first and second plates are placed together to hold a sample, at least three sides of the second plate are within the area of ​​the first plate. The column array has a rectangular or square shape, a flat top, and column lateral dimensions of, for example, 30 to 40 micrometers (μm), 25 to 45 μm, 20 to 50 μm, 10 to 60 μm, or 5 to 70 μm; column heights of, for example, 10 to 30 μm, 5 to 40 μm, 1 to 50 μm, or 0.1 to 100 μm; and distances between the centers of two adjacent columns of, for example, 80 to 110 μm, 60 to 130 μm, 30 to 180 μm, or 30 to 200 μm.

[0507] B) Hemoglobin measurement using OAC - using a wavelength

[0508] In an exemplary experiment, the OAC is, for example, a QMAX device with two plates. The first plate is a flat PMMA substrate with a thickness of 1 mm and a size of 30 mm × 24 mm. The second plate is a 175 μm thick PMMA film with a micropillar array having a size of 24 mm × 22 mm. This pillar array has a pillar size of 30 μm × 40 μm, a pillar-to-pillar edge distance of 80 μm, and a pillar height of 10 μm or 30 μm.

[0509] The sample was fresh whole blood (2.5 μL for 10 μm column height, 5 μL for 30 μm column height). The sample was dropped onto a certain position on the first plate and pressed down with the second plate.

[0510] exist Figure 5 In the optical measurements shown, white LED light is filtered by dual bandpass filters (e.g., 532 to 576 nm green; and 625 to 675 nm red) and illuminates two 45-degree mirror sets. The light then passes through a translucent diffuser to eliminate the coherence of the point source wavefront and ensure that intensity variations are caused only by absorption. Finally, the resulting diffused light passes through a QMAX device (i.e., the sample card) and is collected by a lens for imaging and recording by a camera.

[0511] The LED light source and camera used in this example can both come from, for example, a mobile phone.

[0512] The image captured by the camera, i.e. the recorded image, shows two areas: one area is the column area; the other area is the blood sample area.

[0513] Light absorption in the column region is negligible. Furthermore, the extinction coefficients of oxyhemoglobin [HbO2] and deoxyhemoglobin [Hb] in the wavelength range of 532 nm to 576 nm are similar to...

[0514] therefore,

[0515] like Figure 6 As shown, I is the average intensity in the blood region, and Io is the average intensity at the center of the column region. When calculating the average intensity, a 5μm area near the column boundary is subtracted to reduce the averaging error.

[0516]

[0517] Hemoglobin levels in the blood, ranging from 6 g / dL to 11 g / dL, were measured using a QMAX device and a commercial Abbott Emerald hemocytometer. Results are as follows: Figure 7 The comparisons are shown below. For each concentration, three calories were measured to calculate the standard deviation.

[0518] From the results, when compared with industry standards, namely HemoCue America's commercial instruments ( www.hemocue.us ),For example Compared to the Hb 801 or Hb 301 systems, the repeatability (CV) of hemoglobin measurements on the same blood samples using the QMAX card is approximately 5%, with an R² value of 96%. The Hb 301 system provides rapid and easy laboratory-quality results in 3 seconds or less. The instrument is reportedly optimized for hemoglobin measurements in blood bank donation and public health settings.

[0519] C) Example-3

[0520] Figure 8 shows an example of hemoglobin measurement, an image of light transmission through a thin layer (without lysis) of whole blood in an OAC (e.g., the sample holder described in Figure 1), with the light source being a diffused light source (e.g., the diffuser is placed in front of a point light source), and the image was taken with an iPhone. In Figure 8, light guide spacers are periodically placed on the QMAX card with a vertical periodicity of 120 μm and a horizontal periodicity of 110 μm.

[0521] Figure 9 The options for using are shown. Figure 9 The image is used to identify examples of the sample and reference regions for hemoglobin. The boundaries of the sample and reference regions are marked. Figure 9 In the diagram, the reference region (with its outer boundary shaded) is inside the light guide spacer; D is the distance between the edge of the reference region and the edge of the light guide spacer, which is 10 μm; d is the distance between the edge of the sampling region and the edge of the light guide spacer, which is 30 μm; and T is the distance between the edge of the sampling region and the edge of the reference region, which is 40 μm.

[0522] D) Image Processing

[0523] The image processing algorithm used in hemoglobin absorbance measurements with the disclosed device having a single white light source and a Bayer filter includes the following steps:

[0524] The original image data file (e.g., RAW file) is converted into an image file with two or three color values ​​for each pixel by means of Bayer mode interpolation, but without using (i.e., omitting certain conventional analysis and metrics) such as color space conversion, white balance, gamma, curves, noise reduction, or any combination thereof.

[0525] Detect the optical guide spacer;

[0526] Determine reference regions and sampling regions, and associate specific reference regions with specific sampling regions to create identified individual regions;

[0527] For one or more associated identified individual regions, the total analyte concentration, such as total hemoglobin concentration, for the identified individual region is calculated using the above formula from the intensity of the sample region and the intensity of the reference region; and

[0528] Merge one or more separate regions, for example, using a merging algorithm, to produce a single analyte absorption measurement or value for the analyzed sample.

[0529] The detection and positioning of light guide spacers can be performed, for example, periodically, systematically, or regularly located or placed on a QMAX card. Various object detection algorithms can be employed, including, for example, template matching, speckle detection, and contour detection. For instance, detection can be performed in a single color channel within a color space (e.g., RGB, HSV, HSI, Lab, YCrCb, etc.), such as the green channel in the RGB color space, or the hue channel in the HSV color space, or a combination of two or more color channels, such as using a red-green-blue channel in the RGB color space.

[0530] After detecting and locating the periodic light guide spacing, a reference region (i.e., located inside or within the light guide spacing region) and a sampling region are selected. Examples of the dimensions of the reference region and the sampling region, as well as the distances between the edges of the light guide spacing, the reference region, and the sampling region, are mentioned in the accompanying drawings.

[0531] Reference regions and sampling regions can be associated by their relative positions and distances. When one (or more) reference regions are associated with one (or more) sampling regions to produce a single region, hemoglobin absorption measurements can be calculated using the methods and formulas described above. In some embodiments, a reference region can be associated with a sampling region having, for example, the shortest distance, and hemoglobin absorption measurements can be calculated for each association.

[0532] Merging algorithms can be used for mathematical characterization and further analysis of measurement data. For example, merging each associated hemoglobin absorption measurement from a reference region and a sample region produces a single hemoglobin absorption measurement for the blood sample. Various merging algorithms can be used to characterize measurement data, such as median, mean, maximum, minimum, k-means, etc. Merging can also refer to combining data, but it can also refer to combining information rather than the original data. A common use of merging is for variance estimation. In one example, merging combines two estimates of the variance from samples from two different groups, and taking the weighted average of the merged values ​​provides a single estimate of the common variance.

[0533] In some embodiments, imaging processing may use artificial intelligence, machine learning, or both. In some embodiments, imaging processing may use deep learning.

[0534] E) Using two wavelengths

[0535] The setup was similar to the experiment described above, except that two different bandpass filters were used and two photos were taken.

[0536] After taking the photograph, the blood with two different wavelengths λ1 and λ2, such as 660nm and 940nm, was calculated.

[0537]

[0538]

[0539] We obtained:

[0540]

[0541]

[0542] ε is the extinction coefficient of hemoglobin, [Hb] and [HbO2] are the concentrations of hemoglobin, and L is the length of the optical path through the sample or the gap size of the QMAX device.

[0543] Therefore, total hemoglobin concentration = [HbO2] + [Hb].

[0544] This method can further provide detailed information on the [HbO2] to [Hb] ratio.

[0545] Image processing for calculating HgB absorption measurements from blood samples using two wavelengths.

[0546] In some embodiments, this disclosure provides an imaging method for HgB blood samples. The image can be processed using the following steps, the algorithm described:

[0547] 1. Use a digital image of a hemoglobin sample as input;

[0548] 2. Run a color distribution check. If the check fails, reject the image sample;

[0549] 3. Operation column detection;

[0550] 4. Check the column detection score. If the check fails, reject the image sample;

[0551] 5. After the image passes the column detection score check, the algorithm performs a horizontal column check;

[0552] 6. Run a vertical column check; and

[0553] 7. Calculate the hemoglobin (HgB) value.

[0554] Figure X5 A flowchart illustrating the workflow for image processing in HgB absorption measurements is shown. During color distribution checking, the image is rejected if the average intensity of a color channel is outside a given range. Each channel is checked for different ranges. The image is rejected if the column detection score is outside a given range.

[0555] Figure X6 illustrates the components of HgB image processing, including: horizontal column inspection, vertical column inspection, and intensity ratio calculation. Horizontal clustering identifies horizontal lines passing through the column centers. The algorithm includes the following steps:

[0556] 1. Center the column cluster along the horizontal direction;

[0557] 2. Run a horizontal line fit above the center of the column;

[0558] 3. Identify the dominant horizontal direction;

[0559] 4. Perform a parallelism check on the running lines (if the check fails, reject the image sample);

[0560] 5. After the image undergoes line parallelism checking, the algorithm proceeds to the next step of vertical direction clustering.

[0561] Vertical clustering identifies vertical lines passing through the center of the pillar. The algorithm includes the following steps:

[0562] 1. Center the column cluster along the vertical direction;

[0563] 2. Run a vertical line fit above the center of the column;

[0564] 3. Identify the dominant vertical direction;

[0565] 4. Run line parallelism check (if the check fails, the image sample is rejected);

[0566] The component algorithm for "calculating HgB values" includes the following steps:

[0567] 1. Check the intercolumn distance (if the check fails, reject the image sample);

[0568] 2. After the image is checked for inter-cylinder distance, the algorithm generates cylindrical loops;

[0569] 3. Use a color interference removal matrix to separate the red and green channels;

[0570] 4. Calculate the ratio of blood intensity to column intensity for each column in the red and green channels;

[0571] 5. Check the coefficient of variation (CV) of the intensity ratio (if the check fails, reject the image sample);

[0572] 6. After the image was checked by CV, it was found that the intensity ratio of the red and green channels was moderate;

[0573] 7. Calculate the linear combination of the output intensity ratios of the red and green channels, and output the result as the HgB value;

[0574] For column distances, if the average distance between the centers of each pair of adjacent columns is outside a given range, the image is rejected.

[0575] The red and green channels from a dual-wavelength HgB image may interfere with each other. A color deinterference matrix is ​​applied to derive new red and green channels to reduce color interference.

[0576]

[0577] Where a1, a2, b1, and b2 are the coefficients of the color de-interference matrix. For example, they can be determined using a single-wavelength reference sample.

[0578] After calculating the intensity ratio of each column in the red and green channels, the algorithm checks the CV of the intensity ratio. If the CV of the intensity is outside a given range, the image is rejected.

[0579] The intensity ratio of the red and green channels is r 红 and r 绿 The intermediate value is used for HgB value calculation. The formula used for HgB value calculation is based on a linear combination of the logarithms of these two ratios:

[0580] HgB=Aln(r 红 )+Bln(r 绿 )+C

[0581] For example, using regression analysis of HgB blood samples with known HgB values, three coefficients A, B, and C are pre-determined.

[0582] F) Optical guide spacer, sampling area, and reference area

[0583] In some embodiments, the sampling region boundary has dimensions of 120 μm by 110 μm; the sampling region edge has dimensions of 60 μm × 45 μm; the light guide spacer or post has dimensions of 40 μm × 30 μm; and the reference region has dimensions of 20 μm × 15 μm. In some embodiments, the area of ​​the reference region is half the size of the light guide spacer region, the distance between the edge of the sampling region and the edge of the light guide spacer is half the distance between the edges of the light guide spacer regions, and the area of ​​the sampling region is equal to the periodic interval distance.

[0584] Example

[0585] The following examples illustrate the apparatus and methods for preparing, using, and analyzing the present disclosure according to the general procedures described above.

[0586] Example 1 (Actual)

[0587] A device with a dual-band bandpass filter for analyzing hemoglobin in blood. In the device shown in Figure N1, the light source is a single white light source, such as an LED in a smartphone, the filter is a dual-band bandpass filter that converts or purifies the single white light source into a mixture of two different colors of light, green and red, and the sample is human whole blood expected to contain hemoglobin as the analyte. For example, the dual bandpass filter converts or purifies the single white light before the sample is examined.

[0588] Example 2 (foreshadowing)

[0589] A device with a three-band bandpass filter for analyzing hemoglobin in blood. In the device shown in Figure N5, the light source is a single white light source, such as an LED in a smartphone, and the filter is a three-band bandpass filter that converts or cleans the single white light source into a mixture of three different colors of light, such as red, green, and blue light, and the sample is human whole blood expected to contain hemoglobin as the analyte. For example, after questioning the sample, the three-band bandpass filter converts or cleans the single white light.

[0590] 1. An apparatus comprising:

[0591] The components include a first plate, a second plate, a light guide spacer (LGS), a sampling area, and a reference area, wherein:

[0592] (i) The first plate and the second plate are configured to sandwich a sample for light transmission analysis between the plates in a thin layer, and each plate has a sample contact area on its inner surface that contacts the sample.

[0593] (ii) The light-guiding spacer (LGS) has a cylindrical shape, sandwiched between two plates, wherein each end of the column is in direct contact with one of the plates forming the LGS-plate contact area, and is configured to allow light to pass from the first plate through the LGS to the second plate without passing through the sample.

[0594] (iii) The sampling region is the area where the light sequentially passes through the first plate, the sample, and the second plate, wherein the sampling region does not have the LGS; and

[0595] (iv) The reference area is the region in which light passes through the first plate, the light-guiding spacer and the second plate in sequence but does not pass through the sample;

[0596] The LGS contact area and the lateral cross-section of the LGS are larger than the wavelength of light.

[0597] The optical guide spacer is surrounded by or close to the sample; and

[0598] The sample in the sampling area has a thickness of 500 μm or less.

[0599] 2. An apparatus for analyzing hemoglobin in a sample, comprising:

[0600] The first plate, the second plate, and the optical guide spacer, wherein:

[0601] (i) The first plate and the second plate can be moved relative to each other to form different configurations, including open and closed configurations;

[0602] (ii) Each of the plates includes an inner surface having a sample contact area for contacting a sample containing or suspected of containing hemoglobin; and

[0603] (iii) The light guide spacer has a columnar shape, wherein the top and bottom surfaces of the light guide spacer are substantially flat, and the bottom surface is fixed to the inner surface of one of the plates, wherein the area of ​​the top and bottom surfaces of each spacer and the average transverse cross section are respectively greater than the wavelength of the light used to analyze the sample, wherein the light guide spacer is in the sample contact area.

[0604] An open structure is one in which two plates are separated, the spacing between the plates is not adjustable by spacers, and the sample is deposited on one or both of the plates.

[0605] The closed structure is configured such that the sample is deposited after the open structure; and in the closed structure: at least a portion of the sample is compressed by two plates into a layer of very uniform thickness, wherein the uniform thickness of the layer is defined by the sample contact area of ​​the plates and adjusted by the plates and the optical guide spacer; and

[0606] In the closed configuration: (a) the top surface of at least one spacer in the sample contact area is in direct contact with one of the plates, and the at least one spacer and the areas of the plates above and below the at least one spacer define a reference area, wherein the reference area is transparent to light within the wavelength range; and (b) at least one area in the sample contact area on one plate and its corresponding area on the other plate are not occupied by the light-guiding spacer, thereby defining a sampling area that is transparent to light within the same wavelength range.

[0607] 3. An apparatus for analyzing hemoglobin in an analyte in a sample, comprising:

[0608] The first plate, the second plate, and the optical guide spacer, wherein:

[0609] (i) The first and second plates are configured to hold a sample containing or suspected of containing the analyte, wherein at least a portion of the sample is between and in contact with the two plates; and

[0610] (ii) An optical guide spacer having a columnar shape and a predetermined basic height.

[0611] The top and bottom surfaces of these optical guide spacers are substantially flat, and at least one spacer's top and bottom surfaces are in direct contact with these plates.

[0612] The area of ​​the top and bottom surfaces of each spacer and its average transverse cross-section are both greater than the wavelength of the light used to analyze the sample.

[0613] Wherein: (a) at least one spacer and the area of ​​the plate directly above and below the at least one spacer define a reference area that is transparent to light within a wavelength range and passing through the plates and the spacer, and (b) at least one area of ​​the sample contact area on one plate and its corresponding area on the other plate are not occupied by the light-guiding spacers, thereby defining a sampling area that is transparent to light within the same wavelength range.

[0614] 4. An apparatus for analyzing an analyte in a sample, comprising:

[0615] The first plate, the second plate, and the optical guide spacer, wherein:

[0616] (i) The first plate and the second plate can be moved relative to each other to form different configurations, including open and closed configurations;

[0617] (ii) Each of the plates includes an inner surface having a sample contact area for contacting a sample containing or suspected of containing the analyte; and

[0618] (iii) The light guide spacer has a columnar shape and a predetermined substantially uniform height, wherein the top and bottom surfaces of the light guide spacer are substantially flat and the bottom surface is fixed to the inner surface of one or more plates, wherein the area of ​​the top and bottom surfaces of each spacer and the average transverse cross section are respectively greater than the wavelength of the light used to analyze the sample, wherein at least one of the light guide spacers is in the sample contact area.

[0619] An open structure is one in which two plates are separated, the spacing between the plates is not adjustable by spacers, and the sample is deposited on one or both of the plates.

[0620] The closed structure is configured such that the sample is deposited after the open structure; and in the closed structure: at least a portion of the sample is compressed by two plates into a layer of very uniform thickness, wherein the uniform thickness of the layer is defined by the sample contact area of ​​the plates and adjusted by the plates and the optical guide spacer; and

[0621] In the closed structure, (a) the top surface of at least one spacer in the sample contact area is in direct contact with one of the plates, and the at least one spacer and the plates define a reference area above and below the at least one spacer, wherein the reference area is transparent to light within a certain wavelength range, and (b) at least one area in the sample contact area on one plate and its corresponding area on the other plate are not occupied by the light-guiding spacer, thereby defining a sampling area transparent to light within a wavelength range.

[0622] 5. An apparatus for analyzing hemoglobin in a sample, comprising:

[0623] The first plate, the second plate, and the optical guide spacer, wherein:

[0624] (v) The first plate and the second plate are configured to sandwich the sample in a thin layer;

[0625] The first plate, the second plate, and the optical guide spacer, wherein:

[0626] (i) The first plate and the second plate are configured to sandwich the sample in a thin layer;

[0627] (ii) The light guide spacer has a columnar shape sandwiched between the two plates, wherein each end of the column directly contacts one of the plates such that there is no sample between the end of the column and the corresponding plate, wherein the light guide spacer is surrounded by or close to the sample, and wherein the direct contact area and the average transverse cross section of the column are at least 1 μm^2 (square micrometers) or greater.

[0628] The spacing between the inner surfaces of the plates is 200 μm or less.

[0629] 6. An apparatus for sample analysis, comprising:

[0630] The device, light source, camera, and adapter as described in any of the foregoing device embodiments, wherein...

[0631] (i) The light source is configured to emit light within a wavelength range configured to pass through the reference region;

[0632] (ii) The camera is configured to image the reference region and the sampling region.

[0633] (iii) The adapter is configured to position the device, the light source and the camera relative to each other such that light from the light source passes through the reference area and the sampling area and is imaged by the camera.

[0634] 7. The device as described in any of the foregoing device embodiments, further comprising:

[0635] The processor is configured to process the image captured by the camera and determine the properties of the analyte in the sample based on a comparison of light transmission from the reference region and the sampled region.

[0636] 8. The device as claimed in any of the preceding claims, wherein the camera and the processor are part of a single mobile device.

[0637] 9. The device as claimed in any of the preceding claims, wherein the light source and the processor are part of a single mobile device.

[0638] 10. The device as claimed in any of the preceding claims, wherein the light source, the camera, and the processor are part of a single mobile device.

[0639] 11. The device as described in any of the foregoing device embodiments, wherein the mobile device is a smartphone.

[0640] 12. A method for analyzing samples using transmitted light, comprising the following steps:

[0641] (a) A device having any of the foregoing device embodiments;

[0642] (b) Depositing the sample into an open structure of the device, wherein the sample is suspected of containing an analyte;

[0643] (c) To make the device into the closed configuration;

[0644] (d) It has a light source having a wavelength configured to pass through a reference region of the device;

[0645] (e) It has an imager configured to image a reference region and a sampling region of the device;

[0646] (f) has an adapter configured to position the device, the light source and the camera relative to each other such that light from the light source passes through the reference region and the sampling region and is imaged by the camera;

[0647] (g) The properties of the analyte are determined by comparing the light transmission from the sampling area and the reference area.

[0648] 13. The apparatus, method, or system as claimed in any of the preceding claims, wherein the analyte is hemoglobin.

[0649] 14. The apparatus, method, or system as claimed in any of the preceding claims, wherein the analyte is a certain type of cell.

[0650] 15. The apparatus, method, or system of any of the preceding claims, wherein the thickness of the sample layer is adjusted by the plate and the light guide spacer, and is substantially the same as the uniform height of the light guide spacer;

[0651] 16. The apparatus, method, or system as claimed in any of the preceding claims, wherein the analyte is red blood cells.

[0652] 17. The apparatus, method, or system of any of the preceding claims, wherein the analyte is leukocytes.

[0653] 18. The apparatus, method, or system as claimed in any of the preceding claims, wherein the reference region and the sampling region have the same size.

[0654] 19. The apparatus, method, or system of any of the preceding claims, wherein the reference region is located within a corresponding region of the cross-section of the optical spacer.

[0655] 20. The apparatus, method, or system according to any of the preceding claims, wherein the reference region is less than 0.1 μm², less than 0.2 μm², less than 0.5 μm², less than 1 μm², less than 2 μm², less than 5 μm², less than 10 μm², less than 20 μm², less than 50 μm², less than 100 μm², less than 20 μm², less than 500 μm², less than 1000 μm², less than 20 μm², less than 20 μm², less than 1 ... 00 μm², less than 5000 μm², less than 10000 μm², less than 20000 μm², less than 50000 μm², less than 100000 μm², less than 200000 μm², less than 500000 μm², less than 1 mm², less than 2 mm², less than 5 mm², less than 10 mm², less than 20 mm², or less than 50 mm², or within any two of these values.

[0656] 21. The apparatus, method, or system of any of the preceding claims, wherein the apparatus further comprises a plurality of light guide spacers having substantially uniform height, and wherein at least one of the light guide spacers is located within the sample contact area.

[0657] 22. The apparatus, method, or system of any of the preceding claims, wherein the apparatus further comprises a plurality of light guide spacers having substantially uniform height, wherein the distance between two adjacent light guide spacers is known, and wherein at least one of the light guide spacers is within the sample contact area.

[0658] 23. The apparatus, method, or system of any of the preceding claims, wherein the apparatus further comprises a plurality of light guide spacers having substantially uniform height, wherein the distance between two adjacent light guide spacers is known and substantially constant (i.e., the light guide spacers are substantially a periodic array), and wherein at least one of the light guide spacers is in the sample contact region.

[0659] 24. The apparatus, method, or system of any of the preceding claims, wherein the bottom surface of the light guide spacer is fixed to the inner surface of one of the plates by molding the light guide spacer onto the inner surface of the plates.

[0660] 25. The apparatus, method, or system of any of the preceding claims, wherein the bottom surface of the light guide spacer is fixed to the inner surface of one of the plates and is made of the same material as the inner surface.

[0661] 26. The apparatus, method, or system of any of the preceding claims, wherein the bottom surface of the light guide spacer is fixed to the inner surface of one of the plates and is made of the same material as the inner surface, and the bottom surface of the light guide spacer has no interface with the inner surface of the plate.

[0662] 27. The apparatus, method, or system of any preceding claim, wherein the wavelength of the light is longer than 300 nm, and wherein the wavelength of the light is also less than 20 μm, less than 15 μm, less than 10 μm, less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, less than 1 μm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, or within any two values.

[0663] 28. The apparatus, method, or system of any of the preceding claims, wherein the wavelength of the light is longer than 500 nm, and wherein the wavelength of the light is also less than 600 nm, less than 590 nm, less than 580 nm, less than 570 nm, less than 560 nm, less than 550 nm, less than 540 nm, less than 530 nm, less than 520 nm, less than 510 nm, or within any two values.

[0664] 29. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average lateral cross-section of each photoconductive spacer is less than 1 μm² (square micrometers), 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², 100,000 μm², 200,000 μm², 500,000 μm², 1 mm², 2 mm², 5 mm², 10 mm², 50 mm², or within any two values.

[0665] 30. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average lateral cross-section of each optical spacer is less than 1 μm² (square micrometers), 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², 100,000 μm², 200,000 μm², or within any two values.

[0666] 31. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average lateral cross-section of each optical spacer is less than 1 μm², 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², or within any two values.

[0667] 32. The apparatus, method, or system of any preceding claim, wherein the sample contact area is greater than 100 μm², greater than 200 μm², greater than 400 μm², greater than 600 μm², greater than 800 μm², greater than 1,000 μm², greater than 2,000 μm², greater than 4,000 μm², greater than 6,000 μm², greater than 8,000 μm², greater than 10,000 μm², greater than 20,000 μm², greater than 40,000 μm², greater than 60,000 μm², greater than 80,000 μm², greater than 100,000 μm², greater than 200,000 μm², greater than 250,000 μm², greater than 500,000 μm², or within any two values.

[0668] 33. The apparatus, method, or system as claimed in any of the preceding claims, wherein the predetermined constant interval distance is at least about twice the size of the analyte.

[0669] 34. The apparatus, method, or system of any of the preceding claims, wherein the predetermined constant interval distance is at least 2 times, at least 6 times, at least 8 times, at least 10 times, at least 20 times, at least 40 times, at least 60 times, at least 80 times, or at least 100 times larger than the size of the analyte.

[0670] 35. The apparatus, method, or system according to any of the preceding claims, wherein the height of the light guide spacer is 1 μm, 2 μm, 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1,000 μm, 2,000 μm, 5,000 μm, 10,000 μm, or within any two values.

[0671] 36. The apparatus, method, or system according to any of the preceding claims, wherein the spacers are arranged in a periodic array, and are 1 μm, 2 μm, 5 μm, 10 μm, 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1,000 μm, 2,000 μm, 5,000 μm, 10,000 μm, or within any two values.

[0672] 37. The apparatus, method, or system of any of the preceding claims, wherein the LGS has a columnar shape with substantially flat ends.

[0673] 38. The apparatus, method, or system of any of the preceding claims, wherein one or both ends of the LGS are made from a single piece by bonding, fusion, or other methods of attaching the LGS to one or both ends of the plate.

[0674] 39. The apparatus, method, or system of any of the preceding claims, wherein the shape of the transverse cross-section of the LGS includes, but is not limited to, a circle, a rectangle, a square, a triangle, a polygon, a letter, a number, or a combination thereof.

[0675] 40. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average lateral cross-section of each optical spacer (LGS) is 1 μm², 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², 100,000 μm², 200,000 μm², 500,000 μm², 1 mm², 2 mm², 5 mm², 10 mm², 50 mm², or within a range of any two values.

[0676] 41. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average lateral cross-section of each photoconductive spacer is 1 μm² (square micrometers), 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², 100,000 μm², 200,000 μm², or within any two values.

[0677] 42. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average lateral cross-section of each optical spacer is 1 μm², 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², 10,000 μm², 30,000 μm², or within any two values.

[0678] 43. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average lateral cross-section of each optical spacer is 1 μm², 10 μm², 20 μm², 30 μm², 50 μm², 100 μm², 150 μm², 200 μm², 300 μm², 500 μm², 1000 μm², 2000 μm², 5000 μm², or within any two values.

[0679] 44. The apparatus, method, or system as claimed in any of the preceding claims, wherein the average transverse cross section of each light guide spacer is 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times larger than the wavelength of light passing through the reference region, or within any two values.

[0680] 45. The apparatus, method, or system of any of the preceding claims, wherein the average transverse cross section of each light guide spacer is 1, 2, 3, 5, 10, 20, 50, 100, 200, or 500 times larger than the wavelength of light passing through the reference region, or within any two values.

[0681] 46. ​​The apparatus, method, or system as claimed in any of the preceding claims, wherein the reference region is smaller than the size of the minimum lateral cross-section of the light guide post. One advantage is that it avoids or reduces light scattering from the light guide sidewalls that could affect the reference signal.

[0682] 47. The apparatus, method, or system of any of the preceding claims, wherein the minimum distance between the edge of the light-guiding spacer and the edge of the reference region is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, or within any two values.

[0683] 48. The apparatus, method, or system of any of the preceding claims, wherein the minimum distance between the edge of the light-guiding spacer and the edge of the reference region is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 100 μm, 200 μm, or within any two values.

[0684] 49. The apparatus, method, or system of any of the preceding claims, wherein the minimum distance between the edge of the light-guiding spacer and the edge of the reference region is 1 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, or within any two values.

[0685] 50. The apparatus, method, or system of any preceding claim, wherein the minimum distance between the edge of the light-guiding spacer and the edge of the reference region is greater than the wavelength passing through the reference region by 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times, or within any two values.

[0686] 51. The apparatus, method, or system of any preceding claim, wherein the minimum distance between the edge of the light guide spacer and the edge of the reference region is greater than the wavelength passing through the sampling region by 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times, or within any two values.

[0687] 52. The apparatus, method, or system of any of the preceding claims, wherein the ratio of the area of ​​the reference region to the area of ​​the optical spacer is 3 / 10, 2 / 5, 1 / 2, 3 / 5, 7 / 10, 4 / 5, or within any two values.

[0688] 53. The apparatus, method, or system of any of the preceding claims, wherein the edge of the sampling region is at a certain distance from the edge of the light guide post.

[0689] 54. The apparatus, method, or system as claimed in any of the preceding claims, wherein the area of ​​the sampling region is 3 / 5, 7 / 10, 4 / 5, 9 / 10, 1, 11 / 10, 6 / 5, 13 / 10, 7 / 5, 3 / 2 of the periodic interval distance, or within a range of any two values.

[0690] 55. The apparatus, method, or system of any of the preceding claims, wherein the distance between the edge of the sampling region and the edge of the light guide spacer is 1 / 5, 3 / 10, 2 / 5, 1 / 2, 3 / 5, 7 / 10, 4 / 5, 9 / 10, 1 of the light guide spacer region, or within any two values.

[0691] 56. The apparatus, method, or system of any of the preceding claims, wherein the distance between the edge of the sampling region and the edge of the optical guide spacer is greater than the wavelength passing through the reference region by 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times, or within any two values.

[0692] 57. The apparatus, method, or system of any preceding claim, wherein the distance between the edge of the sampling region and the edge of the optical spacer is greater than the wavelength passing through the sampling region by 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times, or within any two values.

[0693] 58. The apparatus, method, or system of any of the preceding claims, wherein the distance between the edge of the sampling region and the reference region is 1 μm (micrometer), 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, or within any two values.

[0694] 59. The apparatus, method, or system of any of the preceding claims, wherein the distance between the edge of the sampling region and the reference region is 30 μm to 50 μm, 20 μm to 60 μm, 10 μm to 70 μm, 5 μm to 75 μm, or within any two values.

[0695] 60. The apparatus, method, or system of any preceding claim, wherein the distance between the edge of the sampling region and the reference region is greater than the wavelength passing through the reference region by 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times, or within any two values.

[0696] 61. The apparatus, method, or system of any of the preceding claims, wherein the distance between the edge of the sampling region and the reference region is 2 / 5, 1 / 2, 3 / 5, 7 / 10, 4 / 5, 9 / 10, 1, 11 / 10, 6 / 5, 13 / 10, 7 / 5, 3 / 2, 8 / 5, 17 / 10 of the optical spacer region, or within a range between any two values.

[0697] 62. The apparatus, method, or system of any preceding claim, wherein the distance between the edge of the sampling region and the reference region is greater than the wavelength of light passing through the reference region by 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 5000 times, or within any two values.

[0698] 63. The apparatus, method, or system of any of the preceding claims, wherein the distance between the edge of the sampling region and the reference region is 1, 2, 3, 5, 10, 20, 50, 100, 200, or 500 times greater than the wavelength passing through the sampling region.

[0699] 64. The apparatus, device, or method as claimed in any of the preceding claims, wherein the analyte is a biomarker, environmental marker, or food marker.

[0700] 65. The apparatus, device, or method as claimed in any of the preceding claims, wherein the analyte is a biomarker indicating the presence or severity of a disease or condition.

[0701] 66. The apparatus, device, or method as claimed in any of the preceding claims, wherein the analyte is a cell, protein, or nucleic acid.

[0702] 67. The apparatus, device, or method as claimed in any of the preceding claims, wherein the analyte is hemoglobin.

[0703] 68. The apparatus, device, or method as claimed in any of the preceding claims, wherein the analyte comprises proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, organic compounds, bacteria, viruses, cells, tissues, nanoparticles, and other molecules, compounds, mixtures, and substances thereof.

[0704] 69. The apparatus, device, or method according to any of the preceding claims, wherein the sample is in its original, diluted, or processed form as follows: body fluid, feces, amniotic fluid, aqueous humor, vitreous fluid, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, earwax, chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, or exhaled condensate.

[0705] 70. The apparatus, device, or method as claimed in any of the preceding claims, wherein the sample is a raw, diluted, or processed form of blood.

[0706] 71. The apparatus, device, or method as claimed in any of the preceding claims, wherein the sample comprises whole blood.

[0707] 72. The method or apparatus of any of the preceding claims, wherein the spacing distance (SD) is equal to or less than about 150 μm (micrometers).

[0708] 73. The method or apparatus of any of the preceding claims, wherein the spacing distance (SD) is equal to or less than about 100 μm (micrometers).

[0709] 74. The method or apparatus of any of the preceding claims, wherein the fourth power of the spacing distance (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) (ISD) 4 / (hE)) is 5×10 6 μm 3 / GPa or less.

[0710] 75. The method or apparatus of any of the preceding claims, wherein the fourth power of the spacing distance (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) (ISD) 4 / (hE)) is 5×10 5 μm 3 / GPa or less.

[0711] 76. The method or apparatus of any of the preceding claims, wherein the spacer has a columnar shape, a substantially flat-topped surface, a predetermined substantially uniform height and a predetermined constant spacing distance, the spacing distance being at least about 2 times larger than the size of the analyte, wherein the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the spacer contact area to the total plate area, and wherein for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (1).

[0712] 77. The method or apparatus of any preceding claim, wherein the spacer has a columnar shape, a substantially flat-topped surface, a predetermined substantially uniform height, and a predetermined constant spacing distance, the spacing distance being at least about 2 times larger than the size of the analyte, wherein the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the spacer contact area to the total plate area, and wherein for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (1), wherein the fourth power of the spacing distance (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) (ISD) 4 / (hE)) is 5x10 6 μm 3 / GPa or less.

[0713] 78. The apparatus of any of the preceding claims, wherein the ratio of the spacing distance of the spacers to the average width of the spacers is 2 or greater, and the fill factor of the spacers multiplied by the Young's modulus of the spacers is 2 MPa or greater.

[0714] 79. The apparatus, device, or method as claimed in any of the preceding claims, wherein one or both plates include position markers located on or inside the surface of the plates, the position markers providing information about the position of the plates.

[0715] 80. The apparatus, device, or method as claimed in any of the preceding claims, wherein one or both plates include scale markings on or within the surface of the plates, the scale markings providing information on the lateral dimensions of the sample and / or the structure of the plates.

[0716] 81. The apparatus, device, or method as claimed in any of the preceding claims, wherein one or both plates include imaging markers located on or inside the surfaces of the plates, the imaging markers assisting in the imaging of the sample.

[0717] 82. The apparatus, method, or system of any of the preceding claims, wherein the sample is in its original, diluted, or processed form as follows: body fluid, feces, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, earwax, chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal drainage, sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, or exhaled condensate.

[0718] 83. The apparatus, method, or system of any of the preceding claims, wherein the sample is raw, diluted, or processed form of blood.

[0719] 84. The apparatus, method, or system of any of the preceding claims, wherein the sample comprises whole blood.

[0720] 85. The apparatus, method, or system as claimed in any of the preceding claims, wherein the sample is a biological sample, a chemical sample, an environmental sample, or a food sample.

[0721] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise, such as when the word “single” is used. For example, reference to “analyte” includes a single analyte and multiple analytes, reference to “capture agent” includes a single capture agent and multiple capture agents, reference to “detector” includes a single detector and multiple detectors, and reference to “reagent” includes a single reagent and multiple reagents.

[0722] A range may be expressed herein as from “about” one particular value and / or to “about” another particular value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each range are important relative to and independent of the other endpoint. The term “about” or “approximately” may mean within an acceptable margin of error for a particular value as determined by one person skilled in the art, which will depend in part on how the value is measured or determined, such as limitations of the measurement system. For example, “about” may mean within one or more standard deviations, according to practice in the art. Alternatively, “about” may mean a range of at most 20%, at most 10%, at most 5%, or at most 1% of a given value. Or, particularly concerning biological systems or processes, the term may mean within orders of magnitude of the value, within 5 times, and more preferably within 2 times. Where specific values ​​are described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for the specific value. The term "about" has the meaning commonly understood by one of ordinary skill in the art. In some embodiments, the term "about" may refer to ±10%. In some embodiments, the term "about" may refer to ±5%.

[0723] As used herein, the terms “adapter” and “configuration” mean that an element, component, or object is designed and / or intended to perform a given function. Therefore, the use of the terms “adapter” and “configuration” should not be construed as meaning that a given element, component, or object is simply “capable” of performing a given function. Similarly, an object stated as configured to perform a particular function may additionally or optionally be described as operable to perform that function.

[0724] As used herein, when the phrase “for example,” the phrase “as an example,” and / or simply the terms “example” and “exemplary” refer to one or more components, features, details, structures, embodiments, and / or methods used according to this disclosure, they are intended to convey illustrative, non-exclusive examples of components, features, details, structures, embodiments, and / or methods according to this disclosure. Therefore, the described components, features, details, structures, embodiments, and / or methods are not intended to be limiting, essential, or exclusive / exhaustive; and other components, features, details, structures, embodiments, and / or methods, including those structurally and / or functionally similar and / or equivalent, are also within the scope of this disclosure.

[0725] As used herein, when referring to a list of more than one entity, the phrases “at least one” and “one or more” mean any one or more entities in the entity list, and are not limited to at least one of each and every entity specifically listed in the entity list. For example, “at least one of A and B” (or equivalently, “at least one of A or B”, or equivalently, “at least one of A and / or B”) could refer to A alone, B alone, or a combination of A and B.

[0726] As used herein, the term “and / or” between the first entity and the second entity refers to (1) the first entity, (2) the second entity, and (3) one of the first entity and the second entity. Multiple entities listed using “and / or” should be interpreted in the same way, such as “one or more” of entities thus combined. Other entities may optionally exist in addition to those specifically identified by the “and / or” clause, whether related to or unrelated to those specifically identified.

[0727] When referring to numerical ranges herein, the invention includes embodiments that include endpoints, embodiments that exclude two endpoints, and embodiments that include one endpoint while excluding the other. It should be assumed that two endpoints are included unless otherwise stated. Furthermore, unless otherwise stated or obvious from the context and understanding by one of ordinary skill in the art.

[0728] Where any patent, patent application or other reference is incorporated herein by reference and (1) defines a term in a manner inconsistent with any unincorporated portion of this disclosure or other incorporated references and / or (2) otherwise is inconsistent with any unincorporated portion of this disclosure or other incorporated references, the unincorporated portion of this disclosure shall prevail, and the term or the disclosure in which it is incorporated shall prevail only with respect to the reference in which the term is defined and / or the disclosure in which it is incorporated originally existed.

Claims

1. An apparatus for measuring the light absorption of a sample at each of two different wavelengths, the apparatus comprising: A sample holder having a first plate, a second plate, an optical spacer (LGS), at least one sampling area, and at least one reference area, wherein the two plates clamp the sample to be analyzed into a thin layer. A color camera used to measure light, wherein the light has a second intensity First intensity of wavelength mixing Wavelength, wherein the camera includes a first color channel A and a second color channel B; each color channel measures light and generates light signals I of different wavelengths respectively. A and I B ; A non-transient computer-readable medium for storing the algorithm (I), which uses optical signals I of different wavelengths. A and I B The strength is determined by the predetermined channel crosstalk matrix. and and (ii) determine the light absorption of the sample for each of the wavelengths; The sampling region is the area where the light passes sequentially through the first plate, the sample, and the second plate, and the sampling region does not have the LGS; The reference region is the area where light passes through the first plate, the light guide spacer, and the second plate in sequence, but does not pass through the sample.

2. The device of claim 1, wherein the light having two different wavelengths comprises a broadband light source passing through a bandwidth filter, wherein the bandwidth filter allows the light of two wavelengths to pass through while blocking the light of other wavelengths.

3. The device of claim 1 or 2, wherein the light having two different wavelengths comprises two light sources emitted simultaneously, wherein each light source has a different wavelength.

4. The apparatus of claim 1, wherein the sample is blood and the analyte is hemoglobin.

5. The device as claimed in claim 1 or 4, wherein the color camera is a camera on a mobile phone.

6. The device of claim 5, wherein the color camera has at least two color channels, and each color channel contains more than one detection element.

7. The device of claim 6, wherein the first color channel is the red channel of a color camera, and the second color channel is the green channel of the same camera.

8. The device of claim 1, wherein the predetermined channel crosstalk matrix has the capability to be used respectively by using the first strength Wavelength and the second intensity The wavelength is measured for each predetermined matrix element.

9. The device of claim 1, wherein the reference area is less than 0.1 μm. 2 Less than 0.2um 2 Less than 0.5um 2 Less than 1µm 2 Less than 2um 2 Less than 5um 2 Less than 10um 2 Less than 20um 2 Less than 50um 2 Less than 100um 2 Less than 200um 2 Less than 500um 2 Less than 1000um 2 Less than 2000um 2 Less than 5000um 2 Less than 10000um 2 Less than 20000um 2 Less than 50000um 2 , or within the range of any two values.

10. The device of claim 1, wherein the device further comprises a plurality of light guide spacers having substantially uniform height, and wherein at least one of the light guide spacers is located within the sample contact area.

11. The device of claim 1, wherein the device further comprises a plurality of light guide spacers having substantially uniform height, wherein the distance between two adjacent light guide spacers is known, and wherein at least one of the light guide spacers is within the sample contact area.

12. The device of claim 1, wherein the device further comprises a plurality of light guide spacers having substantially uniform height, wherein the distance between two adjacent light guide spacers is known and arranged in a periodic array, and wherein at least one of the light guide spacers is within the sample contact area.

13. The device of claim 1, wherein the bottom surface of the light guide spacer is fixed to the inner surface of one of the first plate or the second plate by molding the light guide spacer on the inner surface of the first plate or the second plate.

14. The device of claim 1, wherein the bottom surface of the light guide spacer is fixed to the inner surface of one of the first plate or the second plate and is made of the same material as the inner surface.

15. The device of claim 1, wherein the bottom surface of the light guide spacer is fixed to the inner surface of one of the first plate or the second plate and is made of the same material as the inner surface, and the bottom surface of the light guide spacer has no interface with the inner surface of the first plate or the second plate.

16. The apparatus of claim 1, wherein the thickness of the sample is 250 μm or less.

17. The device according to any one of claims 13-15, wherein the light guide spacer has a height of 100 μm or less.

18. A method for determining the intensity of each wavelength of light having two different wavelengths using a color camera, the method comprising: A color camera for measuring light is provided, wherein the camera includes a first color channel A and a second color channel B; and wherein the light has a first intensity. Wavelength and Second Intensity Wavelength mixing; Non-transient computer-readable media that provide storage algorithms; The light is measured using the color camera, wherein each color channel of the color camera generates a light signal I of a different wavelength. A and I B ;as well as Using the algorithm and the signal I A and I B To determine the strength separately and as well as The algorithm described therein includes a predetermined channel crosstalk matrix, which divides the signal I... A and I B With strength and Related.

19. The method of claim 18, wherein the color camera is a camera on a mobile phone.

20. The method of claim 18 or 19, wherein the color camera has at least two color channels, and each color channel contains more than one detection element.

21. The method of claim 20, wherein the first color channel is the red channel of a color camera, and the second color channel is the green channel of the same camera.

22. The method of claim 18, wherein the predetermined channel crosstalk matrix has, respectively, the first strength Wavelength and the second intensity The wavelength is measured for each predetermined matrix element.

23. A method for measuring the light absorption of a sample at each of two different wavelengths, the method comprising: Provide the device according to any one of claims 1-17; Place the sample to be analyzed in the sample holder; The illumination beam passes through the sample holder, and the light is measured using the color channel of a color camera; The algorithm was used to calculate the intensity of the sample region and the reference region, respectively. and And determine the light absorption of the sample for each wavelength.

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