Cell counting system and method

Abstract

The present invention provides a novel sample chamber, unit, and multiwell plate, as well as a system and method thereof, which incorporates measurement assurance for cell counting methods, and calibration and / or quality assurance for the measurement and analysis of various types of living cells, such as cell number, cell size, cell concentration, cell subpopulation, cell morphology, and cell viability.

Description

Technical Field

[0001] Priority Claim and Related Patent Applications This application claims the benefit of priority from U.S. Provisional Application No. 62 / 895,704, filed on September 4, 2019, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present invention generally relates to the measurement and analysis of biological samples. More particularly, the present invention relates to novel cell counting chambers, units and multi-well plates, as well as systems and methods for calibrating and / or quality assurance of the measurement and analysis of various types of living cells, including the measurement of cell number, cell size, cell concentration, cell sub-populations, cell morphology and cell viability.

Background Art

[0003] Important aspects in medical diagnosis and biomedical research include the detection, identification, quantification, and characterization of various cells and biomolecules of interest by examining biological samples such as blood, cerebrospinal fluid, cell cultures, and urine. Healthcare providers and biomedical researchers routinely analyze biological samples with respect to the microscopic presence, cell number, and concentration of cells and biomolecules.

[0004] In clinical trials, for example, the concentration of various blood cells such as red blood cells and white blood cells can provide extremely important information regarding the health status of a patient. In cell-based assays, cell counting is an important parameter. This is used to determine the appropriate seeding density of cell cultures, normalize protein-based assays, or determine the number of cells required for downstream assays such as flow cytometry.

[0005] Traditionally, cell counting has been performed manually using a hemocytometer under a light microscope. In the last decade, automated cell counters have become commercially available and have been well-received in biological laboratories. These cell counters are primarily designed for detecting single samples, and the operator must place a disposable slide in the instrument each time cells are counted. While existing technology is a significant improvement over manual counting, reducing operating time and operator variability, it still requires considerable time when analyzing a large number of samples.

[0006] Calibration and quality assurance methods remain inadequate and underdeveloped. In cell therapy, cell counts are used to control the dose of cells administered to patients. In both molecular biological research and clinical diagnosis and treatment, measuring cell viability, i.e., measuring and calculating the fractions of dead and living cells, is crucial. Biological samples vary widely in concentration and composition. Without accurate calibration and proper measurement assurance, it can be difficult to know whether a particular sample is suitable for image-based automated cell counting, and how reliable the resulting data is.

[0007] In addition to efficiently providing cell count measurements, the counting instrument and method include built-in calibration and measurement assurance functions, making it valuable that both the sample and the method are suitable for reliable and accurate counting.

[0008] Therefore, there is a continuing need for novel and improved devices and methods that are suitable for application to diverse cell types, biological samples, and conditions, capable of rapidly calibrating and ensuring the measurement of automated cell counts, and that provide reliable quality of cell count data. [Overview of the project]

[0009] The present invention provides a novel sample chamber, analysis unit, multiwell plate, and cell counting system that incorporates functions for high-throughput, automated calibration and assurance of cell measurement and analysis, such as cell number, concentration, subpopulation, morphology, size, viability, cell cycle, and surface markers.

[0010] The sample chamber, analysis unit, and multiwell plate are equipped with clearly defined microscale markings, objects, or features (sometimes collectively referred to as "features" or "microscale features") along the imaging path, thereby simplifying measurement calibration and quality assurance of results, which are important for biomedical applications.

[0011] In one embodiment, the present invention generally relates to a sample chamber suitable for holding a liquid sample for optical imaging. The sample chamber includes (a) an inlet for introducing a liquid sample into the sample chamber for observation or analysis; (b) an imaging chamber for holding the liquid sample, the imaging chamber having fluid communication with the inlet and including one or more light-transmitting windows suitable for observing or analyzing a liquid sample in the imaging chamber; and (c) an outlet having fluid communication with the imaging chamber for discharging air or for discharging the liquid sample. The imaging chamber is characterized by at least one of the following: a chamber height that varies continuously or stepwise, passing through at least a portion of one or more light-transmitting windows, and a defined feature having one or more known dimensions, area or volume, and being optically accessible through one or more light-transmitting windows, and the defined feature exhibiting one or more fluorescent colors at one or more intensities.

[0012] In another embodiment, the present invention generally relates to a sample analysis unit, which includes (i) a mixing well for preparing a liquid sample for analysis, and (ii) a sample chamber, as disclosed herein, positioned in spatial proximity to the mixing well.

[0013] In yet another aspect, the present invention generally relates to a sample analysis unit. The sample analysis unit includes (i) a mixing well for preparing a liquid sample for analysis, and (ii) a first sample chamber suitable for holding the liquid sample for optical imaging. The first sample chamber includes (a) a first inlet for introducing a liquid sample into the first sample chamber for observation or analysis, (b) a first imaging chamber for holding the liquid sample, the first imaging chamber having fluid communication with the first inlet and including a first or first pair of light-transmitting windows suitable for observing or analyzing the liquid sample in the first imaging chamber, and (c) a first outlet having fluid communication with the first imaging chamber for discharging air or for draining the liquid sample. The first imaging chamber is characterized by one or more of the following: (i) a height of the first chamber that varies either continuously or stepwise, passing through at least a portion of the first or a first set of light-transmitting windows; (ii) a first defined feature having one or more known offset values ​​from the focal plane of the first imaging chamber, the feature being optically accessible through the first or a first set of light-transmitting windows; and (iii) a defined first feature having one or more known dimensions, area or volume, the first feature being optically accessible through the first or a first set of light-transmitting windows; and (iv) the first defined feature exhibiting one or more fluorescence colors at one or more intensities.

[0014] In yet another aspect, the present invention generally relates to a multiwell plate or apparatus including a sample analysis unit disclosed herein.

[0015] In yet another aspect, the present invention generally relates to a multiwell plate comprising a plurality of sample analysis units, each sample analysis unit comprising (i) a mixing well for preparing a liquid sample for analysis, and (ii) one or more sample chambers suitable for holding the liquid sample for optical imaging. Each sample chamber comprises (a) an inlet for introducing a liquid sample into the sample chamber for observation or analysis, (b) an imaging chamber for holding the liquid sample, the imaging chamber comprising one or more light-transmitting windows that are in fluid communication with the inlet and suitable for observing or analyzing the liquid sample in the imaging chamber, and (c) an outlet that is in fluid communication with the imaging chamber for discharging air or for discharging the liquid sample. The imaging chamber is characterized by a defined feature having one or more known offset values ​​from the focal plane of the imaging chamber and being optically accessible through one or more light-transmitting windows, and / or a defined feature having one or more known dimensions, area or volume and being optically accessible through one or more light-transmitting windows. The defined characteristics exhibit one or more fluorescent colors at one or more intensities.

[0016] In yet another aspect, the present invention generally relates to a system for analyzing biological samples, comprising a sample analysis unit or multiwell plate as disclosed herein.

[0017] In yet another aspect, the present invention generally relates to a method for evaluating the focus setting of an imaging system for analyzing cell counts. The method includes: manually or automatically adjusting the focal position until one or more defined features of the imaging chamber appear at the best focus; recording the best focal position according to the defined features; manually or automatically adjusting the focus toward a trial focal position of cells in a sample where cells are estimated to be at the best focus for cell counting; recording the trial focal position of cells according to the sample; performing image acquisition to measure cell counts; comparing the focal position recorded according to the defined features with the focal position recorded according to the sample; and determining whether the difference between the two recorded focal positions is acceptable to confirm or reject the cell count measurement.

[0018] In yet another aspect, the present invention relates generally to a method for obtaining a focus setting for an imaging system for analyzing cell counts. The method includes the steps of manually or automatically adjusting the focal position until one or more defined features of an imaging chamber appear in the best focus; manually or automatically adjusting the focal position by a predetermined offset value from the focal position of one or more defined features; and performing image acquisition to measure cell counts.

[0019] In one embodiment, the focal position is adjusted manually in each case.

[0020] In one embodiment, the focal position is automatically adjusted in each case.

[0021] In yet another aspect, the present invention generally relates to a method for calibrating cell size measurements of an imaging system for analyzing cell counts. The method includes the steps of: measuring the size of cells in a sample; measuring the size of one or more defined features of an imaging chamber having known sizes; comparing the measured size of the defined features with their known sizes; determining a calibration coefficient based on the comparison of the known and measured sizes of the features; applying the calibration coefficient to the cell size measurements; and communicating the adjustment value of the cell size measurements.

[0022] In yet another aspect, the present invention generally relates to a method for evaluating cell size measurements of an imaging system for analyzing cell counts. The method includes the steps of: measuring the size of cells in a sample; measuring the size of one or more defined features of an imaging chamber having known sizes; comparing the measured size of the defined features with the known size; and determining whether the difference between the measured size of the features and the known size is acceptable, thereby confirming or rejecting the cell size measurement.

[0023] In yet another aspect, the present invention generally relates to a method for simultaneously evaluating the focus of an imaging system and a measured cell size for analyzing cell counts. The method includes: manually or automatically adjusting the focal position until one or more defined features of an imaging chamber appear at the best focus; measuring the size of cells in a sample; measuring the size of one or more defined features of an imaging chamber having known sizes; comparing the measured size of the defined features with their known sizes; determining whether the difference between the two measured values ​​is acceptable, thereby confirming or rejecting the focus setting and the measured cell size; and repeating these steps until an acceptable size measurement is obtained.

[0024] In yet another aspect, the present invention generally relates to a method for evaluating the suitability of a sample for analyzing cell counts without diluting or concentrating the sample. The method includes providing the sample in one or more sample chambers having the height of a plurality of chambers, recording the cell count of the sample at the height of the plurality of chambers, comparing the recorded cell counts, determining whether they are proportional to the height of the chambers, and determining or rejecting the measured cell count value.

[0025] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell counts without diluting or concentrating the sample. The method includes providing the sample in one or more sample chambers having the height of a plurality of chambers, recording the cell count of the sample at the height of the plurality of chambers, analyzing the measured value of the recorded cell counts, determining the height of the chambers in which the measured value of the cell counts is proportional to the height of the chambers, and determining the measured value of the corresponding cell counts.

[0026] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell counts without diluting or concentrating the sample. The method includes providing the sample in one or more sample chambers having the height of a plurality of chambers, recording the cell count of the sample at the height of the plurality of chambers, analyzing the measured value of the recorded cell counts, determining the height of the chambers in which the measured value of the cell counts is proportional to the height of the chambers, and determining the measured value of the corresponding cell counts.

[0027] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell counts without diluting or concentrating the sample. The method includes providing the sample in one or more sample chambers having the height of a plurality of chambers, recording the cell count of the sample at the height of the plurality of chambers, analyzing the measured value of the recorded cell counts, determining which measured values are proportional to the known height of the chambers, determining the measured values of the cell counts that are sufficiently proportional to the height of the chambers and rejecting those that are not.

[0028] In yet another aspect, the present invention generally relates to a method for measuring and analyzing the cell count without diluting or concentrating a sample. The method includes providing a sample in one or more sample chambers having a height of a plurality of chambers, recording the cell count of the sample at the height of the plurality of chambers, scoring the measured cell count based on how well the measured cell count matches an expected value for a known height of the chamber, and determining and rejecting measurements that do not meet a predetermined specification.

[0029] In yet another aspect, the present invention generally relates to a method for calibrating fluorescence measurements of an imaging system for analyzing cell counts. The method includes measuring the fluorescence of cells in a sample, measuring the fluorescence of one or more defined features of an imaging chamber having a known fluorescence, comparing the measured fluorescence of the defined features with their known fluorescence, determining a calibration factor based on the comparison of the measured fluorescence and the known fluorescence of the defined features, applying the calibration factor to the measured value of the fluorescence of the cells, and communicating an adjusted value of the fluorescence measurement.

[0030] In yet another aspect, the present invention generally relates to a method for evaluating fluorescence measurements of an imaging system for analyzing cell counts. The method includes measuring the fluorescence of cells in a sample, measuring the fluorescence of one or more defined features of an imaging chamber having a known fluorescence, comparing the measured fluorescence of the defined features with their known fluorescence to determine or reject the fluorescence measurement value.

[0031] In yet another aspect, the present invention generally relates to a method for calibrating and / or evaluating cell count analysis, including a step of calibrating or evaluating according to one or more methods disclosed in the specification.

[0032] In yet another aspect, the present invention generally relates to a method for measuring the cell count using a sample chamber disclosed herein, a sample analysis unit disclosed herein, a multi-well plate disclosed herein, or a system for analyzing a biological sample disclosed herein. [Brief explanation of the drawing]

[0033] [Figure 1] This is a schematic diagram illustrating the thickness of several exemplary chambers. [Figure 2] This is a schematic diagram of an example counting unit and an assembly of counting chambers of different thicknesses, used together. [Figure 3] This is a schematic diagram of exemplary markings or features for calibrating or evaluating the focus of a system. [Figure 4] This is a schematic diagram of exemplary markings or features for calibrating or evaluating cell size. [Figure 5] This is a schematic diagram of exemplary markings or features for calibrating or evaluating the fluorescence of cells. [Figure 6] This is an exemplary demonstration of cell counting proportional to the chamber thickness (which varies in stages). (A) Exemplary counting chambers of various thicknesses. (B) Demonstration of proportionality. [Figure 7A] This is an exemplary demonstration of cell counting proportional to the chamber thickness (continuously varying). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 7B] This is an exemplary demonstration of cell counting proportional to the chamber thickness (continuously varying). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 7C] This is an exemplary demonstration of cell counting proportional to the chamber thickness (continuously varying). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 7D] This is an exemplary demonstration of cell counting proportional to the chamber thickness (continuously varying). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 8] This is an example of a cell counting chamber sharing a common filling port. [Figure 9]These are exemplary cell counting chambers of different thicknesses that share a common filling port. [Figure 10] This is an exemplary demonstration of bead counting proportional to the effective concentration using the apparatus shown in Figure 9. [Figure 11] This is an exemplary demonstration of the relationship between the focal point and the resulting image. [Figure 12] This is an exemplary demonstration of the imaged object, its size histogram, and its circularity histogram. [Figure 13] This is an exemplary demonstration of the imaged fluorescent beads. [Modes for carrying out the invention]

[0034] The present invention relates to a novel sample chamber, unit, and multiwell plate with built-in functions that enable the evaluation of counting methods, as well as the calibration and / or quality assurance of various types of measurement and analysis of living cells, including, for example, measurements of cell number, cell size, cell concentration, cell subpopulation, cell morphology, and cell viability, and to a system and method thereof.

[0035] Calibration and evaluation of cell number proportions A key feature of the present invention is that the confirmation of proportionality in cell numbers is achieved without the need to dilute the sample or repeatedly measure samples at different concentrations. Conventionally, samples have been diluted to confirm appropriate proportionality in order to bring reliability to counting methods and protocols. The disclosed method evaluates the proportionality in cell numbers by measuring diverse regions of an imaging chamber having various, however specified, chamber thicknesses, instead of creating a series of concentrations to confirm the proportionality. In particular, the number of particles in the field of view is adjusted by a change in chamber thickness proportional to the volume.

[0036] As schematically illustrated in Figure 1, multiple thicknesses may be generated within a single counting chamber. The change in chamber thickness may be stepwise (Figure 1A) or continuous (Figure 1B). As illustrated in Figure 1C (longitudinal direction from inlet to outlet) and Figure 1D (shortitudinal direction from one edge of chamber to the other), the change in chamber thickness may be along either axis of the chamber.

[0037] As schematically illustrated in Figure 2, multiple counting chambers of various thicknesses may be used together. The multiple chambers may be manufactured as a single unit used as a set. In Figure 2A, four chambers of four different thicknesses and a single inlet are combined into one unit. In Figure 2B, three chambers of three different thicknesses and a single inlet are combined into one unit. Furthermore, the multiple chambers may be joined to a device with multiple chambers (e.g., a 96-well plate).

[0038] Figure 6 illustrates an exemplary demonstration of cell counting proportional to chamber thickness, where the thickness varies in a stepped manner. A set of counting chambers of varying thicknesses is used, each slide incorporating a different combination of adhesive spacer layers (Figure 6A). When filled with the same bead / cell suspension sample, the number of cells to be counted is proportional to the chamber thickness (Figure 6B).

[0039] Chambers of varying thicknesses may be produced using multiple thicknesses of adhesive, multiple layers of adhesive, and / or structures formed from UV-curable polymers using imprint lithography.

[0040] Figure 7 illustrates an exemplary demonstration of cell counting proportional to the chamber thickness, where the thickness changes continuously. Figure 7A shows two counting chambers with continuously varying chamber heights (left: varying in the short direction, right: varying in the long direction), constructed using multiple adhesive spacer layers. The adhesive areas are highlighted in blue, with darker blue indicating thicker adhesive. The adhesive layers surrounding the counting chambers formed steps, but the chamber ceiling transitioned smoothly from the thicker side to the thinner side, resulting in a continuously tapered chamber thickness. Two configurations were created in which the chamber thickness change occurred in both the short direction (X direction) and the long direction (Y direction) of the counting chamber.

[0041] In one experiment, a solution of 5 pm beads was filled into the chamber (2e6 beads / mL), and a histogram of bead positions was obtained. The slope and R² value obtained from the linear fitting of the histogram yielded a proportional metric. The histogram of X positions shown in Figure 7B was obtained from slide A of one of the counting chambers, and the histogram of Y positions shown in Figure 7C was obtained from slide B of one of the chambers.

[0042] Cell counting chambers may be configured to share a common entry (filling) port. A multi-chambered apparatus may contain independent chambers or chambers joined by one or more common sample filling ports. In Figure 2C, the multiwell plate features 10 sets of 3-chamber units. In Figure 2D, the multiwell plate features 4 sets of 4-chamber units and 2 sets of 3-chamber units.

[0043] Figure 8 shows a multi-well plate illustrating this multi-chamber configuration. Tests demonstrated that the cell suspension flowed simultaneously and consistently into multiple counting chambers. Figure 8A shows the cut adhesive layer (with backing paper still attached) defining the cell counting chambers. Figure 8B shows the adhesive layer with the surface sheet (with protective insulating film still attached) superimposed, indicating the alignment of the filling and discharging ports. Figure 8C shows the underside of the completed cell counting chambers, attached to the bottom of a 96-well plate. Multiple plates were tested for proper fluid flow and consistency of cell counting. No significant differences in concentration measurements were observed in counting chambers with different configurations when the bead solution was added to multiple filling ports.

[0044] In Figure 9, the multiwell plate contains four sample-filling ports, each connected to four counting chambers of different thicknesses. Figure 9A shows a top view, while Figure 9B shows a bottom view of the plate. Each chamber extends across four sample viewing windows. The plate can accommodate four samples and may generate four replicate linearity curves at four effective dilution points for each sample, as shown in Figure 9C.

[0045] The imaging system can rapidly count cells filled in a chamber created at the bottom of a standard 96-well plate. Custom software functions may be incorporated to analyze the cell count ratio and return a determination of the sample's suitability for the instrument's automatic counting. The software may also automatically analyze the count at various chamber heights and indicate to the user whether the cell suspension should be further diluted or concentrated for better counting. Furthermore, automatic exclusion of parts of the chamber with too many or too few cells may be achieved.

[0046] Calibration and evaluation of the system's focus Another feature of the present invention is an embedded microscale marking or feature, such as two-dimensional or three-dimensional markings on the surface of the chamber, which, with known offset values, can be reliably and consistently focused on cell counting and viability determination. This feature of the present invention enables proper and consistent focusing, which is essential for image-based cell counting. Changes in the appearance of cells under bright-field illumination and fluorescence irradiation can degrade the accuracy of cell counting algorithms, but consistent focusing avoids this.

[0047] To assist the system's focusing and ensure consistent focus, markings or features are embedded at known positions on the floor of the imaging chamber. For example, features spaced apart across the field of view can be used to assess whether the chamber is horizontal. Features characterized by high luminance contrast at the system's resolution level can accurately determine the focal position. For example, as schematically illustrated in Figure 3, features containing areas that exhibit high contrast when at the ideal focal position (both coarse and fine detail) can provide a more sensitive method for determining the relative focus. Furthermore, the 3D focal point can utilize various length and height scales, allowing the algorithm to comparatively determine changes over a wide range of distances.

[0048] To determine the focal position where a feature is in optimal focus, an automated focusing method may be implemented. This can be achieved by combining a focus score with an optimization algorithm. Careful calibration based on how the feature image changes with the focal position may lead to rapid calibration of the system's focus. With precisely fabricated microscale features, such calibration can achieve near-instantaneous registration of the focal position.

[0049] Once the focal position is registered in the system instrument, an offset value is set, and the ideal focal position for a specific type of cell can be recorded. This may be done manually by the user based on visual inspection, automatically based on the focal score, or by using the built-in offset value for a specific assay. Once the ideal focal position and reference marking plane for the cell are established, the instrument can quickly move to the ideal focus and acquire the next image.

[0050] Calibration and evaluation of cell size measurements Another feature of the present invention is the use of markings or features with clearly defined sizes, which allows the system to calibrate and evaluate cell size measurements. Cell counting instruments communicate the size of the detected object. The size communicated by the segmentation algorithm varies with the focal position and image brightness. Out-of-focus cells may appear larger and this may be communicated, but they may also appear with less contrast and be communicated as smaller in size by the texture algorithm. For fluorescence images, brighter illumination tends to yield larger cell measurements. Also, poor cell size measurements may affect density measurements.

[0051] Size calibration involves obtaining an accurate conversion from pixels to microns and evaluating the accuracy of the segmented blob size. Optical diffraction can affect the appearance of markings of different sizes. Features with known separation over larger distances yield more accurate estimates regarding the pixel-to-micron conversion coefficient. For the pixel-to-micron conversion, it is advantageous to use markings or features that are as spaced as possible in the image without 2D distortion or rotation. The accuracy of the segmented blob size best includes features of similar size and optical behavior to the cells being counted.

[0052] Figure 4 is a schematic diagram of microscale features of known dimensions, which can be used to calibrate segmentation algorithms. Checkerboard patterns may be used to calibrate cameras. Algorithms have been devised that are very suitable for finding the lines of a checkerboard pattern with sub-pixel accuracy. Another example is a cluster of dots, where the distance between the dots is clearly defined. Features such as 5 pm dots in a defined pattern can be used both to establish a calibrated coordinate system for measuring cell positions and to evaluate the accuracy of segmented sizes.

[0053] Calibration and evaluation of cell fluorescence measurements Another feature of the present invention is the use of objects or features with clearly defined fluorescence, thereby enabling the system to calibrate appropriate fluorescence irradiation and evaluate fluorescence measurements. Both count and size measurements in fluorescence imaging mode are determined by the intensity of light received by the camera. Intensity is determined by the brightness of the light source, the transmittance of the filter, and the fluorescence intensity of the cells themselves. Fluorescently labeled cells indicate that the emitted intensity is distributed. If cell segmentation from the background is performed using an intensity threshold, the number of counted cells will vary with the intensity of the excitation light. If the same threshold is set for the analysis of experiments performed under different lighting conditions, the number will vary for this reason alone. By well-controlling the fluorescent objects or features incorporated into the counting device, the intensity can be normalized to that of the marker, leading to better consistency of counts across experiments and between instruments. This also allows for comparisons between experiments performed with different exposure times.

[0054] For example, as schematically illustrated in Figure 5, the intensity of imaged cells may be correlated to or normalized to the brightness of a clearly characterized fluorescent object or feature embedded in or added to the cell counting chamber or slide. This allows for comparison of results between instruments and between experiments. Furthermore, system instruments can utilize these features to simply confirm that irradiation is functioning correctly, thereby confidently attributing cells with low or absent counts to the sample rather than the instrument.

[0055] Figure 5 shows two simple patterns of fluorescence features. In Figure 5A, a fluorescent checkerboard pattern is provided, which allows calibration of the scale (e.g., the size and / or spacing of the squares), fluorescence intensity (e.g., using the brightness of the fluorescent paint), and focal point. Figure 5B shows an alternative pattern consisting of fluorescent dots of known size and various colors. Multiple fluorescent channels (brightness and focal position) can be calibrated by coloring multiple dots. In addition, the scale calibration and the origin of the spatial coordinate system are provided by a known distance between pairs of dots. In Figure 5C, multiple small markings are placed around the counting chamber. Furthermore, multiple fluorescent colored markers can be used to find focal planes of different wavelengths. To reduce the manufacturing cost of such features, clearly characterized fluorescent beads may be used in lien to direct marking of the cell chamber.

[0056] Combined markings for multiplexed calibration and measurement accuracy assurance. Focus registration, proportionality verification, size calibration, and fluorescence intensity benchmarking may be achieved together. For example, a known object or feature of a known size may be printed in fluorescent ink on the outer surface of the bottom of the slide. The system instrument can focus on the object or feature and apply a known offset value to shift the focus to the focal plane of the cell. The system instrument may adjust the brightness or gain of the camera's light source to verify and, if necessary, complement the intensity of the fluorescent feature that meets the specifications. It may also verify a measured distance between objects or features to ensure that the image calibration is within an acceptable boundary. Such markings may be etched, printed, or otherwise added to the cell counting chamber (e.g., by imprint lithography).

[0057] Therefore, in one embodiment, the present invention generally relates to a sample chamber suitable for holding a liquid sample for optical imaging. The sample chamber includes (a) an inlet for introducing a liquid sample into the sample chamber for observation or analysis; (b) an imaging chamber for holding the liquid sample, the imaging chamber having fluid communication with the inlet and including one or more light-transmitting windows suitable for observing or analyzing the liquid sample inside the imaging chamber; and (c) an outlet having fluid communication with the imaging chamber for discharging air or for discharging the liquid sample. The imaging chamber is characterized by one or more of the following: a defined feature having a chamber height that changes either continuously or stepwise, passing through at least a portion of one or more light-transmitting windows, and having one or more known offset values ​​from the focal plane of the imaging chamber, wherein the feature is optically accessible through one or more light-transmitting windows; and a defined feature having one or more known dimensions, area, or volume, wherein the feature is optically accessible through one or more light-transmitting windows, and the defined feature exhibits one or more fluorescence colors at one or more intensities.

[0058] In one embodiment, at least a portion of the chamber height is continuously varied.

[0059] In one embodiment, at least a portion of the chamber height is varied in steps.

[0060] In one embodiment, the height of the chamber is varied both continuously and in steps (partially continuously and partially in steps).

[0061] In one embodiment, the height of the chamber varies in the range of approximately 1 pm to approximately 5,000 pm (for example, approximately 1 pm to approximately 5,000 pm, approximately 5 pm to approximately 5,000 pm, approximately 10 pm to approximately 5,000 pm, approximately 50 pm to approximately 5,000 pm, approximately 100 pm to approximately 5,000 pm, approximately 500 pm to approximately 5,000 pm, approximately 1 pm to approximately 1,000 pm, approximately 1 pm to approximately 500 pm, approximately 1 pm to approximately 100 pm, approximately 1 pm to approximately 50 pm, and approximately 1 pm to approximately 10 pm).

[0062] In one embodiment, the height of the chamber varies in the range of approximately 10 pm to approximately 500 pm (for example, approximately 10 pm to approximately 500 pm, approximately 25 pm to approximately 500 pm, approximately 50 pm to approximately 500 pm, approximately 100 pm to approximately 500 pm, approximately 10 pm to approximately 250 pm, approximately 10 pm to approximately 100 pm, approximately 10 pm to approximately 50 pm, and approximately 10 pm to approximately 25 pm).

[0063] In one embodiment, at least some of the defined features are three-dimensional and feature the height of one or more defined features.

[0064] In one embodiment, all defined features are three-dimensional and are characterized by the height of one or more defined features.

[0065] In one embodiment, the heights of one or more defined features are the same.

[0066] In some embodiments, the height of one or more defined features is different.

[0067] In one embodiment, the height of one or more defined features is in the range of approximately 0.1 μm to approximately 5,000 μm (for example, approximately 0.1 μm to approximately 1,000 μm, approximately 0.1 μm to approximately 500 μm, approximately 0.1 μm to approximately 200 μm, approximately 0.1 μm to approximately 100 μm, approximately 0.1 μm to approximately 50 μm, approximately 0.1 μm to approximately 25 μm, approximately 0.1 μm to approximately 10 μm, approximately 0.1 μm to approximately 5 μm, approximately 0.1 μm The microparticle sizes are approximately m~1μm, 1μm~5,000μm, 10μm~5,000μm, 50μm~5,000μm, 100μm~5,000μm, 500μm~5,000μm, 1,000μm~5,000μm, 1μm~500μm, 2μm~200μm, 2μm~100μm, 5μm~100μm, and 5μm~50μm.

[0068] In one embodiment, a defined feature having one or more known offset values ​​is inscribed in the bottom of the imaging chamber.

[0069] In one embodiment, a defined feature having one or more known offset values ​​is inscribed in the ceiling of the imaging chamber.

[0070] In one embodiment, a defined feature having one or more known dimensions, area, or volume is inscribed within the bottom of the imaging chamber.

[0071] In one embodiment, a defined feature having one or more known dimensions, area, or volume is inscribed within the ceiling of the imaging chamber.

[0072] In one embodiment, the defined features include a plurality of coarse and fine features.

[0073] In one embodiment, the defined features exhibit one or more predetermined patterns.

[0074] In one embodiment, the defined feature exhibits one or more fluorescent colors and / or one or more fluorescent intensities.

[0075] In one embodiment, the imaging chamber is configured to have a volume in the range of about 0.1 μL to about 200 pL (for example, about 0.5 pL to about 200 μL, about 1 pL to about 200 μL, about 10 pL to about 200 pL, about 50 pL to about 200 pL, about 0.1 pL to about 100 pL, about 0.1 pL to about 50 pL, about 0.1 pL to about 20 pL, about 0.1 pL to about 10 pL, about 0.1 pL to about 5 pL, about 0.1 pL to about 1 pL).

[0076] In one embodiment, the imaging chamber is configured to have a volume in the range of about 4 μL to about 100 pL (for example, about 10 pL to about 100 pL, about 20 pL to about 100 pL, about 50 pL to about 100 pL, about 4 pL to about 50 pL, about 4 pL to about 20 pL, about 4 pL to about 10 pL).

[0077] In one embodiment, the imaging chamber includes a single light-transmitting window.

[0078] In one embodiment, the imaging chamber includes two or more light-transmitting windows.

[0079] In one embodiment, the imaging chamber has three, four, or five light-transmitting windows.

[0080] In another embodiment, the present invention generally relates to a sample analysis unit, which includes (i) a mixing well for preparing a liquid sample for analysis, and (ii) a sample chamber, as disclosed herein, positioned in spatial proximity to the mixing well.

[0081] In yet another aspect, the present invention generally relates to a sample analysis unit. The sample analysis unit includes (i) a mixing well for preparing a liquid sample for analysis, and (ii) a first sample chamber suitable for holding the liquid sample for optical imaging. The first sample chamber includes (a) a first inlet for introducing a liquid sample into the first sample chamber for observation or analysis, (b) a first imaging chamber for holding the liquid sample, the first imaging chamber having fluid communication with the first inlet and including a first or first pair of light-transmitting windows suitable for observing or analyzing the liquid sample in the first imaging chamber, and (c) a first outlet having fluid communication with the first imaging chamber for discharging air or for draining the liquid sample. The first imaging chamber is characterized by one or more of the following: (i) a height of the first chamber that changes either continuously or stepwise, passing through at least a portion of the first or a first set of light-transmitting windows; (ii) a first defined feature having one or more known offset values ​​from the focal plane of the first imaging chamber, the feature being optically accessible through the first or a first set of light-transmitting windows; and (iii) a defined first feature having one or more known dimensions, area or volume, the first feature being optically accessible through the first or a first set of light-transmitting windows; and (iv) the first defined feature exhibiting one or more fluorescence colors at one or more intensities.

[0082] In one embodiment, the sample analysis unit further includes a second sample chamber suitable for holding a liquid sample for optical imaging. The second sample chamber includes (a) a second inlet for introducing a liquid sample into the second sample chamber for observation or analysis; (b) a second imaging chamber for holding the liquid sample, the second imaging chamber having fluid communication with the second inlet and including a second or second pair of light-transmitting windows suitable for observing or analyzing the liquid sample in the second imaging chamber; and (c) a second outlet having fluid communication with the second imaging chamber for discharging air or for draining the liquid sample. The second imaging chamber is characterized by one or more of the following: (i) a height of the second chamber that varies either continuously or stepwise, passing through at least a portion of the second or a second set of light-transmitting windows; (ii) a second defined feature having one or more known offset values ​​from the focal plane of the second imaging chamber, the second feature being optically accessible through the second or a second set of light-transmitting windows; and (iii) a defined second feature having one or more known dimensions, area or volume, the second feature being optically accessible through the second or a second set of light-transmitting windows; and (iv) the second defined feature exhibiting one or more fluorescence colors at one or more intensities.

[0083] In one embodiment, the sample analysis unit further includes a third sample chamber suitable for holding a liquid sample for optical imaging. The third sample chamber includes (a) a third inlet for introducing a liquid sample into the third sample chamber for observation or analysis; (b) a third imaging chamber for holding the liquid sample, the third imaging chamber having fluid communication with the third inlet and including a third or third pair of light-transmitting windows suitable for observing or analyzing the liquid sample in the third imaging chamber; and (c) a third outlet having fluid communication with the third imaging chamber for discharging air or for draining the liquid sample. The third imaging chamber is characterized by one or more of the following: (i) a height of the third chamber that varies either continuously or stepwise, passing through at least a portion of the third or a set of third light-transmitting windows; (ii) a third defined feature having one or more known offset values ​​from the focal plane of the third imaging chamber, the third feature being optically accessible through the third or a set of third light-transmitting windows; and (iii) a defined third feature having one or more known dimensions, area or volume, the third feature being optically accessible through the third or a set of third light-transmitting windows; and (iv) a third defined feature exhibiting one or more fluorescence colors at one or more intensities.

[0084] In one embodiment, the sample analysis unit further includes a fourth sample chamber suitable for holding a liquid sample for optical imaging. The fourth sample chamber includes (a) a fourth inlet for introducing a liquid sample into the fourth sample chamber for observation or analysis; (b) a fourth imaging chamber for holding the liquid sample, the fourth imaging chamber having fluid communication with the fourth inlet and including a fourth or fourth pair of light-transmitting windows suitable for observing or analyzing the liquid sample in the fourth imaging chamber; and (c) a fourth outlet having fluid communication with the fourth imaging chamber for discharging air or for draining the liquid sample. The fourth imaging chamber is characterized by one or more of the following: (i) a height of the fourth chamber that varies either continuously or stepwise, passing through at least a portion of the fourth or a set of fourth light-transmitting windows; (ii) a fourth defined feature having one or more known offset values ​​from the focal plane of the fourth imaging chamber, wherein the fourth feature is optically accessible through the fourth or a set of fourth light-transmitting windows; (iii) a defined fourth feature having one or more known dimensions, area or volume, wherein the fourth feature is optically accessible through the fourth or a set of fourth light-transmitting windows; and (iv) a fourth defined feature exhibiting one or more fluorescence colors at one or more intensities.

[0085] In one embodiment, the sample analysis unit further includes one or more additional sample chambers suitable for holding a liquid sample for optical imaging.

[0086] In one embodiment of the sample analysis unit, the first, second, third, fourth, and any other inlets all share the same inlet.

[0087] In one embodiment, each of the first, second, third, fourth, and any other imaging chambers is characterized by a uniform chamber height, while the first, second, third, fourth, and any other imaging chambers have different chamber heights.

[0088] In one embodiment, each of the first, second, third, and fourth imaging chambers, as well as any other, is characterized by non-uniform chamber heights.

[0089] In some embodiments, the first, second, third, fourth, and any other defined features are the same.

[0090] In some embodiments, the first, second, third, fourth, and any other defined features are different.

[0091] In one embodiment, each imaging chamber is configured to have a volume of approximately 0.1 μL to approximately 200 pL (for example, approximately 0.5 pL to approximately 200 pL, approximately 1 pL to approximately 200 pL, approximately 10 pL to approximately 200 pL, approximately 50 pL to approximately 200 pL, approximately 0.1 pL to approximately 100 pL, approximately 0.1 pL to approximately 50 pL, approximately 0.1 pL to approximately 20 pL, approximately 0.1 pL to approximately 10 pL, approximately 0.1 pL to approximately 5 pL, approximately 0.1 pL to approximately 1 pL).

[0092] In one embodiment, each imaging chamber is configured to have a volume of approximately 4 pL to approximately 100 pL (for example, approximately 10 pL to approximately 100 pL, approximately 20 pL to approximately 100 pL, approximately 50 μL to approximately 100 pL, approximately 4 μL to approximately 50 pL, approximately 4 pL to approximately 20 pL, and approximately 4 pL to approximately 10 pL).

[0093] In one embodiment, each of the first or first set of light-transmitting windows, the second or second set of light-transmitting windows, the third or third set of light-transmitting windows, and the fourth or fourth set of light-transmitting windows includes a single light-transmitting window.

[0094] In one embodiment, each of the first or first set of light-transmitting windows, the second or second set of light-transmitting windows, the third or third set of light-transmitting windows, and the fourth or fourth set of light-transmitting windows includes two or more light-transmitting windows.

[0095] In one embodiment, each of the first or first set of light-transmitting windows, the second or second set of light-transmitting windows, the third or third set of light-transmitting windows, and the fourth or fourth set of light-transmitting windows includes three, four, or five light-transmitting windows.

[0096] In yet another aspect, the present invention generally relates to a multiwell plate or apparatus including a sample analysis unit disclosed herein.

[0097] In yet another embodiment, the present invention generally relates to a multiwell plate comprising a plurality of sample analysis units, wherein each sample analysis unit comprises (i) a mixing well for preparing a liquid sample for analysis, and (ii) one or more sample chambers suitable for holding the liquid sample for optical imaging. Each sample chamber comprises (a) an inlet for introducing a liquid sample into the sample chamber for observation or analysis, (b) an imaging chamber for holding the liquid sample, the imaging chamber comprising one or more light-transmitting windows that are in fluid communication with the inlet and suitable for observing or analyzing the liquid sample in the imaging chamber, and (c) an outlet that is in fluid communication with the imaging chamber for discharging air or for draining the liquid sample. The imaging chamber is characterized by a defined feature having one or more known offset values ​​from the focal plane of the imaging chamber and being optically accessible through one or more light-transmitting windows, and / or a defined feature having one or more known dimensions, volume or capacity and being optically accessible through one or more light-transmitting windows. The defined characteristics exhibit one or more fluorescent colors at one or more intensities.

[0098] In some embodiments, the multiwell plate includes two or more sample analysis units. In some embodiments, the multiwell plate includes four or more sample analysis units. In some embodiments, the multiwell plate includes six or more sample analysis units. In some embodiments, the multiwell plate includes eight or more sample analysis units. In some embodiments, the multiwell plate includes ten or more sample analysis units. In some embodiments, the multiwell plate includes twelve or more sample analysis units.

[0099] In embodiments with a multiwell plate, each sample analysis unit includes two or more sample chambers. In embodiments with a multiwell plate, each sample analysis unit includes three or more sample chambers. In embodiments with a multiwell plate, each sample analysis unit includes four or more sample chambers.

[0100] In some embodiments of the multiwell plate, all defined features are the same.

[0101] In some embodiments, the defined features are not all the same.

[0102] In one embodiment, each imaging chamber is configured to have a volume of approximately 0.1 μL to approximately 200 pL (for example, approximately 0.5 pL to approximately 200 pL, approximately 1 pL to approximately 200 pL, approximately 10 pL to approximately 200 pL, approximately 50 pL to approximately 200 pL, approximately 0.1 pL to approximately 100 pL, approximately 0.1 pL to approximately 50 pL, approximately 0.1 pL to approximately 20 pL, approximately 0.1 pL to approximately 10 pL, approximately 0.1 pL to approximately 5 pL, approximately 0.1 pL to approximately 1 pL).

[0103] In one embodiment, each imaging chamber is configured to have a volume of approximately 4 pL to approximately 100 pL (for example, approximately 10 pL to approximately 100 pL, approximately 20 pL to approximately 100 pL, approximately 50 pL to approximately 100 pL, approximately 4 pL to approximately 50 pL, approximately 4 pL to approximately 20 pL, and approximately 4 pL to approximately 10 pL).

[0104] In yet another aspect, the present invention generally relates to a system for analyzing biological samples, comprising a sample analysis unit or multiwell plate as disclosed herein.

[0105] In one embodiment, the system further includes at least one fluorescent light source, at least one brightfield light source, at least one optical system for focusing the fluorescent and / or brightfield light beams, a detection device, and a computing unit.

[0106] In one embodiment, the system includes two or more fluorescent light sources.

[0107] In one embodiment, the system includes two or more bright-field light sources.

[0108] In yet another aspect, the present invention generally relates to a method for evaluating the focus setting of an imaging system for analyzing cell counts. The method includes the steps of: adjusting the focal position until one or more defined features of an imaging chamber appear in the best focus; recording the focal position according to the defined features; adjusting the focal position until the cells of a sample appear in the best focus; recording the focal position according to the sample; measuring cell counts; comparing the focal position recorded according to the defined features with the focal position recorded according to the sample; and determining whether the difference between the two recorded focal positions is acceptable, thereby confirming or rejecting the cell count measurement.

[0109] In yet another aspect, the present invention generally relates to a method for evaluating the focus setting of an imaging system for analyzing cell counts. The method includes: manually or automatically adjusting the focus position until one or more defined features of the imaging chamber appear at the best focus; recording the focus position according to the defined features; manually or automatically adjusting the focus toward a trial focus position of cells where cells are estimated to be at the best focus for cell counting; recording the trial focus position of cells according to a sample; performing image acquisition to measure the number of cells; comparing the focus position recorded according to the defined features with the focus position recorded according to the sample; and determining whether the difference between the two recorded focus positions is acceptable to confirm or reject the cell count measurement.

[0110] In one embodiment, if a cell count measurement is rejected, the method further includes: manually or automatically adjusting the focus toward a new trial focal position of the cell where the cell is estimated to be at the best focal point for cell counting; recording the new trial focal position of the cell according to the sample; acquiring an image to measure the cell number; comparing the focal position recorded according to a defined feature with the focal position recorded according to the sample; determining whether the difference between the two recorded focal positions is acceptable and confirming or rejecting the cell count measurement; and repeating the focus adjustment as necessary to generate an acceptable focal position.

[0111] In yet another aspect, the present invention relates generally to a method for obtaining a focus setting for an imaging system for analyzing cell counts. The method includes the steps of manually or automatically adjusting the focal position until one or more defined features of an imaging chamber appear in the best focus; manually or automatically adjusting the focal position by a predetermined offset value from the focal position of one or more defined features; and performing image acquisition to measure cell counts.

[0112] In yet another aspect, the present invention generally relates to a method for calibrating cell size measurements of an imaging system for analyzing cell counts. The method includes the steps of: measuring the size of cells in a sample; measuring the size of one or more defined features of an imaging chamber having known sizes; comparing the measured size of the defined features with their known sizes; determining a calibration coefficient based on the comparison of the known and measured sizes of the features; applying the calibration coefficient to the cell size measurements; and communicating the adjustment value of the cell size measurements.

[0113] In yet another aspect, the present invention generally relates to a method for evaluating cell size measurements of an imaging system for analyzing cell counts. The method includes the steps of: measuring the size of cells in a sample; measuring the size of one or more defined features of an imaging chamber having known sizes; comparing the measured size of the defined features with the known size; and determining whether the difference between the measured size of the features and the known size is acceptable, thereby confirming or rejecting the cell size measurement.

[0114] In one embodiment, if a cell size measurement is rejected, the method further includes the steps of manually or automatically adjusting one or more instrument settings; measuring the size of cells in a sample; measuring the size of one or more defined features of an imaging chamber having known sizes; comparing the measured size of the defined features with their known sizes; determining whether the difference between the measured size of the features and their known sizes is acceptable, thereby confirming or rejecting the cell size measurement; and repeating these steps until an acceptable size measurement is obtained.

[0115] In yet another aspect, the present invention generally relates to a method for simultaneously evaluating the focus of an imaging system and a measured cell size for analyzing cell counts. The method includes: manually or automatically adjusting the focal position until one or more defined features of an imaging chamber appear at the best focus; measuring the size of cells in a sample; measuring the size of one or more defined features of an imaging chamber having known sizes; comparing the measured sizes of the defined features with their known sizes; determining whether the difference between the two measured values ​​is acceptable to confirm or reject the focus setting and the measured cell size; and repeating these steps until an acceptable size measurement is obtained.

[0116] In yet another aspect, the present invention generally relates to a method for evaluating the suitability of a sample for analyzing cell counts without diluting or concentrating the sample. The method includes the steps of: placing a sample in one or more sample chambers having a plurality of chamber heights; recording the number of cells in the sample at the plurality of chamber heights; and comparing the recorded cell counts to determine whether they are proportional to the chamber heights, thereby confirming or rejecting the cell count measurements.

[0117] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell counts without diluting or concentrating a sample. The method includes the steps of: placing a sample in one or more sample chambers having a plurality of chamber heights; recording the number of cells in the sample at the plurality of chamber heights; and analyzing the recorded cell count measurements to determine the chamber height at which the cell count measurement is proportional to the chamber height, and determining the corresponding cell count measurement.

[0118] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell counts without diluting or concentrating a sample. The method includes the steps of: placing a sample in one or more sample chambers having a plurality of chamber heights; recording the number of cells in the sample at the plurality of chamber heights; and analyzing the recorded cell count measurements to determine the chamber height at which the cell count measurement is proportional to the chamber height, and determining the corresponding cell count measurement.

[0119] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell counts without diluting or concentrating a sample. The method includes steps of: placing a sample in one or more sample chambers having multiple chamber heights; recording the cell counts of the sample at the multiple chamber heights; analyzing the recorded cell count measurements to determine which measurements are proportional to known chamber heights; and confirming the cell count measurements that are sufficiently proportional to the chamber heights and rejecting those that are not.

[0120] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell counts without diluting or concentrating a sample. The method includes steps of: placing a sample in one or more sample chambers having multiple chamber heights; recording the number of cells in the sample at the multiple chamber heights; scoring the obtained cell count measurements based on how well they match the expected values ​​for known chamber heights; and confirming the measurements that meet predetermined specifications and rejecting those that do not.

[0121] In one embodiment, the method further includes the step of excluding cell count measurements recorded at chamber heights where the cell count measurements are not proportional to the chamber height.

[0122] In one embodiment, the method further includes the step of excluding cell count measurements recorded at chamber heights where the cell count measurements are not proportional to the chamber height. In another embodiment, the method further includes the step of generating one or more quality scores for the cell count measurements based on the proportional results. For example, if the apparatus has six different chamber heights, the sample measurements that yield proportional numbers across all six chamber heights are of higher quality than the measurements that exclude three of the six chamber heights due to the lack of proportionality.

[0123] In yet another aspect, the present invention generally relates to a method for calibrating fluorescence measurements of an imaging system for analyzing cell counts. The method includes the steps of: measuring the fluorescence of cells in a sample; measuring the fluorescence of one or more defined features of an imaging chamber having known fluorescence; comparing the measured fluorescence of the defined features with their known fluorescence; determining a calibration coefficient based on the comparison between the measured fluorescence of the defined features and the known fluorescence; applying the calibration coefficient to the cell fluorescence measurements; and communicating the adjustment values ​​for the fluorescence measurements.

[0124] In yet another aspect, the present invention generally relates to a method for evaluating fluorescence measurements of an imaging system for analyzing cell counts. The method includes the steps of: measuring the fluorescence of cells in a sample; measuring the fluorescence of one or more defined features of an imaging chamber having known fluorescence; and confirming or rejecting fluorescence measurements by comparing the measured fluorescence of the defined features with their known fluorescence.

[0125] In one embodiment, if a fluorescence measurement is rejected, the method includes the steps of manually or automatically adjusting one or more instrument settings; measuring the fluorescence of cells in a sample; measuring the fluorescence of one or more defined features of an imaging chamber having known fluorescence; comparing the measured fluorescence of the defined features with the known fluorescence to determine or reject the fluorescence measurement; and repeating these steps until the fluorescence measurement of the defined features becomes acceptable.

[0126] In yet another aspect, the present invention generally relates to a method for calibrating and / or evaluating cell counting analysis, comprising a step of performing calibration or evaluation, relating to one or more methods disclosed in the specification.

[0127] In one embodiment, at least some of the one or more defined features are three-dimensional and feature the height of one or more defined features.

[0128] In one embodiment, all defined features are three-dimensional and are characterized by the height of one or more defined features.

[0129] In one embodiment, the heights of one or more defined features are the same.

[0130] In some embodiments, the height of one or more defined features is different.

[0131] In one embodiment, the height of one or more defined features is in the range of approximately 0.1 μm to approximately 5,000 μm (for example, approximately 0.1 μm to approximately 1,000 μm, approximately 0.1 μm to approximately 500 μm, approximately 0.1 μm to approximately 200 μm, approximately 0.1 μm to approximately 100 μm, approximately 0.1 μm to approximately 50 μm, approximately 0.1 μm to approximately 25 μm, approximately 0.1 μm to approximately 10 μm, approximately 0.1 μm to approximately 5 μm, approximately 0.1 μm The microparticle sizes are approximately m~1μm, 1μm~5,000μm, 10μm~5,000μm, 50μm~5,000μm, 100μm~5,000μm, 500μm~5,000μm, 1,000μm~5,000μm, 1μm~500μm, 2μm~200μm, 2μm~100μm, 5μm~100μm, and 5μm~50μm.

[0132] In one embodiment, the defined feature is inscribed in the bottom of the imaging chamber.

[0133] In one embodiment, a defined feature having one or more known offset values ​​is inscribed in the ceiling of the imaging chamber.

[0134] In one embodiment, a defined feature having one or more known dimensions, area, or volume is inscribed within the bottom of the imaging chamber.

[0135] In one embodiment, a defined feature having one or more known dimensions, area, or volume is inscribed within the ceiling of the imaging chamber.

[0136] In one embodiment, the defined features include a plurality of coarse and fine features.

[0137] In one embodiment, the defined feature represents one or more predetermined patterns or geometric shapes.

[0138] In one embodiment, the defined feature exhibits one or more fluorescent colors and / or one or more fluorescent intensities.

[0139] In one embodiment, the imaging chamber is configured to have a volume of approximately 0.1 μL to approximately 200 pL (for example, approximately 0.5 pL to approximately 200 μL, approximately 1 pL to approximately 200 μL, approximately 10 pL to approximately 200 pL, approximately 50 pL to approximately 200 pL, approximately 0.1 pL to approximately 100 pL, approximately 0.1 pL to approximately 50 pL, approximately 0.1 pL to approximately 20 pL, approximately 0.1 pL to approximately 10 pL, approximately 0.1 pL to approximately 5 pL, approximately 0.1 pL to approximately 1 pL).

[0140] In one embodiment, the imaging chamber is configured to have a volume of approximately 4 pL to approximately 100 pL (for example, approximately 10 pL to approximately 100 pL, approximately 20 pL to approximately 100 pL, approximately 50 pL to approximately 100 pL, approximately 4 pL to approximately 50 pL, approximately 4 pL to approximately 20 pL, and approximately 4 pL to approximately 10 pL).

[0141] In yet another aspect, the present invention generally relates to a method for measuring cell counts using a sample chamber, a sample analysis unit, a multiwell plate, or a system for analyzing biological samples as disclosed herein.

[0142] The present invention is highly suitable for high-throughput cell counting instruments and associated software with self-calibration and measurement assurance functions. An example of such a system utilizes five excitation filters (375, 475, 530, 540, 630 nm) and six emission filters (450, 525, 600, 610 LP, 660, 695 nm) for transmitted bright-field and reflected fluorescence channels. It also employs an infinity-corrected optical objective for high-resolution and high-quality imaging. The exemplary system operates in XYZ mode, enabling imaging and analysis of cells in standard microplates (6-1536 wells), T25 and T75 flasks, as well as glass and chamber slides. This improves cell counting time to 24 samples per minute for bright-field analysis (trypan blue) and 24 samples per 3 minutes for fluorescence analysis (acridine orange and propidium iodide).

[0143] In addition, the device may be integrated with a liquid handler to perform a fully automated, high-throughput cell counting process.

[0144] An exemplary system is constructed with software designed to image and analyze different types of consumables, such as cell counting plates, 6- to 1536 well standard microplates, glass slides, T75 and T25 flasks, and chamber slides, as disclosed herein. This may be used to measure cell concentration, cell size, and morphology, such as perimeter, roundness, area, major / minor axis, compactness, elongation, eccentricity, sphericity, convexity, aspect ratio, and robustness. In fluorescence-based assays, fluorescence intensity can be measured. For high-throughput cell counting and analysis, plate handlers, liquid handlers, and further accessories integrated into the system are intended for full automation.

[0145] The software analyzes the captured images to determine cell concentration, cell size and morphology, and the percentage of fluorescently labeled populations (acridine orange, propidium iodide (PI), 4',6-diamidino-2-phenylindole (DAPI), Hoechst, 7-aminoactinomycin D (7AAD), Sytox Green, Sytox Red, DRAQ5 / 7, nuclear green / red / blue / far red, trypan) The system assesses various aspects of cell cycles (such as blue), transduction efficiency (green fluorescent protein (GFP), red fluorescent protein (RFP), mCherry, blue fluorescent protein (BFP), mCardinal, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), etc.), apoptosis (annexin V-FITC, -PE and PI, or caspase 3 / 7), autophagy (LC3II-FITC, -GFP), cell cycle (PI, Hoechst, DAPI, BrdU (bromodeoxyuridine), EdU (5-ethynyl-2'-deoxyuridine)), cellular senescence (β-gal green), vitality (calcein AM, CFDA-AM (5-carboxyfluorescein diacetate acetoxymethyl ester), FDA (fluorescein diacetate), CFDA), ROS, mitochondrial potential and health, surface marker staining, and intracellular staining.

[0146] The system according to the present invention may be designed to perform assays in oncology, immuno-oncology, virology, cell therapy, cell line development, regenerative medicine (stem cell research), brewing research, and renewable energy. It may be used to analyze cell lines (cancer cells NCI-60 and others), primary cells (PBMCs, spleen cells, leukocyte apheresis, apheresis, thymocytes), stem cells, platelets, erythrocytes, yeast and algae, CHO cells, etc. The system may be used to perform assays such as cell growth and proliferation, viability, changes in cell size due to activation, transduction efficiency, apoptosis, autophagy, cell cycle, cellular senescence, ROS, mitochondrial potential and health, population analysis of surface markers, and population analysis of intracellular staining.

[0147] In addition, the system can determine population proportions based on size, morphology, or fluorescent labeling. Population proportions can be determined by comparing the number or concentration of cells in a target cell population based on size, morphology, or fluorescent labeling, by identifying the total number of cells in bright-field or any other fluorescent channel.

[0148] The following examples are illustrative of the embodiment of the present invention and are not limiting in any way.

[0149] Figure 10 shows exemplary results obtained using the apparatus of Figure 9 and samples of bead solutions with three different concentrations. The number of beads counted in the image is plotted against the effective concentration, which is the actual concentration of the sample multiplied by the chamber thickness. There may be proportional deviations between the high and low ends of the effective concentration range. Therefore, the numbers for low-concentration and high-concentration samples may be discarded. A sufficient number of samples that fall within the linear range were used to record the samples, and the confirmed values ​​were returned, excluding non-linear points. In addition, the concentration range that the instrument can reliably measure can be increased by using multiple chamber thicknesses.

[0150] Figure 11 shows that various focal positions lead to various images of the object being imaged. In (A), the goal is to make surface details visible by imaging a microscopic object. The object rests on a translucent surface, but if the focus is on the surface itself, the details disappear (B). For example, for a similar object of this size, the goal is to focus 182 pm away from the surface. A key feature is that it is easily recognizable as being 548 pm from the surface on which the object of interest rests (grid lines, panel C). The grid can be reliably focused on, and once focused on the grid, the position of the objective lens can be indicated. Subsequently, the objective lens can be moved with an offset value of 548 pm to the surface of the sample, and then further to 182 pm to the best focus on the object (total offset value is 730 pm). If dirt or scratches cause confusion in the focusing algorithm and incorrectly focus on the wrong position, resulting in an image of the grid or object (D), a focus offset value that is too different from the expected 730 pm will be transmitted, and the focus will be rejected.

[0151] Figure 12 shows exemplary demonstrations of the imaging targets and their size and roundness histograms. Panel A shows a portion of the image of a suspension of Jurkat cells mixed with borosilicate glass beads of known size. The inset shows a region of the same sample image with slightly blurred focus. The beads have a test mean particle size of 17.3 pm and a standard deviation of 2.0 pm. Targets found by the algorithm are highlighted in green. Panel B shows the size histogram of targets identified in a well-focused image. The peak value at 20 pm corresponds to a bead. The beads were selected using a gate of 16–25 pm. For these targets, the algorithm returned a mean particle size of 19.96 pm and a standard deviation of 1.94 pm.

[0152] This result indicates that the algorithm was reporting a size approximately 15% larger than the actual size of the features (beads). Similarly, the reported cell sizes may also have been affected. Also plotted in (B) is a histogram of the object sizes identified by the algorithm on an image of the sample with the overall focus slightly blurred. While clear peak values ​​corresponding to beads have been excluded, images may be rejected for cell counting. Panel C shows the circularity histogram obtained from a well-focused image. Histograms of pre-identified objects, such as beads, are also plotted. For spherical objects, the peak value does not occur at 1.0, as expected. This is a special case where a feature with known dimensions (Figure 4) was used to detect size-calibrated tissue. In this case, discrepancies may arise due to incorrect length calibration along one of the image dimensions. Additionally, distortion from the system's optical elements may be present. Distortion can be another reason for rejection.

[0153] Figure 13 shows an example of imaging with fluorescent beads. (A) and (B) show images of two Jurkat samples containing similar fluorescent beads. In each pair of images, one cell and one bead were observed. The cells were stained with propidium iodide, which labels dead cells. In the first pair (A), the fluorescence of the cells was very faint. Without the presence of the beads, one might question whether the excitation illumination intensity or the camera exposure time was appropriate for this assay. The beads allow us to confirm that the faintness of the cells is due to not fluorescing strongly and not a problem with the instrument or settings.

[0154] In the second set of images (B), Jurkat cells absorbed propidium iodide and fluoresced strongly. The fluorescence images of these cells were taken with a longer exposure time than the fluorescence images of the first cells (A), but the images were difficult to reliably compare without a common reference feature. The presence of standard beads allows for a more consistent cell count by comparing the two images.

[0155] Panel C demonstrates the calibration of the bead intensity in Panel B. In this case, a simple luminance profile of one dimension was measured. By adjusting the luminance of the second fluorescence image with a coefficient of 0.8, it was found that the bead intensity of Panel A and the bead intensity matched well.

[0156] The disclosures by the applicant are described herein in preferred embodiments with reference to the figures, where the same numbers represent the same or similar elements. Throughout this specification, any reference to “one embodiment,” “embodiment,” or similar expression means that any specific features, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Thus, throughout this specification, any occurrence of the phrase “in one embodiment,” “in an embodiment,” or similar expression may all refer to the same embodiment, though not necessarily.

[0157] The features, structures, or properties disclosed by the applicant may be combined in any suitable manner in one or more embodiments. Numerous specific details are referenced in this description to provide a full understanding of embodiments of the invention. However, it will be apparent to those skilled in the art that the applicant's structures and / or methods may be carried out without using one or more of the specific details, or using other methods, components, materials, etc. In other examples, well-known structures, materials, or operations are not shown or described in detail so as not to obscure the aspects of this disclosure.

[0158] In the specification and the attached claims, the singular forms "a," "an," and "the" include the plural form unless otherwise clearly specified in the context.

[0159] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure, but preferred methods and materials are described herein. The methods detailed herein may be carried out in any logically possible order, in addition to the specific order disclosed.

[0160] Embedding by citation This disclosure includes references and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, and web content. All such documents are incorporated herein by reference in their entirety for all purposes. Any material, or any part thereof, that is said to be incorporated herein by reference but which conflicts with existing definitions, references, or other disclosure materials expressly provided herein is incorporated only insofar as it does not create a conflict between the incorporated material and the disclosure material. If a conflict arises, the conflict should be resolved in a manner that favors this disclosure as a preferred disclosure.

[0161] Equivalents Representative examples are intended to aid in illustrating the present invention and are not intended to limit the scope of the invention, nor should they be construed as such. In fact, various modifications of the invention and many further embodiments, in addition to those shown and described herein, will be apparent to those skilled in the art from the entirety of this specification, including the examples contained herein and references to scientific and patent literature. The examples include important additional information, illustrations, and guidance that can be adapted to the practice of the invention in various embodiments and their equivalents.

Claims

1. A sample chamber suitable for holding a liquid sample for optical imaging, wherein the sample chamber is (a) an inlet for introducing the liquid sample into the sample chamber for observation or analysis, (b) An imaging chamber for holding the liquid sample, the imaging chamber having fluid communication with the inlet and including one or more light-transmitting windows suitable for observing or analyzing the liquid sample inside the imaging chamber, (c) A fluid outlet connected to the imaging chamber for discharging air or for discharging the liquid sample, The aforementioned imaging chamber is The height of the chamber, which varies either continuously or in steps, passing through at least a portion of the one or more light-transmitting windows, A defined feature having one or more known offset values ​​from the focal plane of the imaging chamber, wherein the feature is optically accessible through one or more light-transmitting windows. A defined feature having one or more known dimensions, area, or volume, wherein the feature is optically accessible through one or more light-transmitting windows. The aforementioned features include a sample chamber characterized by one or more of the following: a feature that exhibits one or more fluorescent colors at one or more intensities; and a sample chamber characterized by one or more of the following.

2. The sample chamber according to claim 1, wherein at least a portion of the height of the chamber is continuously varied.

3. The sample chamber according to claim 1, wherein at least a portion of the height of the chamber is varied in steps.

4. The sample chamber according to any one of claims 1 to 3, wherein the height of the chamber is both continuously varied and stepped.

5. The sample chamber according to any one of claims 2 to 4, wherein the height of the chamber varies in the range of about 1 pm to about 5,000 pm.

6. The sample chamber according to any one of claims 2 to 4, wherein the height of the chamber varies in the range of about 10 pm to about 500 pm.

7. A sample chamber according to any one of claims 1 to 6, wherein at least some of the defined features are three-dimensional and characterized by the height of one or more defined features.

8. A sample chamber according to any one of claims 1 to 6, wherein all of the aforementioned defined features are three-dimensional and characterized by the height of one or more defined features.

9. The sample chamber according to claim 7 or 8, wherein the height of one or more of the defined features is the same.

10. A sample chamber according to claim 7 or 8, wherein the heights of one or more of the defined features are different.

11. The sample chamber according to any one of claims 7 to 10, wherein the height of one or more of the specified features is in the range of about 0.1 pm to about 5,000 pm.

12. The sample chamber according to claim 11, wherein the height of one or more of the defined features is in the range of about 1 pm to about 500 pm.

13. The sample chamber according to claim 12, wherein the height of one or more of the defined features is in the range of about 2 pm to about 200 pm.

14. The sample chamber according to any one of claims 1 to 13, wherein the defined feature having one or more known offset values ​​is inscribed in the bottom of the imaging chamber.

15. The sample chamber according to any one of claims 1 to 14, wherein the defined feature having one or more known offset values ​​is inscribed in the ceiling portion of the imaging chamber.

16. The sample chamber according to any one of claims 1 to 15, wherein the defined features having one or more known dimensions, area, or volume are inscribed in the bottom of the imaging chamber.

17. The sample chamber according to any one of claims 1 to 15, wherein the defined features having one or more known dimensions, area, or volume are inscribed in the ceiling portion of the imaging chamber.

18. The sample chamber according to any one of claims 1 to 17, wherein the aforementioned defined features include a plurality of coarse and fine features.

19. The sample chamber according to any one of claims 1 to 18, wherein the aforementioned defined feature exhibits one or more predetermined patterns or geometric shapes.

20. The aforementioned feature is a sample chamber according to any one of claims 1 to 19, which exhibits one or more fluorescent colors and / or one or more fluorescent intensities.

21. The sample chamber according to any one of claims 1 to 20, wherein the imaging chamber is configured to have a volume of about 0.1 pL to about 200 pL.

22. The sample chamber according to claim 21, wherein the imaging chamber is configured to have a volume of about 4 pL to about 100 pL.

23. The sample chamber according to any one of claims 1 to 22, wherein the imaging chamber is provided with a single light-transmitting window.

24. The sample chamber according to any one of claims 1 to 22, wherein the imaging chamber comprises two or more light-transmitting windows.

25. The sample chamber according to any one of claims 1 to 22, wherein the imaging chamber comprises three, four, or five light-transmitting windows.

26. A sample analysis unit, wherein the sample analysis unit is (i) A mixing well for preparing a liquid sample for analysis, (ii) A sample analysis unit comprising a sample chamber according to any one of claims 1 to 25, which is spatially adjacent to the mixing well.

27. A sample analysis unit, wherein the sample analysis unit is (i) A mixing well for preparing a liquid sample for analysis, (ii) A first sample chamber suitable for holding a liquid sample for optical imaging, wherein the first sample chamber is (a) A first inlet for introducing the liquid sample into the first sample chamber for observation or analysis, (b) A first imaging chamber for holding the liquid sample, the first imaging chamber comprising a first or first set of light-transmitting windows that are in fluid communication with the first inlet and suitable for observing or analyzing the liquid sample inside the first imaging chamber, (c) A first sample chamber comprising a first outlet for discharging air or for discharging the liquid sample, which is in fluid communication with the first imaging chamber, The first imaging chamber is The height of the first chamber, which varies either continuously or in steps, passing through at least a portion of the first or first set of light-transmitting windows, A first defined feature having one or more known offset values ​​from the focal plane of the first imaging chamber, wherein the feature is optically accessible through the first or first set of light-transmitting windows, A first defined feature having one or more known dimensions, area, or volume, wherein the first feature is optically accessible through the first or first set of light-transmitting windows. A sample analysis unit characterized by one or more of the following: the first defined feature exhibits one or more fluorescent colors at one or more intensities.

28. The system further includes a second sample chamber suitable for holding a liquid sample for optical imaging, wherein the second sample chamber is (a) A second inlet for introducing the liquid sample into the second sample chamber for observation or analysis, (b) A second imaging chamber for holding the liquid sample, the second imaging chamber comprising a second or second set of light-transmitting windows that are in fluid communication with the second inlet and suitable for observing or analyzing the liquid sample in the second imaging chamber, (c) A second outlet, which is in fluid communication with the second imaging chamber, for discharging air or for discharging the liquid sample, The second imaging chamber described above is The height of the second chamber, which varies either continuously or in steps, passing through at least a portion of the second or second set of light-transmitting windows, A second defined feature having one or more known offset values ​​from the focal plane of the second imaging chamber, wherein the second feature is optically accessible through the second or second set of light-transmitting windows. A second defined feature having one or more known dimensions, area, or volume, wherein the second feature is optically accessible through the second or second set of light-transmitting windows. The sample analysis unit according to claim 27, wherein the second defined feature is characterized by exhibiting one or more fluorescent colors at one or more intensities, and one or more of the second defined feature.

29. The system further includes a third sample chamber suitable for holding a liquid sample for optical imaging, wherein the third sample chamber is (a) A third inlet for introducing the liquid sample into the third sample chamber for observation or analysis, (b) A third imaging chamber for holding the liquid sample, the third imaging chamber comprising a third or third set of light-transmitting windows that are in fluid communication with the third inlet and suitable for observing or analyzing the liquid sample inside the third imaging chamber, (c) A third outlet, which is in fluid communication with the third imaging chamber, for discharging air or for discharging the liquid sample, The third imaging chamber described above is The height of the third chamber, which varies either continuously or in steps, passing through at least a portion of the third or third set of light-transmitting windows, A third defined feature having one or more known offset values ​​from the focal plane of the third imaging chamber, wherein the third feature is optically accessible through the third or third set of light-transmitting windows. A third defined feature having one or more known dimensions, area, or volume, wherein the third feature is optically accessible through the third or third set of light-transmitting windows. The sample analysis unit according to claim 28, wherein the third defined feature is characterized by exhibiting one or more fluorescent colors at one or more intensities, and one or more of the third defined feature.

30. The system further includes a fourth sample chamber suitable for holding a liquid sample for optical imaging, wherein the fourth sample chamber is (a) A fourth inlet for introducing the liquid sample into the fourth sample chamber for observation or analysis, (b) A fourth imaging chamber for holding the liquid sample, the fourth imaging chamber comprising a fourth or fourth set of light-transmitting windows that are in fluid communication with the fourth inlet and suitable for observing or analyzing the liquid sample inside the fourth imaging chamber, (c) A fourth outlet, which is in fluid communication with the fourth imaging chamber, for discharging air or for discharging the liquid sample, The fourth imaging chamber is, The height of the fourth chamber, which varies either continuously or in steps, and which passes through at least a portion of the fourth or fourth set of light-transmitting windows, A fourth defined feature having one or more known offset values ​​from the focal plane of the fourth imaging chamber, wherein the fourth feature is optically accessible through the fourth or a fourth set of light-transmitting windows. A fourth defined feature having one or more known dimensions, area, or volume, wherein the fourth feature is optically accessible through a fourth or fourth set of light-transmitting windows. The sample analysis unit according to claim 29, wherein the fourth defined feature is characterized by exhibiting one or more fluorescent colors at one or more intensities, and one or more of the fourth defined feature.

31. The sample analysis unit according to claim 30, further comprising one or more separate sample chambers suitable for holding a liquid sample for optical imaging.

32. The sample analysis unit according to any one of claims 27 to 31, wherein the first, second, third, and fourth, as well as any other sample chambers, share the same inlet.

33. The sample analysis unit according to any one of claims 27 to 32, wherein each of the first, second, third, and fourth imaging chambers, as well as any other imaging chamber, is characterized by a uniform chamber height, but the first, second, third, and fourth imaging chambers have different chamber heights.

34. The sample analysis unit according to any one of claims 27 to 33, wherein each of the first, second, third, fourth, and any other imaging chambers is characterized by a non-uniform chamber height.

35. The sample analysis unit according to any one of claims 27 to 34, wherein the first, second, third, fourth, and any other imaging chambers incorporate the same defined features.

36. The sample analysis unit according to any one of claims 27 to 34, wherein the first, second, third, fourth, and any other imaging chambers incorporate different defined features.

37. The sample analysis unit according to any one of claims 27 to 36, wherein each of the imaging chambers is configured to have a volume of about 0.1 μL to about 200 μL.

38. The sample analysis unit according to claim 37, wherein each of the imaging chambers is configured to have a volume of about 4 pL to about 100 pL.

39. A sample analysis unit according to any one of claims 27 to 38, wherein each of the first or first set of light-transmitting windows, the second or second set of light-transmitting windows, the third or third set of light-transmitting windows, and the fourth or fourth set of light-transmitting windows each includes a single light-transmitting window.

40. A sample analysis unit according to any one of claims 27 to 38, wherein each of the first or first set of light-transmitting windows, the second or second set of light-transmitting windows, the third or third set of light-transmitting windows, and the fourth or fourth set of light-transmitting windows each includes two or more light-transmitting windows.

41. A sample analysis unit according to any one of claims 27 to 38, wherein each of the first or first set of light-transmitting windows, the second or second set of light-transmitting windows, the third or third set of light-transmitting windows, and the fourth or fourth set of light-transmitting windows each includes three, four, or five light-transmitting windows.

42. A multiwell plate or apparatus comprising a sample analysis unit according to any one of claims 26 to 41.

43. A multiwell plate containing multiple sample analysis units, each sample analysis unit is: (i) A mixing well for preparing a liquid sample for analysis, (ii) One or more sample chambers suitable for holding a liquid sample for optical imaging, wherein each sample chamber is (a) an inlet for introducing the liquid sample into the sample chamber for observation or analysis, (b) An imaging chamber for holding the liquid sample, the imaging chamber having fluid communication with the inlet and including one or more light-transmitting windows suitable for observing or analyzing the liquid sample inside the imaging chamber, (c) One or more sample chambers, each having a fluid-connected outlet for discharging air or for discharging the liquid sample, The aforementioned imaging chamber is A defined feature having one or more known offset values ​​from the focal plane of the imaging chamber, wherein the feature is optically accessible through one or more light-transmitting windows, and / or A defined feature having one or more known dimensions, area, or volume, wherein the feature is optically accessible through one or more light-transmitting windows. The aforementioned feature is a multiwell plate characterized by exhibiting one or more fluorescent colors at one or more intensities.

44. A multiwell plate according to claim 43, comprising two or more sample analysis units.

45. A multiwell plate according to claim 44, comprising six or more sample analysis units.

46. A multiwell plate according to claim 45, comprising 10 or more sample analysis units.

47. Each of the sample analysis units comprises two or more sample chambers, according to any one of claims 43 to 46, in the multiwell plate.

48. Each of the sample analysis units comprises three or more sample chambers, as described in claim 47, for the multiwell plate.

49. Each of the sample analysis units comprises four or more sample chambers, as described in claim 48, for the multiwell plate.

50. The multiwell plate according to any one of claims 43 to 49, wherein the aforementioned features are the same.

51. The multiwell plate according to any one of claims 43 to 50, wherein each of the imaging chambers is configured to have a volume of about 0.1 μL to about 200 pL.

52. The multiwell plate according to claim 51, wherein each of the imaging chambers is configured to have a volume of about 4 pL to about 100 pL.

53. The multiwell plate according to any one of claims 43 to 52, wherein the imaging chamber comprises a single light-transmitting window.

54. The multiwell plate according to any one of claims 43 to 52, wherein the imaging chamber comprises two or more light-transmitting windows.

55. The multiwell plate according to any one of claims 43 to 52, wherein the imaging chamber comprises three, four, or five light-transmitting windows.

56. A system for analyzing a biological sample, comprising a sample analysis unit or a multiwell plate according to any one of claims 43 to 55.

57. At least one fluorescent light source, At least one bright-field light source, A system for focusing the light beam of a fluorescence beam and / or a bright-field light beam, The system according to claim 56, further comprising a detection device.

58. The system according to claim 57, further comprising a processing unit.

59. The system according to any one of claims 56 to 58, comprising two or more fluorescent light sources.

60. The system according to any one of claims 56 to 59, comprising two or more bright-field light sources.

61. A method for evaluating the focus setting of an imaging system for analyzing cell counts, wherein the method is: A step of manually or automatically adjusting the focal position until one or more defined features of the imaging chamber appear at the best focus, A step of recording the best focal position according to the defined characteristics, A step of manually or automatically adjusting the focus toward the trial focal position of the cells in the sample, where the cells are estimated to be at the best focal point for cell counting, A step of recording the trial focal position of the cells according to the sample, The process involves acquiring images to measure the number of cells, A step of comparing the focal position recorded according to the defined characteristics with the focal position recorded according to the sample, A method comprising the step of determining whether the difference between two recorded focal positions is acceptable, thereby confirming or rejecting a cell count measurement.

62. If the cell count measurement is rejected, the method is: A step of manually or automatically adjusting the focus toward a new trial focal position of the cells in the sample, where the cells are estimated to be at the best focal point for cell counting; A step of recording a new trial focal position of the cell according to the sample, The process involves acquiring images to measure the number of cells, A step of comparing the focal position recorded according to the defined characteristics with the focal position recorded according to the sample, A step of determining whether the difference between the two recorded focal positions is acceptable, and confirming or rejecting the cell count measurement, The method according to claim 61, further comprising the step of repeatedly adjusting the focus as necessary to generate an acceptable focal position.

63. A method for obtaining the focus setting of an imaging system for analyzing cell counts, the method being: A step of manually or automatically adjusting the focal position until one or more defined features of the imaging chamber appear at the best focus, A step of manually or automatically adjusting the focal position of one or more defined features by a predetermined offset value from the focal position; A method comprising the step of acquiring an image in order to measure the cell count.

64. The method according to any one of claims 61 to 63, wherein the focal position is manually adjusted in each case.

65. The method according to any one of claims 61 to 63, wherein the focal position is automatically adjusted in each case.

66. A method for calibrating cell size measurements of an imaging system for analyzing cell counts, wherein the method is: A step to measure the size of cells in the sample, A step of measuring the size of one or more defined features of an imaging chamber having a known size, A step of comparing the measured size of the specified features with their known sizes, A step of determining the calibration coefficient based on a comparison of the known size of the aforementioned feature with the measured size, A step of applying the calibration coefficient to the cell size measurement, A method comprising the step of communicating an adjustment value for a cell size measurement.

67. A method for evaluating cell size measurements of an imaging system in order to analyze cell counts, wherein the method is A step to measure the size of cells in the sample, A step of measuring the size of one or more defined features of an imaging chamber having a known size, A step of comparing the measured size of the aforementioned defined features with their known sizes, A method comprising the step of determining whether the difference between the measured size of the feature and the known size is acceptable, thereby confirming or rejecting the measured cell size.

68. If the aforementioned cell size measurement is rejected, The process of manually or automatically adjusting the settings of one or more devices, A step to measure the size of cells in the sample, A step of measuring the size of one or more defined features of an imaging chamber having a known size, A step of comparing the measured size of the specified features with their known sizes, A step of determining whether the difference between the measured size of the aforementioned feature and the known size is acceptable, and thereby confirming or rejecting the measured cell size; The method according to claim 67, further comprising the step of repeating these steps until an acceptable size measurement is obtained.

69. A method for simultaneously evaluating the focus of an imaging system and a cell size measurement for analyzing cell counts, wherein the method is: A step of manually or automatically adjusting the focal position until one or more defined features of the imaging chamber appear at the best focus, A step to measure the size of cells in the sample, A step of measuring the size of one or more defined features of the imaging chamber having a known size, A step of comparing the measured size of the specified features with their known sizes, A step of determining whether the difference between the two aforementioned measurements is acceptable, and confirming or rejecting the focus setting and the cell size measurement, A method comprising the step of repeating these steps until an acceptable size measurement is obtained.

70. A method for evaluating the suitability of a sample for analyzing cell counts without diluting or concentrating the sample, the method being: A step of placing a sample in one or more sample chambers having multiple chamber heights, A step of recording the number of cells in the sample at different heights of multiple chambers, A method comprising the steps of comparing the recorded cell counts and determining whether they are proportional to the height of the chamber, thereby confirming or rejecting the measured cell count.

71. A method for measuring and analyzing cell counts without diluting or concentrating a sample, wherein the method is: A step of placing a sample in one or more sample chambers having multiple chamber heights, A step of recording the number of cells in the sample at the height of the plurality of chambers, A method comprising the steps of: analyzing the recorded cell count measurements; determining the height of the chamber at which the cell count measurement is proportional to the height of the chamber; and determining the corresponding cell count measurement.

72. A method for measuring and analyzing cell counts without diluting or concentrating a sample, wherein the method is: A step of placing a sample in one or more sample chambers having multiple chamber heights, A step of recording the number of cells in the sample at the height of the plurality of chambers, A method comprising the steps of analyzing the recorded cell count measurements, determining the range of cell counts in which the cell count measurements are proportional to the height of the chamber, and determining the corresponding cell count measurements.

73. A method for measuring and analyzing cell counts without diluting or concentrating a sample, wherein the method is: A step of placing a sample in one or more sample chambers having multiple chamber heights, A step of recording the number of cells in the sample at the height of the plurality of chambers, The process involves analyzing the recorded cell count measurements and determining which measurements are proportional to the known chamber height, A method comprising the steps of determining a cell count that is sufficiently proportional to the height of the chamber and rejecting those that are not.

74. A method for measuring and analyzing cell counts without diluting or concentrating a sample, wherein the method is: A step of placing a sample in one or more sample chambers having multiple chamber heights, A step of recording the number of cells in the sample at the height of the plurality of chambers, A step of scoring the obtained cell count measurement based on how well the cell count matches the expected value for the known chamber height, A method comprising the steps of determining whether a measurement value meets a specified specification and rejecting any that does not.

75. The method according to any one of claims 71 to 74, further comprising the step of excluding cell count measurements recorded at chamber heights where the cell count measurements are not proportional to the chamber height.

76. The method according to any one of claims 71 to 75, further comprising the step of generating one or more quality scores for the measured cell count based on the proportional result.

77. A method for calibrating fluorescence measurements of an imaging system for analyzing cell counts, the method being: A step of measuring the fluorescence of cells in a sample, A step of measuring the fluorescence of one or more defined features of an imaging chamber having known fluorescence, A process of comparing the measured fluorescence of defined characteristics with known fluorescence, A step of determining a calibration coefficient based on a comparison of the measured fluorescence of the defined characteristics with the known fluorescence, A step of applying a calibration coefficient to the fluorescence measurement value of the cell, A method comprising the step of conveying an adjusted value for the measured fluorescence.

78. A method for evaluating fluorescence measurements of an imaging system to analyze cell counts, wherein the method is: A step of measuring the fluorescence of cells in a sample, A step of measuring the fluorescence of one or more defined features of an imaging chamber having known fluorescence, A method comprising the steps of determining or rejecting measured fluorescence values ​​of the defined features by comparing them with known fluorescence values.

79. If the aforementioned fluorescence measurement is rejected, The process of manually or automatically adjusting the settings of one or more devices, A step of measuring the fluorescence of cells in a sample, A step of measuring the fluorescence of one or more defined features of an imaging chamber having known fluorescence, A step of determining or rejecting the measured fluorescence of the defined characteristic by comparing it with the known fluorescence of the defined characteristic, The method according to claim 78, comprising the step of repeating these steps until the measured fluorescence of the defined feature is acceptable.

80. A method for calibrating and / or evaluating cell count analysis, comprising a step of calibration or guaranteeing measured values, according to one or more of claims 61 to 79.

81. The method according to any one of claims 61 to 80, wherein at least some of the one or more defined features are three-dimensional and feature the height of one or more defined features.

82. The method according to any one of claims 61 to 80, wherein all of the aforementioned defined features are three-dimensional and characterized by the height of one or more defined features.

83. The method according to claim 81 or 82, wherein the heights of one or more of the defined features are the same.

84. The method according to claim 81 or 82, wherein the heights of one or more of the defined features are different.

85. The method according to any one of claims 61 to 84, wherein the height of one or more of the specified features is in the range of about 0.1 pm to about 5,000 pm.

86. The method according to claim 85, wherein the height of one or more of the specified features is in the range of about 1 pm to about 500 pm.

87. The method according to claim 86, wherein the height of one or more of the specified features is in the range of about 2 pm to about 200 pm.

88. The method according to any one of claims 61 to 87, wherein the aforementioned feature is inscribed in the bottom of the imaging chamber.

89. The method according to any one of claims 61 to 88, wherein the defined feature having one or more known offset values ​​is inscribed in the ceiling of the imaging chamber.

90. The method according to any one of claims 61 to 89, wherein the defined feature having one or more known dimensions, area, or volume is inscribed in the bottom of the imaging chamber.

91. The method according to any one of claims 61 to 89, wherein the defined feature having one or more known dimensions, area, or volume is inscribed in the ceiling of the imaging chamber.

92. The method according to any one of claims 61 to 91, wherein the aforementioned features include a plurality of coarse and fine features.

93. The method according to any one of claims 61 to 92, wherein the aforementioned feature represents one or more predetermined patterns or geometric shapes.

94. The method according to any one of claims 61 to 93, wherein the defined features include one or more fluorescent colors and / or fluorescence intensity levels.

95. The method according to any one of claims 61 to 94, wherein the imaging chamber is configured to have a volume of about 0.1 pL to about 200 pL.

96. The method according to claim 95, wherein the imaging chamber is configured to have a volume of about 4 μL to about 100 μL.

97. A method for measuring the number of cells using a sample chamber according to any one of claims 1 to 25, a sample analysis unit according to any one of claims 26 to 41, a multiwell plate according to any one of claims 42 to 55, or a system for analyzing a biological sample according to any one of claims 56 to 60.

Images