Cell count measurement system and method

Abstract

The present invention provides novel sample chambers, units and multi-well plates, and systems and methods thereof, with built-in calibration and / or quality assurance for measurement assurance of cell counting methods and for measurement and analysis of various types of biological cells, such as cell number, cell size, cell concentration, cell subpopulations, cell morphology, cell viability, etc.

Description

[Technical field]

[0001] Priority claims and related patent applications This application claims the benefit of priority from U.S. Provisional Application No. 62 / 895,704, filed September 4, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates generally to the measurement and analysis of biological samples, and more particularly to novel cell counting chambers, units and multi-well plates, and systems and methods thereof, for calibrating and / or quality assuring the measurement and analysis of various types of biological cells, including the measurement of cell number, cell size, cell concentration, cell subpopulations, cell morphology and cell viability. [Background technology]

[0003] An important aspect of medical diagnostics and biomedical research involves the detection, identification, quantification, and characterization of various cells and biomolecules of interest by testing biological samples such as blood, spinal fluid, cell cultures, and urine. Health care providers and biomedical researchers routinely analyze biological samples for the microscopic presence, cell number, and concentration of cells and biomolecules.

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

[0005] Traditionally, cell counting is performed manually using a hemocytometer under an optical microscope. In the last decade, automated cell counting devices have become commercially available and have been well received in biological laboratories. These cell counting devices are primarily designed to detect single samples, and require the operator to place a disposable slide on the instrument each time cells are counted. Although the existing techniques are a significant improvement over manual counting, reducing the operation time and operator variability, they still require a large amount of time when a large number of samples need to be analyzed.

[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 biology research and clinical diagnostics and therapy, the measurement of cell viability, i.e. the measurement and calculation of the fraction of dead and live cells, is important. Biological samples vary widely in concentration and composition. Without good calibration and proper measurement assurance, it can be difficult to know whether a particular sample is amenable to automated image-based cell counting and how reliable the data obtained are.

[0007] It is valuable to have counting instruments and methods that not only efficiently provide a measurement of cell number, but also include built-in calibration and measurement assurance features, so that both the sample and the method are suitable for reliable and accurate count measurements.

[0008] Thus, there continues to be a need for new and improved devices and methods that can rapidly calibrate and certify automated cell counting measurements suitable for application to a wide variety of cell type biological samples and conditions, providing confidence in the quality of the cell counting data. Summary of the Invention

[0009] The present invention provides novel sample chambers, analysis units, multi-well plates and cell counting systems with built-in automated calibration and assurance capabilities for high-throughput cellular measurements and analysis, e.g., cell number, concentration, subpopulations, morphology, size, viability, cell cycle, surface markers, etc.

[0010] Sample chambers, analytical units and multi-well plates are provided with microscale markings, targets or features (sometimes collectively referred to as "features" or "microscale features") that are clearly defined in the imaging path, thereby simplifying measurement calibration and quality assurance of results, which is important in biomedical applications.

[0011] In one aspect, 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 a liquid sample, the imaging chamber being in fluid communication with the inlet and including one or more optically transparent windows suitable for observing or analyzing the liquid sample in the imaging chamber; and (c) an outlet in fluid communication with the imaging chamber for evacuating air or for draining the liquid sample. The imaging chamber is characterized by at least one of: a chamber height that varies, either continuously or in steps, through at least a portion of the one or more optically transparent windows; a defined feature having one or more known offsets from a focal plane of the imaging chamber, the feature being optically accessible through the one or more optically transparent windows; and a defined feature having one or more known dimensions, areas, or volumes, the defined feature being optically accessible through the one or more optically transparent windows, the defined feature exhibiting one or more fluorescent colors at one or more intensities.

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

[0013] In yet another aspect, the present invention generally relates to a sample analysis unit, the sample analysis unit including (i) a mixing well for preparing a liquid sample for analysis, and (ii) a first sample chamber suitable for holding a liquid sample for optical imaging, the first sample chamber including (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 a liquid sample, the first imaging chamber in fluid communication with the first inlet and including a first or first set of optically transparent windows suitable for observing or analyzing the liquid sample in the first imaging chamber, and (c) a first outlet in fluid communication with the first imaging chamber for evacuating air or for draining the liquid sample. The first imaging chamber is characterized by one or more of: (i) a first chamber height that varies, either continuously or in steps, through at least a portion of the first or first set of optically transmissive windows; (ii) a first defined feature having one or more known offsets from a focal plane of the first imaging chamber, the feature being optically accessible through the first or first set of optically transmissive windows; (iii) a first defined feature having one or more known dimensions, area or volume, the first feature being optically accessible through the first or first set of optically transmissive windows; and (iv) the first defined feature exhibits one or more fluorescent colors at one or more intensities.

[0014] In yet another aspect, the invention generally relates to a multi-well plate or device that includes the sample analysis unit disclosed herein.

[0015] In yet another aspect, the present invention generally relates to a multi-well plate including a plurality of sample analysis units, each of which includes (i) a mixing well for preparing a liquid sample for analysis, and (ii) one or more sample chambers suitable for holding a liquid sample for optical imaging. Each 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 a liquid sample, the imaging chamber in fluid communication with the inlet and including one or more optically transparent windows suitable for observing or analyzing the liquid sample in the imaging chamber, and (c) an outlet in fluid communication with the imaging chamber for venting air or for allowing the liquid sample to flow out. The imaging chamber is characterized by a defined feature having one or more known offsets from a focal plane of the imaging chamber, the defined feature being optically accessible through one or more optically transparent windows, and / or a defined feature having one or more known dimensions, areas, or volumes, the defined feature being optically accessible through one or more optically transparent windows. The defined features 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 that includes a sample analysis unit or a multi-well plate as disclosed herein.

[0017] In yet another aspect, the present invention generally relates to a method for evaluating focus settings of an imaging system for analyzing cell counting, the method comprising the steps of manually or automatically adjusting the focus position until one or more defined features of an imaging chamber appear in best focus, recording the best focus position according to the defined features, manually or automatically adjusting the focus towards a trial focus position of cells of the sample where the cells are presumed to be in the best focus for cell counting, recording the trial focus position of the cells according to the sample, performing image acquisition to measure cell counting, 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 or not to confirm or reject the measured cell counting value.

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

[0019] In an embodiment, the focus position is adjusted manually in each case.

[0020] In an embodiment, the focus 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 including measuring a size of cells in a sample, measuring a size of one or more defined features of an imaging chamber having a known size, comparing the measured size of the defined features to the known sizes, determining a calibration factor based on the comparison of the known and measured sizes of the features, applying the calibration factor to the cell size measurements, and communicating an adjustment to 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 including measuring the size of cells in a sample, measuring the size of one or more defined features of an imaging chamber having a known size, comparing the measured size of the defined features to the known size, and determining whether a difference between the measured size of the features and the known size is acceptable to confirm or reject the cell size measurement.

[0023] In yet another aspect, the present invention generally relates to a method for simultaneously evaluating focus and cell size measurements of an imaging system for analyzing cell counting, the method including the steps of manually or automatically adjusting the focus position until one or more defined features of the imaging chamber appear in best focus, measuring the size of cells in a sample, measuring the size of one or more defined features of the imaging chamber having known sizes, comparing the measured sizes of the defined features to the known sizes, determining whether the difference between the two measurements is acceptable to confirm or reject the focus setting and cell size measurements, and repeating these steps until an acceptable size measurement is obtained.

[0024] In yet another aspect, the invention generally relates to a method for assessing the suitability of a sample for cell count analysis without diluting or concentrating the sample, the method comprising providing the sample in one or more sample chambers having multiple chamber heights, recording cell counts of the sample at the multiple chamber heights, and comparing the recorded cell counts to determine whether they are proportional to the chamber heights to confirm or reject the cell count measurements.

[0025] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample, the method including providing a sample in one or more sample chambers having multiple chamber heights, recording cell counts of the sample at the multiple chamber heights, and analyzing the recorded cell count measurements to determine a chamber height at which the cell count measurements are proportional to the chamber height and determine a corresponding cell count measurement.

[0026] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample, the method including providing a sample in one or more sample chambers having multiple chamber heights, recording cell counts of the sample at the multiple chamber heights, and analyzing the recorded cell count measurements to determine a chamber height at which the cell count measurements are proportional to the chamber height and determine a corresponding cell count measurement.

[0027] In yet another aspect, the invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample, the method including providing a sample in one or more sample chambers having multiple chamber heights, recording 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 cell count measurements that are sufficiently proportional to chamber heights and rejecting those that are not.

[0028] In yet another aspect, the invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample. The method includes providing a sample in one or more sample chambers having multiple chamber heights, recording the cell counts of the sample at the multiple chamber heights, scoring the resulting cell count measurements based on how well the cell counts match expectations for a known chamber height, and determining measurements that meet predefined specifications and rejecting those that do not.

[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 including 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 to the known fluorescence, determining a calibration factor based on the comparison of the measured fluorescence of the defined features to the known fluorescence, applying the calibration factor to the measurements of the fluorescence of the cells, and communicating an adjustment to 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 comprising 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 comparing the measured fluorescence of the defined features to the known fluorescence to confirm or reject the fluorescence measurement.

[0031] In yet another aspect, the present invention generally relates to a method for calibrating and / or evaluating a cell counting analysis, comprising performing the calibration or evaluation according to one or more of the methods disclosed herein.

[0032] In yet another aspect, the present invention generally relates to a method of measuring 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 description of the drawings]

[0033] [Figure 1] 1 is a schematic diagram of an exemplary multiple chamber thickness. [Diagram 2] 1 is a schematic diagram of an exemplary counting unit and an assembly of counting chambers of different thicknesses used together; [Diagram 3] 1A-1C are schematic diagrams of exemplary markings or features for calibrating or assessing the focus of a system. [Figure 4] 1 is a schematic diagram of exemplary markings or features for calibrating or assessing cell size. [Diagram 5] 1A-1C are schematic diagrams of exemplary markings or features for calibrating or assessing cellular fluorescence. [Figure 6] 1 is an exemplary demonstration of cell counting proportional to chamber thickness (varied stepwise). (A) Exemplary counting chambers of various thicknesses. (B) Demonstration of proportionality. [Figure 7A] 1 is an exemplary demonstration of cell counting proportional to chamber thickness (continuously varied). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 7B] 1 is an exemplary demonstration of cell counting proportional to chamber thickness (continuously varied). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 7C] 1 is an exemplary demonstration of cell counting proportional to chamber thickness (continuously varied). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 7D] 1 is an exemplary demonstration of cell counting proportional to chamber thickness (continuously varied). (A) Exemplary counting chambers of various thicknesses. (C) Demonstration of proportionality. (D) Demonstration of proportionality. [Figure 8] 1 is an exemplary cell counting chamber sharing a common fill port. [Figure 9]1A-1C are exemplary cell counting chambers of different thicknesses sharing a common fill port. [Figure 10] 10 is an exemplary demonstration of bead counting proportional to effective concentration using the apparatus of FIG. 9. [Figure 11] 1 is an exemplary demonstration of focus positions and resulting images. [Figure 12] 1 is an exemplary demonstration of an imaged object and its size and circularity histograms. [Figure 13] 1 is an exemplary demonstration of imaged fluorescent beads. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention is based on novel sample chambers, units and multi-well plates, systems and methods that have built-in functionality to enable the evaluation of counting methods and the calibration and / or quality assurance of measurements and analyses of various types of biological cells, including, for example, measurements of cell number, cell size, cell concentration, cell subpopulations, cell morphology and cell viability.

[0035] Calibration and evaluation of cell number proportionality A feature of the present invention is that confirmation of proportionality of cell numbers is achieved without the need to dilute samples or repeatedly measure samples at different concentrations. Traditionally, samples are diluted to confirm proper proportionality to provide confidence in counting methods and protocols. Instead of creating a series of concentrations to confirm proportionality, the disclosed approach evaluates proportionality of cell numbers through measuring various areas of an imaging chamber with different but defined chamber thicknesses. In particular, the number of particles in the field of view is modulated by the change in chamber thickness proportional to the volume.

[0036] Multiple thicknesses may be produced within a single counting chamber, as illustrated diagrammatically in Figure 1. The change in chamber thickness may be stepwise (Figure 1A) or continuous (Figure 1B). The change in chamber thickness may be along either axis of the chamber, as illustrated in Figure 1C (longitudinal from inlet to outlet) and Figure 1D (transverse from one edge of the chamber to another edge of the chamber).

[0037] Multiple counting chambers of various thicknesses may be used together, as illustrated diagrammatically in Figure 2. Multiple chambers may be produced as a single unit to be used as a set. In Figure 2A, four chambers of four thicknesses are set together with a single inlet. In Figure 2B, three chambers of three thicknesses are set together with a single inlet. Multiple chambers may also be bonded to a multi-chambered device (e.g., a 96-well plate).

[0038] Figure 6 shows an exemplary demonstration of cell counting proportional to chamber thickness, where the thickness is varied in a stepwise fashion. A set of counting chambers of various thicknesses is used, each slide incorporating a different combination of adhesive spacer layers (Figure 6A). When filled with samples of the same bead / cell suspension, the number of objects counted is proportional to the chamber thickness (Figure 6B).

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

[0040] FIG. 7 shows an exemplary demonstration of cell counting proportional to chamber thickness, where the thickness varies continuously. FIG. 7A shows two counting chambers made using multiple adhesive spacer layers with continuously varying chamber heights (left: lateral variation; right: longitudinal variation). The adhesive areas are highlighted in blue, with darker blue indicating thicker adhesive. The adhesive layer surrounding the counting chamber formed a step, while the ceiling of the chamber transitioned smoothly from the thick to thin side of the chamber, resulting in a continuous tapering of the chamber thickness. Two configurations were made in which the chamber thickness variations occurred in both the lateral (X-direction) and longitudinal (Y-direction) directions of the counting chamber.

[0041] In one experiment, a solution of 5 pm beads was loaded into the chamber (2e6 beads / mL) and a histogram of bead positions was obtained. The slope and R2 value obtained from a linear fit of the histograms provide a proportionality 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 in Figure 7C was obtained from slide B of one of the chambers.

[0042] The cell counting chambers may be configured to share a common entry (fill) port. A multi-chambered device may contain independent chambers or may contain chambers joined by one or more common sample fill ports. In FIG. 2C, the multi-well plate features ten sets of three-chamber units. In FIG. 2D, the multi-well plate features four sets of four-chamber units and two sets of three-chamber units.

[0043] FIG. 8 shows a multi-well plate illustrating this multi-chamber approach. Testing showed that the suspension of cells flowed consistently into multiple counting chambers simultaneously. FIG. 8A shows the cut-out adhesive layer (backing paper not removed) defining the cell counting chambers. FIG. 8B shows the adhesive layer overlaid with the top sheet (protective insulating film not removed) showing the fill and drain ports aligned. FIG. 8C shows the underside of a completed cell counting chamber that is glued to the bottom of a 96-well plate. Multiple plates were tested for proper fluid flow and cell count consistency. No significant differences in concentration measurements were observed in counting chambers of different configurations when a solution of beads was added to multiple fill ports.

[0044] In Figure 9, a multi-well plate contains four sample loading ports connected to four counting chambers, each of different thickness. Figure 9A represents a top view, while Figure 9B represents a bottom view of the plate. Each chamber extends across four sample viewing windows. The plate may accommodate four samples and 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 loaded into chambers made at the bottom of a standard 96-well plate. Custom software functions can be incorporated to analyze the measured proportions of cell numbers and return a determination of the sample's suitability for automatic counting by the instrument. The software can also automatically analyze the numbers at various chamber heights and indicate to the user if the cell suspension should be further diluted or concentrated for better counting. Automatic exclusion can also be achieved for parts of the chamber with too many or too few targets.

[0046] Calibrating and Evaluating the System's Focus Another feature of the invention is built-in micro-scale markings or features, such as 2D or 3D markings on the surface of the chamber, with known offset values ​​to ensure consistent focusing for cell counting and viability determination. This feature of the invention allows for proper and consistent focusing, which is essential for image-based cell counting. Consistent focusing avoids the varying appearance of cells in brightfield and fluorescent illumination, which can reduce the accuracy of cell counting algorithms.

[0047] Markings or features are built into the imaging chamber at known locations relative to the floor to aid in the system's focus and ensure consistent focus. For example, features spaced across the field of view can assess whether the chamber is level. Features that feature high brightness contrast at the system's resolution level can accurately determine focus position. For example, features that contain areas of high contrast when in ideal focus position (coarse and fine details), as illustrated diagrammatically in FIG. 3, can provide a more sensitive method of determining relative focus. Additionally, three-dimensional focus objects can use a variety of length and height scales, allowing the algorithm to contrast and determine changes over a wide range of distances.

[0048] A focusing method that can be automated may be implemented to determine the focus position where the feature is in best focus. This can be achieved by combining focus scores and optimization algorithms. Careful calibration based on how the image of the feature changes with focus position may lead to rapid calibration of the system's focus. With precisely fabricated microscale features, such calibration can result in near-instantaneous registration of focus position.

[0049] Once the focus position is registered with the system instrument, an offset value can be set and the ideal focus position for a particular cell type recorded. This may be done manually by the user based on visual inspection, automatically based on focus scores, or by using the built-in offset value for the particular assay. Once the ideal focus position of the cells and the plane of the fiducial markings 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 well-defined sizes, which allows the system to calibrate and evaluate cell size measurements. Cell counting instruments report the size of detected objects. The size reported by segmentation algorithms varies with focus position and image brightness. Defocused cells may appear larger and may be reported as such, but may show less contrast, leading to a smaller size being reported by texture algorithms. For fluorescent images, brighter illumination tends to give larger cell measurements. Also, poor cell size measurements may affect concentration measurements.

[0051] Size calibration involves obtaining an accurate pixel to micron conversion and evaluating the accuracy of the segmented blob size. Light diffraction can affect the appearance of markings of different sizes differently. Features that are known to be spaced over a larger distance will give a more accurate estimate of the pixel to micron conversion factor. For pixel to micron conversion, it is advantageous to utilize markings or features that are as spaced apart 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 as the cells being counted.

[0052] FIG. 4 is a schematic of a microscale feature of known dimensions, with which the segmentation algorithm can be calibrated. A checkerboard pattern may be used for camera calibration. Algorithms have been devised that are well suited to find the checkerboard lines with sub-pixel accuracy. Another example is a cluster of dots, with well-defined distances between them. Features such as 5 pm dots of a defined pattern can be used both to establish a calibrated coordinate system for measuring cell positions and to assess the accuracy of the segmented sizes.

[0053] Calibration and evaluation of cell fluorescence measurements Another feature of the invention is the use of objects or features with well-defined fluorescence, which allows the system to calibrate the appropriate fluorescence illumination and evaluate the fluorescence measurements. Both counts and size measurements in the fluorescence imaging mode depend on the intensity of the light received by the camera. The intensity depends on the brightness of the light source, the transmittance of the filters, and the fluorescence intensity of the cells themselves. Fluorescently labeled cells show a distribution of emitted intensities. If an intensity threshold is used to segment cells from the background, the number of counted cells will vary with the intensity of the excitation light. For this reason alone, the number will vary if the same threshold is set for the analysis of experiments performed under different lighting conditions. A good control of the fluorescent objects or features captured by the counting device allows normalization to the intensity of the marker, leading to a better consistency of counts from experiment to experiment and from instrument to instrument. This also allows comparisons between experiments performed with different exposure times.

[0054] For example, using well-characterized fluorescent targets or features embedded or added to the cell counting chamber or slide, the intensity of the imaged cells may be related or normalized to the brightness of the target or feature, as illustrated diagrammatically in Figure 5. This allows results to be compared between instruments and between experiments. Also, the system instrument can use these features to simply determine that illumination is functioning properly, so that low or absent cell counts can be confidently attributed to the sample and not the instrument.

[0055] FIG. 5 shows two simple patterns of fluorescent features. In FIG. 5A, a fluorescent checkerboard is provided, by which the scale (e.g., size and / or spacing of the squares), the fluorescence intensity (e.g., using the brightness of the fluorescent paint), and the focus can be calibrated. FIG. 5B shows an alternative pattern consisting of differently colored fluorescent dots of known size. By coloring the dots, multiple fluorescent channels (brightness and focus position) can be calibrated. In addition, the known distance between pairs of dots provides the calibration of the scale and the origin of the spatial coordinates. In FIG. 5C, multiple small markings are placed at positions around the counting chamber. Also, multiple fluorescent color markers allow the focal planes of different wavelengths to be found. To reduce the manufacturing cost of such features, well-characterized fluorescent beads may be utilized in lien with the direct marking of the cell chamber.

[0056] Combined marking for multiplexed calibration and measurement assurance Registering focus, checking proportionality, calibrating size, and benchmarking fluorescence intensity may be accomplished together. For example, an object or feature of known size may be printed in fluorescent ink on the bottom outer surface of a slide. The system instrument may focus on the object or feature and apply a known offset value to move the focus to the focal plane of the cells. The system instrument may adjust the brightness or gain of the camera light source to check and, if necessary, compensate for the intensity of the fluorescent feature to fall within specifications. Also, a certain distance measured between the objects or features may be checked to ensure that the image calibration is within acceptable boundaries. Such markings may be etched, printed, or otherwise added to the cell counting chamber (e.g., imprint lithography).

[0057] Thus, in one aspect, the invention generally relates to a sample chamber suitable for holding a liquid sample for optical imaging, the sample chamber including: (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 in fluid communication with the inlet and including one or more optically transmissive windows suitable for observing or analyzing the liquid sample in the imaging chamber, and (c) an outlet in fluid communication with the imaging chamber for venting air or for allowing the liquid sample to flow out. The imaging chamber is characterized by one or more of: a chamber height that varies, either continuously or in steps, and that passes at least a portion of one or more optically transmissive windows; a defined feature having one or more known offsets from a focal plane of the imaging chamber, the feature being optically accessible through the one or more optically transmissive windows; and a defined feature having one or more known dimensions, areas or volumes, the feature being optically accessible through the one or more optically transmissive windows, the defined feature exhibiting one or more fluorescent colors at one or more intensities.

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

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

[0060] In some embodiments, the chamber height is varied both continuously and stepwise (partly continuously and partly stepwise).

[0061] In some embodiments, the height of the chamber ranges from about 1 pm to about 5,000 pm (e.g., from about 1 pm to about 5,000 pm, from about 5 pm to about 5,000 pm, from about 10 pm to about 5,000 pm, from about 50 pm to about 5,000 pm, from about 100 pm to about 5,000 pm, from about 500 pm to about 5,000 pm, from about 1 pm to about 1,000 pm, from about 1 pm to about 500 pm, from about 1 pm to about 100 pm, from about 1 pm to about 50 pm, from about 1 pm to about 10 pm).

[0062] In certain embodiments, the chamber height varies in a range of about 10 pm to about 500 pm (e.g., about 10 pm to about 500 pm, about 25 pm to about 500 pm, about 50 pm to about 500 pm, about 100 pm to about 500 pm, about 10 pm to about 250 pm, about 10 pm to about 100 pm, about 10 pm to about 50 pm, about 10 pm to about 25 pm).

[0063] In some embodiments, at least some of the defined features are three-dimensional, characterized by the height of one or more defined features.

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

[0065] In one embodiment, the height of one or more defined features is the same.

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

[0067] In certain embodiments, the height of the one or more defined features is in the range of about 0.1 μm to about 5,000 μm (e.g., about 0.1 μm to about 1,000 μm, about 0.1 μm to about 500 μm, about 0.1 μm to about 200 μm, about 0.1 μm to about 100 μm, about 0.1 μm to about 50 μm, about 0.1 μm to about 25 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 ... m to about 1 μm, about 1 μm to about 5,000 μm, about 10 μm to about 5,000 μm, about 50 μm to about 5,000 μm, about 100 μm to about 5,000 μm, about 500 μm to about 5,000 μm, about 1,000 μm to about 5,000 μm, about 1 μm to about 500 μm, about 2 μm to about 200 μm, about 2 μm to about 100 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm).

[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 certain embodiments, a defined feature having one or more known dimensions, area, or volume is inscribed in the bottom of the imaging chamber.

[0071] In one embodiment, a defined feature having one or more known dimensions, area or volume is inscribed in 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 characteristics exhibit one or more predefined patterns.

[0074] In certain embodiments, the defined characteristics indicate one or more fluorescent colors and / or one or more fluorescent intensities.

[0075] In certain embodiments, the imaging chamber is configured to have a volume in the range of about 0.1 μL to about 200 pL (e.g., 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 certain embodiments, the imaging chamber is configured to have a volume in the range of about 4 μL to about 100 pL (e.g., 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 some embodiments, the imaging chamber includes a single optically transparent window.

[0078] In some embodiments, the imaging chamber includes two or more optically transparent windows.

[0079] In some embodiments, the imaging chamber includes three, four, or five light-transmissive windows.

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

[0081] In yet another aspect, the present invention generally relates to a sample analysis unit, the sample analysis unit including (i) a mixing well for preparing a liquid sample for analysis, and (ii) a first sample chamber suitable for holding a liquid sample for optical imaging, the first sample chamber including (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 a liquid sample, the first imaging chamber in fluid communication with the first inlet and including a first or first set of optically transparent windows suitable for observing or analyzing the liquid sample in the first imaging chamber, and (c) a first outlet in fluid communication with the first imaging chamber for evacuating air or for draining the liquid sample. The first imaging chamber is characterized by one or more of: (i) a first chamber height that varies, either continuously or in steps, through at least a portion of the first or first set of optically transmissive windows; (ii) a first defined feature having one or more known offsets from a focal plane of the first imaging chamber, the feature being optically accessible through the first or first set of optically transmissive windows; (iii) a first defined feature having one or more known dimensions, area or volume, the first feature being optically accessible through the first or first set of optically transmissive windows; and (iv) the first defined feature exhibits one or more fluorescent colors at one or more intensities.

[0082] In an embodiment, the sample analysis unit further includes a second sample chamber suitable for holding a liquid sample for optical imaging, the second sample chamber including: (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 a liquid sample, the second imaging chamber in fluid communication with the second inlet and including a second or second set of optically transparent windows suitable for observing or analyzing the liquid sample in the second imaging chamber, and (c) a second outlet in fluid communication with the second imaging chamber for evacuating air or for allowing the liquid sample to flow out. The second imaging chamber is characterized by one or more of: (i) a second chamber height that varies, either continuously or in steps, through at least a portion of the second or set of optically transmissive windows; (ii) a second defined feature having one or more known offsets from a focal plane of the second imaging chamber, the second feature being optically accessible through the second or set of optically transmissive windows; (iii) a second defined feature having one or more known dimensions, areas or volumes, the second feature being optically accessible through the second or set of optically transmissive windows; and (iv) the second defined feature exhibits one or more fluorescent colors at one or more intensities.

[0083] In an embodiment, the sample analysis unit further includes a third sample chamber suitable for holding a liquid sample for optical imaging, the third sample chamber including: (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 a liquid sample, the third imaging chamber in fluid communication with the third inlet and including a third or third set of optically transparent windows suitable for observing or analyzing the liquid sample in the third imaging chamber, and (c) a third outlet in fluid communication with the third imaging chamber for evacuating air or for allowing the liquid sample to flow out. The third imaging chamber is characterized by one or more of: (i) a third chamber height that varies, either continuously or in steps, through at least a portion of the third or third set of optically transmissive windows; (ii) a third defined feature having one or more known offsets from a focal plane of the third imaging chamber, the third feature being optically accessible through the third or third set of optically transmissive windows; (iii) a third defined feature having one or more known dimensions, areas or volumes, the third feature being optically accessible through the third or third set of optically transmissive windows; and (iv) the third defined feature exhibits one or more fluorescent colors at one or more intensities.

[0084] In an embodiment, the sample analysis unit further includes a fourth sample chamber suitable for holding a liquid sample for optical imaging, the fourth sample chamber including: (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 a liquid sample, the fourth imaging chamber in fluid communication with the fourth inlet and including a fourth or fourth set of optically transmissive windows suitable for observing or analyzing the liquid sample in the fourth imaging chamber, and (c) a fourth outlet in fluid communication with the fourth imaging chamber for evacuating air or for allowing the liquid sample to flow out. The fourth imaging chamber is characterized by one or more of: (i) a fourth chamber height that varies, either continuously or in steps, through at least a portion of the fourth or fourth set of optically transmissive windows; (ii) a fourth defined feature having one or more known offsets from a focal plane of the fourth imaging chamber, the fourth feature being optically accessible through the fourth or fourth set of optically transmissive windows; (iii) a fourth defined feature having one or more known dimensions, areas or volumes, the fourth feature being optically accessible through the fourth or fourth set of optically transmissive windows; and (iv) a fourth defined feature exhibiting one or more fluorescent colors at one or more intensities.

[0085] In an embodiment, the sample analysis unit further comprises one or more further 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 further 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 an embodiment, each of the first, second, third, fourth, and any additional imaging chambers is characterized by a non-uniform chamber height.

[0089] In an embodiment, the first, second, third, fourth, and any other defined characteristics are the same.

[0090] In an embodiment, the first, second, third, fourth, and any other defined characteristics are different.

[0091] In certain embodiments, each of the imaging chambers is configured to have a volume of about 0.1 μL to about 200 pL (e.g., about 0.5 pL to about 200 pL, about 1 pL to about 200 pL, 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).

[0092] In certain embodiments, each of the imaging chambers is configured to have a volume of about 4 pL to about 100 pL (e.g., about 10 pL to about 100 pL, about 20 pL to about 100 pL, about 50 μL to about 100 pL, about 4 μL to about 50 pL, about 4 pL to about 20 pL, about 4 pL to about 10 pL).

[0093] In some embodiments, the first or first set of light-transmissive windows, the second or second set of light-transmissive windows, the third or third set of light-transmissive windows, and the fourth or fourth set of light-transmissive windows each comprise a single light-transmissive window.

[0094] In some embodiments, the first or first set of light-transmissive windows, the second or second set of light-transmissive windows, the third or third set of light-transmissive windows, and the fourth or fourth set of light-transmissive windows each include two or more light-transmissive windows.

[0095] In some embodiments, the first or first set of light-transmissive windows, the second or second set of light-transmissive windows, the third or third set of light-transmissive windows, and the fourth or fourth set of light-transmissive windows each include three, four, or five light-transmissive windows.

[0096] In yet another aspect, the invention generally relates to a multi-well plate or device that includes the sample analysis unit disclosed herein.

[0097] In yet another aspect, the present invention generally relates to a multi-well plate including a plurality of sample analysis units, where each sample analysis unit includes (i) a mixing well for preparing a liquid sample for analysis, and (ii) one or more sample chambers suitable for holding a liquid sample for optical imaging. Each 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 a liquid sample, the imaging chamber in fluid communication with the inlet and including one or more optically transparent windows suitable for observing or analyzing the liquid sample in the imaging chamber, and (c) an outlet in fluid communication with the imaging chamber for evacuating air or for exiting the liquid sample. The imaging chamber is characterized by defined features having one or more known offsets from the focal plane of the imaging chamber, optically accessible through one or more optically transparent windows, and / or defined features having one or more known dimensions, volumes, or capacities, optically accessible through one or more optically transparent windows. The defined features exhibit one or more fluorescent colors at one or more intensities.

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

[0099] In some embodiments of the multiwell plate, each of the sample analysis units includes two or more sample chambers. In some embodiments of the multiwell plate, each of the sample analysis units includes three or more sample chambers. In some embodiments of the multiwell plate, each of the sample analysis units includes four or more sample chambers.

[0100] In some embodiments of the multi-well plate, all of the defined characteristics are the same.

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

[0102] In certain embodiments, each of the imaging chambers is configured to have a volume of about 0.1 μL to about 200 pL (e.g., about 0.5 pL to about 200 pL, about 1 pL to about 200 pL, 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).

[0103] In certain embodiments, each of the imaging chambers is configured to have a volume of about 4 pL to about 100 pL (e.g., 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).

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

[0105] In certain embodiments, 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 light beam and / or the brightfield light beam, a detection device, and a computing unit.

[0106] In some embodiments, the system includes two or more fluorescent light sources.

[0107] In some embodiments, 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 a focus setting of an imaging system for analyzing cell counting, the method comprising the steps of: adjusting a focus position until one or more defined features of an imaging chamber appear in best focus, recording the focus position according to the defined features, adjusting the focus position until cells of the sample appear in best focus, recording the focus position according to the sample, performing a cell count measurement, comparing the focus position recorded according to the defined features with the focus position recorded according to the sample, and determining whether a difference between the two recorded focus positions is acceptable to confirm or reject the cell count measurement.

[0109] In yet another aspect, the present invention generally relates to a method for evaluating focus settings of an imaging system for analyzing cell counting, the method comprising the steps of manually or automatically adjusting the focus position until one or more defined features of an imaging chamber appear in best focus, recording the focus position according to the defined features, manually or automatically adjusting the focus towards a trial focus position of the cells where the cells are presumed to be in the best focus for cell counting, recording the trial focus position of the cells according to the sample, performing image acquisition to measure cell number, 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 or not to confirm or reject the measured cell count.

[0110] In one embodiment, if the cell counting measurement is rejected, the method further includes the steps of manually or automatically adjusting the focus towards a new trial focus position of the cells where the cells are presumed to be at the best focus for cell counting, recording the new trial focus position of the cells according to the sample, performing image acquisition to measure the cell number, comparing the focus position recorded according to the defined characteristics with the focus position recorded according to the sample, determining whether the difference between the two recorded focus positions is acceptable or not, confirming or rejecting the cell counting measurement, and repeating the focus adjustment as necessary to produce an acceptable focus position.

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

[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 including measuring a size of cells in a sample, measuring a size of one or more defined features of an imaging chamber having a known size, comparing the measured size of the defined features to the known sizes, determining a calibration factor based on the comparison of the known and measured sizes of the features, applying the calibration factor to the cell size measurements, and communicating an adjustment to 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 including measuring the size of cells in a sample, measuring the size of one or more defined features of an imaging chamber having a known size, comparing the measured size of the defined features to the known size, and determining whether a difference between the measured size of the features and the known size is acceptable to confirm or reject the cell size measurement.

[0114] In one embodiment, if the cell size measurement is rejected, the method further includes manually or automatically adjusting one or more instrument settings, measuring the size of the cells in the sample, measuring the size of one or more defined features of the imaging chamber having a known size, comparing the measured size of the defined features to the known sizes, determining whether the difference between the measured size of the features and the known size is acceptable to confirm or reject 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 focus and cell size measurements of an imaging system for analyzing cell counting, the method including the steps of manually or automatically adjusting the focus position until one or more defined features of the imaging chamber appear in best focus, measuring the size of cells in a sample, measuring the size of one or more defined features of the imaging chamber having known sizes, comparing the measured sizes of the defined features to the known sizes, determining whether the difference between the two measurements is acceptable to confirm or reject the focus setting and cell size measurements, and repeating these steps until an acceptable size measurement is obtained.

[0116] In yet another aspect, the invention generally relates to a method for assessing the suitability of a sample for cell count analysis without diluting or concentrating the sample, the method comprising providing the sample in one or more sample chambers having multiple chamber heights, recording cell counts of the sample at the multiple chamber heights, and comparing the recorded cell counts to determine whether they are proportional to the chamber heights to confirm or reject the cell count measurements.

[0117] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample, the method including providing a sample in one or more sample chambers having multiple chamber heights, recording cell counts of the sample at the multiple chamber heights, and analyzing the recorded cell count measurements to determine a chamber height at which the cell count measurements are proportional to the chamber height and determine a corresponding cell count measurement.

[0118] In yet another aspect, the present invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample, the method including providing a sample in one or more sample chambers having multiple chamber heights, recording cell counts of the sample at the multiple chamber heights, and analyzing the recorded cell count measurements to determine a chamber height at which the cell count measurements are proportional to the chamber height and determine a corresponding cell count measurement.

[0119] In yet another aspect, the invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample, the method including providing a sample in one or more sample chambers having multiple chamber heights, recording 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 cell count measurements that are sufficiently proportional to chamber heights and rejecting those that are not.

[0120] In yet another aspect, the invention generally relates to a method for measuring and analyzing cell count without diluting or concentrating a sample. The method includes providing a sample in one or more sample chambers having multiple chamber heights, recording the cell counts of the sample at the multiple chamber heights, scoring the resulting cell count measurements based on how well the cell counts match expectations for a known chamber height, and determining measurements that meet predefined specifications and rejecting those that do not.

[0121] In certain embodiments, the method further comprises the step of excluding cell number measurements recorded at chamber heights where the cell number measurements are not proportional to the chamber height.

[0122] In some embodiments, the method further comprises excluding cell count measurements recorded at chamber heights where the cell count measurements are not proportional to the chamber height.In some embodiments, the method further comprises generating one or more quality scores for the cell count measurements based on the proportionality results.For example, if there are six different chamber heights in the device, the measurements of the sample that produce proportional numbers across all six chamber heights are of higher quality than measurements that exclude three of the six chamber heights due to non-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 including 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 to the known fluorescence, determining a calibration factor based on the comparison of the measured fluorescence of the defined features to the known fluorescence, applying the calibration factor to the measurements of the fluorescence of the cells, and communicating an adjustment to the fluorescence measurement.

[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 comprising 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 comparing the measured fluorescence of the defined features to the known fluorescence to confirm or reject the fluorescence measurement.

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

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

[0127] In some embodiments, at least some of the one or more defined features are three-dimensional, characterized by a height of the one or more defined features.

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

[0129] In one embodiment, the height of one or more defined features is the same.

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

[0131] In certain embodiments, the height of the one or more defined features is in the range of about 0.1 μm to about 5,000 μm (e.g., about 0.1 μm to about 1,000 μm, about 0.1 μm to about 500 μm, about 0.1 μm to about 200 μm, about 0.1 μm to about 100 μm, about 0.1 μm to about 50 μm, about 0.1 μm to about 25 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 ... m to about 1 μm, about 1 μm to about 5,000 μm, about 10 μm to about 5,000 μm, about 50 μm to about 5,000 μm, about 100 μm to about 5,000 μm, about 500 μm to about 5,000 μm, about 1,000 μm to about 5,000 μm, about 1 μm to about 500 μm, about 2 μm to about 200 μm, about 2 μm to about 100 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm).

[0132] In some embodiments, 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 certain embodiments, a defined feature having one or more known dimensions, area, or volume is inscribed in the bottom of the imaging chamber.

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

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

[0137] In some embodiments, the defined features exhibit one or more predefined patterns or geometric shapes.

[0138] In certain embodiments, the defined characteristics indicate one or more fluorescent colors and / or one or more fluorescent intensities.

[0139] In certain embodiments, the imaging chamber is configured to have a volume of about 0.1 μL to about 200 pL (e.g., 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).

[0140] In certain embodiments, the imaging chamber is configured to have a volume of about 4 pL to about 100 pL (e.g., 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).

[0141] In yet another aspect, the present invention generally relates to a method of measuring cell number 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.

[0142] The present invention is well suited for high throughput cell counting instruments and associated software with self-calibration and measurement assurance capabilities. One example of such a system utilizes five excitation filters (375, 475, 530, 540, 630 nm) for the transmitted brightfield channel and epifluorescence channel, and six emission filters (450, 525, 600, 610LP, 660, 695). It also uses an infinity corrected optical objective for high resolution and high quality imaging. The exemplary system operates in XYZ to image and analyze cells in standard microplates (6-1536 wells), T25 and T75 flasks, and glass and chamber slides. This improves cell counting times to 24 samples per minute for brightfield analysis (trypan blue) and 24 samples per 3 minutes for fluorescence analysis (acridine orange and propidium iodide).

[0143] Additionally, the instrument 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 consumable types, e.g., cell counting plates as disclosed herein, 6-1536 well standard microplates, glass slides, T75, T25 flasks, chamber slides. This may be utilized to measure cell concentration, cell size and morphology, e.g., perimeter, circularity, area, major / minor diameter, compactness, elongation, eccentricity, sphericity, convexity, aspect ratio, robustness, etc. For fluorescence-based assays, fluorescence intensity can be measured. Additional accessories integrated with plate handlers, liquid handlers for high-throughput cell counting and analysis are intended for full automation.

[0145] The software analyzes the captured images to determine cell concentration, cell size and morphology, percentage of fluorescently labeled populations such as viability labels (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 blue, etc.), 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 of the present invention may be designed to perform assays in oncology, immuno-oncology, virology, cell therapy, cell line development, regenerative medicine (stem cell research), oenology research and renewable energy. It may be utilized to analyze cell lines (cancer cells NCI-60 and others), primary cells (PBMC, splenocytes, leukapheresis, apheresis, thymocytes), stem cells, platelets, red blood cells, 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 upon activation, transduction efficiency, apoptosis, autophagy, cell cycle, cell senescence, ROS, mitochondrial potential and health, population analysis of surface markers, population analysis of intracellular stains, etc.

[0147] Additionally, the system can determine population percentages based on size, morphology, or fluorescent labeling by comparing the number or concentration of cells in a target cell population based on size, morphology, or fluorescent labeling with the total number of cells determined in bright field or any other fluorescent channel.

[0148] The following examples are intended to illustrate the practice of the present invention and are not intended to be limiting in any respect.

[0149] FIG. 10 shows exemplary results obtained using the apparatus of FIG. 9 and samples of solutions of beads 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 a proportional deviation at the high and low ends of the effective concentration range. Thus, the number of low and high concentration samples may be rejected. With a sufficient number of numbers falling within the linear range, samples were recorded to return a confirmed number value, excluding the non-linear points. Multiple chamber thicknesses can also increase the concentration range that the instrument can reliably measure.

[0150] In FIG. 11 it can be seen that different focus positions lead to different images of the imaging object. In (A) the goal is to image a microscopic object to make surface details visible. The object rests on a transparent 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. The feature is easily recognizable in a plane known to be 548 pm from the surface on which the object of interest rests (grid lines, panel C). The grid can be reliably focused and once focused on the grid the position of the objective lens can be indicated. The objective lens can then be moved with an offset of 548 pm to reach the surface of the sample and then 182 pm to reach the best focus of the object (total offset of 730 pm). If dirt or scratches confuse the focus algorithm and it accidentally focuses in the wrong place to image a grid or object (D), a focus offset value that is too different from the expected 730 pm will be reported and the focus will be rejected.

[0151] An exemplary demonstration of imaged objects and their size and circularity histograms is shown in Figure 12. Panel A shows a portion of an image of a suspension of Jurkat cells mixed with borosilicate glass beads of known size. The inset shows a region of the image of the same sample slightly out of focus. The beads have a test mean particle size of 17.3 pm with a standard deviation of 2.0 pm. Objects found by the algorithm are highlighted in green. In B, a size histogram of objects identified in the image with good focus is shown. The peak value at 20 pm corresponds to the beads. The beads were selected using a gate between 16 and 25 pm. For these objects, the algorithm returned a mean particle size of 19.96 pm with a standard deviation of 1.94 pm.

[0152] The results showed that the algorithm reported a size of the features (beads) that was approximately 15% larger than their actual size. Similarly, the reported size of the cells may have been affected. Also plotted in (B) is a size histogram of the objects found by the algorithm in an image of the sample with a slight overall defocus. A clear peak corresponding to beads was excluded, but the image may be rejected for cell counting. Panel C shows a circularity histogram obtained from a well-focused image. Also plotted is a histogram of those pre-identified objects such as beads. For spherical objects, the peak value does not occur at 1.0, as would be expected. This is a special case of using features of known dimensions (Figure 4) to detect size-calibrated tissues. In this case, discrepancies may occur due to incorrect length calibration along one of the image dimensions. There may also be distortions resulting from the optics of the system, which may be another cause of rejection.

[0153] In Figure 13, an example of imaging fluorescent beads is shown. In (A) and (B), images of two Jurkat samples containing similar fluorescent beads are shown. In each set of images, one cell and one bead were seen. The cells were stained with propidium iodide, which marks dead cells. In the first set (A), the cells were very faintly fluorescent. Without the presence of beads, one may wonder whether the intensity of the excitation illumination was appropriate for this assay, or whether the camera exposure time was appropriate for this assay. The beads allow us to confirm that the dimness of the cells is due to them not being strongly fluorescent, and not due to an issue with the instrument or settings.

[0154] In the second set of images (B), Jurkat cells have absorbed propidium iodide and are highly fluorescent. Although the fluorescent images of these cells were taken with a longer exposure time than the fluorescent images of the first cells (A), the images are difficult to reliably compare without a common reference feature. The presence of a standard bead allows the two images to be compared to allow for more consistent cell counts.

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

[0156] Applicant's disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent like or similar elements. Reference throughout this specification to "one embodiment," "an embodiment," or similar phrases means that the specific features, structures, or characteristics described in connection with an embodiment are included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," or similar phrases throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0157] Applicant's disclosed features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are cited to provide a thorough understanding of the embodiments of the invention. However, it will be apparent to one of ordinary skill in the art that Applicant's configurations and / or methods may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring aspects of the disclosure.

[0158] In the specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

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

[0160] INCORPORATION BY REFERENCE In this disclosure, references and citations are made to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes. Any material, or part thereof, that is said to be incorporated herein by reference but that contradicts existing definitions, references, or other disclosed materials expressly set forth herein is incorporated to the extent that no contradiction occurs between the incorporated material and the disclosed material. In case of any contradiction, the contradiction should be resolved in favor of the present disclosure as the preferred disclosure.

[0161] Equivalent The representative examples are intended to help illustrate the invention and are not intended, nor should they be construed, as limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of the specification, including the examples contained herein and references to the scientific and patent literature. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of the invention in its various embodiments and equivalents.

Claims

1. 1. A sample chamber suitable for holding a liquid sample for optical imaging, the sample chamber comprising: (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 in fluid communication with the inlet and including one or more optically transmissive windows suitable for observing or analyzing the liquid sample within the imaging chamber; (c) an outlet in fluid communication with the imaging chamber for evacuating air or for allowing the liquid sample to flow out; The imaging chamber includes: one or more known offsets from a focal plane of the imaging chamber; and One or more known dimensions, areas or volumes characterized by the defined feature having the defined feature is inscribed in a bottom and / or ceiling of the imaging chamber and is optically accessible through the one or more optically transmissive windows; The defined feature exhibits one or more fluorescent colors at one or more intensities.

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

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

4. A sample chamber according to claim 2 or 3, wherein the height of the chamber varies in the range of 1 μm to 5,000 μm.

5. A sample chamber according to any one of claims 1 to 4, wherein at least some of the defined features are three-dimensional and characterized by a height of one or more defined features.

6. The sample chamber of claim 5 , wherein the height of the one or more defined features is the same.

7. The sample chamber of claim 5 , wherein the heights of the one or more defined features are different.

8. A sample chamber according to any one of claims 5 to 7, wherein the height of the one or more defined features is in the range 0.1 μm to 5,000 μm.

9. A sample chamber according to any preceding claim, wherein the defined feature having one or more known offset values ​​is inscribed in a bottom or ceiling of the imaging chamber.

10. A sample chamber according to any one of claims 1 to 9, wherein the defined feature having one or more known dimensions, area or volume is inscribed in a bottom or ceiling of the imaging chamber.

11. A sample chamber according to any one of claims 1 to 10, wherein the defined features comprise a plurality of coarse and fine features or exhibit one or more predefined patterns or geometries.

12. A sample chamber according to any preceding claim, wherein the defined characteristics indicate one or more fluorescent colours and / or one or more fluorescent intensities.

13. The sample chamber of any one of claims 1 to 12, wherein the imaging chamber is configured to have a volume of between 0.1 μL and 200 μL.

14. A sample chamber according to any preceding claim, wherein the imaging chamber comprises a single optically transparent window.

15. The sample chamber of any one of claims 1 to 13, wherein the imaging chamber comprises two or more optically transparent windows.

16. A sample analysis unit, the sample analysis unit comprising: (i) a mixing well for preparing a liquid sample for analysis; (ii) a first sample chamber according to any one of claims 1 to 15, arranged in spatial proximity to the mixing well.

17. and a second sample chamber adapted to hold a liquid sample for optical imaging, said second sample chamber comprising: (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 in fluid communication with the second inlet and including a second or second set of optically transmissive windows suitable for observing or analyzing the liquid sample in the second imaging chamber; (c) a second outlet in fluid communication with the second imaging chamber for evacuating air or for allowing the liquid sample to flow out; The second imaging chamber comprises: one or more known offsets from a focal plane of the second imaging chamber; and One or more known dimensions, areas or volumes and characterized by a second defined feature having 17. The sample analysis unit of claim 16, wherein the second defined feature is inscribed in a bottom and / or ceiling of the imaging chamber and is optically accessible through the second or second set of optically transmissive windows.

18. and a third sample chamber adapted to hold a liquid sample for optical imaging, said third sample chamber comprising: (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 in fluid communication with the third inlet and including a third or third set of optically transmissive windows suitable for observing or analyzing the liquid sample in the third imaging chamber; (c) a third outlet in fluid communication with the third imaging chamber for evacuating air or for allowing the liquid sample to flow out; The third imaging chamber comprises: one or more known offsets from a focal plane of the third imaging chamber; and One or more known dimensions, areas or volumes and characterized by a third defined feature having 20. The sample analysis unit of claim 17, wherein the third defined feature is inscribed in a bottom and / or ceiling of the imaging chamber and is optically accessible through the third or third set of optically transparent windows.

19. and a fourth sample chamber adapted to hold a liquid sample for optical imaging, said fourth sample chamber comprising: (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 in fluid communication with the fourth inlet and including a fourth or fourth set of optically transmissive windows suitable for observing or analyzing the liquid sample in the fourth imaging chamber; (c) a fourth outlet in fluid communication with the fourth imaging chamber for evacuating air or for allowing the liquid sample to flow out; The fourth imaging chamber comprises: one or more known offsets from a focal plane of the fourth imaging chamber; and One or more known dimensions, areas or volumes The invention is characterized by a fourth defined feature having 20. The sample analysis unit of claim 18, wherein the fourth defined feature is inscribed in a bottom and / or ceiling of the imaging chamber and is optically accessible through a fourth or fourth set of optically transmissive windows.

20. 20. The sample analysis unit of claim 19, further comprising one or more further sample chambers suitable for holding a liquid sample for optical imaging.

21. 21. The sample analysis unit of claim 19 or 20, wherein the first sample chamber, the second sample chamber, the third sample chamber and the fourth sample chamber, and any further sample chambers share the same inlet.

22. A multi-well plate or device comprising a sample analysis unit according to any one of claims 16 to 21.

23. 23. The multi-well plate of claim 22, comprising two or more sample analysis units.

24. 24. The multi-well plate of claim 23, comprising 10 or more sample analysis units.

25. A system for analyzing a biological sample, comprising a multi-well plate according to any one of claims 22 to 24.

26. at least one fluorescent light source; at least one bright field light source; at least one optical system for constricting the fluorescent light beam and / or the bright field light beam; 26. The system of claim 25, further comprising a detection device.

27. 27. The system of claim 26, further comprising a computing unit.

28. A system as claimed in any one of claims 25 to 27, comprising two or more fluorescent light sources.

29. A system described in any one of claims 25 to 28, comprising two or more bright field light sources.

30. A method for measuring cell number using a sample chamber according to any one of claims 1 to 15, a sample analysis unit according to any one of claims 16 to 21, a multiwell plate according to any one of claims 22 to 24, or a system for analyzing a biological sample according to any one of claims 25 to 29.

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