CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application No. 63/369,573 filed July 27, 2022, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

[0002] The present disclosure relates generally to fluorescence imaging and more specifically to standardization and normalization of fluorescence imaging.

BACKGROUND

[0003] Fluorescence imaging is commonly used for non-invasively imaging various biological cells. This allows to research biological processes of, for example, living cells, or other like biological matter. To perform analysis on such cells there are various systems that are commercially available to provide a platform for cell protein analysis. In certain cases, this is performed using Western analysis. For example, various systems for single cell Western blot (scWestern) analysis are sold under the tradename ProteinSimple including a representative example sold under the trademark MILO™ which uses a multiwell design having 6,400 individual wells per chip and is designed to statistically capture a single cell per well. Typically, the diameter of the wells is adapted to accommodate single cells that are sown on these multiwell chips at a dilution that statistically should provide a single cell per well.

[0004] An example of a portion of a chip 100 having wells 110 therein is shown in Fig. 1. Wells 110 are depressions within the substrate of the chip and are organized in rows and columns. For example, a well 110-i-j, where each of ‘i’ and ‘j’ are integers equal to or greater than T, is position in row T and in column ‘j’- For example, but not by way of limitation, a multiwell chip having 6,400 wells may be organized 80-by-80 with 1 <i,j<80. The same multiwell chip may be arranged differently as a 100-by-64 with 1 <i<100 and 1 <j<64 (or vice versa, for that matter). [0005] Each such chip is operative individually and used with respect to certain microscopy equipment for performing fluorescence imaging. In some cases, a plurality of such chips are mounted on a holder that is manipulated under the view of the microscope and images are taken thereof. However, because of a lack of standardization, normalization tools, and methods the use is restricted to qualitative analysis as one set of measurements cannot be effectively compared to another because it is not clear that an apple-to-apple comparison is taking place. Specifically, it is difficult to compare images that may have been captured under different lighting conditions, resolution, and alignment.

[0006] It would therefore be advantageous to provide a solution that overcomes these known deficiencies of the prior art.

SUMMARY

[0007] A summary of several example embodiments of the disclosure follows. This summary is provided for the convenience of the reader to provide a basic understanding of such embodiments and does not wholly define the breadth of the disclosure. This summary is not an extensive overview of all contemplated embodiments and is intended to neither identify key or critical elements of all embodiments nor to delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more embodiments in a simplified form as a prelude to the more detailed description that is presented later. For convenience, the term “certain embodiments” may be used herein to refer to a single embodiment or multiple embodiments of the disclosure.

[0008] Some example embodiments disclosed herein include a cell capture chip that comprises a substrate, a plurality of wells arranged in a two-dimensional matrix of rows and columns on the substrate, each well designed to capture one or more cells therein, and, a plurality of calibration elements, arranged in a two-dimensional matrix of rows and columns on the substrate, wherein the plurality of calibration elements are disposed between the rows and the columns at a distance from each other such that at least two calibration elements fit within a single field-of-view.

[0009] Some example embodiments disclosed herein also include a device for holding a plurality of cell capture chips that comprises a holder for holding therein a plurality of single-cell capture devices, wherein each single-cell capture device comprises a plurality of wells arranged in a two-dimensional matrix of rows and columns on the substrate, each well designed to capture one or more cells therein, and, a plurality of calibration elements disposed on the holder at predetermined distances from each other such that at least two calibration elements fit within a single field-of-view, wherein each of the plurality of calibration elements is visible when the plurality of single-cell capture devices are placed within the holder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The subject matter disclosed herein is particularly pointed out and distinctly claimed in the claims at the conclusion of the specification. The foregoing and other objects, features, and advantages of the disclosed embodiments will be apparent from the following detailed description taken in conjunction with the accompanying drawings.

[0011] Figure 1 an illustration of a portion of a carrier chip having wells therein.

[0012] Figure 2 is an illustration of a portion of a first carrier chip having a plurality of wells and therebetween a calibration element according to an embodiment.

[0013] Figure 3 is an illustration of a portion of the first carrier chip having a plurality of wells and a plurality of calibration elements according to an embodiment.

[0014] Figure 4 is an illustration of a holder holding therein one or more first carrier chips according to an embodiment.

DETAILED DESCRIPTION

[0015] It is important to note that the embodiments disclosed herein are only examples of the many advantageous uses of the innovative teachings herein. In general, statements made in the specification of the present application do not necessarily limit any of the various claimed embodiments. Moreover, some statements may apply to some inventive features but not to others. In general, unless otherwise indicated, singular elements may be plural and vice versa with no loss of generality. In the drawings, like numerals refer to like parts through several views.

[0016] A carrier chip for fluorescent microscopy imaging contains a plurality of wells, each well designed for the purpose of biological processes research, analysis and/or characterization of, for example, live cells. The wells are organized in rows and columns. In between rows, and at predetermined distances in both the X and Y directions certain elements are provided that enable the standardization and normalization of image data captured using a microscope. The distances for placement of the elements are such that a plurality of such elements in different locations are visible within a field-of-view of the microscope. The elements may include fluorescent calibration beads, image resolution targets, position and alignment markers and/or flat field light distribution calibration markers. Such carrier chips enable the extraction of normalized and standardized fluorescent data, that enable comparison between different images taken on the same system as well as different systems as well as reliable temporal imagery.

[0017] Fig. 2 is an example illustration of a portion 200 of a first carrier chip having a plurality of wells 110 and therebetween a calibration element 220 according to an embodiment. The wells 110 may accept therein one or more cells. The calibration element 220, that enables to determine a plurality of cells within a well 110, may have a plurality of positions 221 , for example, 221-1 -1 through 221-3-4 therein. It should be understood that this case of 12 positions is merely an example and should not be viewed as a limitation on the scope of the invention.

[0018] In an embodiment, the minimum number of positions is equal to or greater than the minimum number of calibration elements that need to be present in a field-of-view of a microscope so that when processing a captured image, the orientation, alignment, and position can be correctly determined. The ability to calibrate in the manner described herein allows for handling one, two, three, or more cells within a well, based on any desired analysis and furthermore, using different carriers, which may further be of different types, as calibration additional elements provided and processes described herein, allow for such to be performed.

[0019] In an embodiment, the positions 221 may have embedded therein fluorescent calibration beads, that are attached to the surface of the carrier chip at the predetermined positions 221. In one embodiment these positions 221 are cavities within the surface of the carrier chip 200 and adapted to accept the fluorescent beads. The positions 221 may be etched depending on particular needs during the production of the carrier chip 200, and the attaching may provide a unique pattern of positions that uniquely identify the location of the calibration element 220 within the carrier chip. In an embodiment, the position 221-1-1 is always etched (or otherwise never having a fluorescent bead placed therein) which allows to determine the orientation of the carrier chip if a plurality of calibration elements are detected in the microscope field-of-view, or for that matter in an image taken by microscopic imaging. An etched position 221 could be viewed as equivalent to a logical ‘0’ while an unetched position 221 could be viewed as a logical ‘T. Hence various numbers or patterns, as the case may be, can be shown.

[0020] In an embodiment one position, other than the position 221-1 may be required to always be active, i.e. , unetched, or at logical ‘T. For example, this can be the position 221-1 -4. In another embodiment the position 221-1 may be used for generating a parity check. In such a case the position 221-1-4 is either etched or unetched depending upon the parity generation value for a binary number encoded in the element 220.

[0021] Etching of fluorescent beads from positions 221 at the manufacturing stage of the carrier chip can determine a unique pattern. This unique pattern may be imaged as the pattern it represents or, using the ON and OFF, or T and ‘O’, as a representation of a binary number. As noted above, the use of 12 positions of which one or two positions are used for other reasons, may allow the representation of a binary number of, for example, 10 digits, or the range of 0-1023 (2 ^A 10). The more positions used the larger the number. The numbering scheme for each region of the carrier chip 200 allows for effective stitching of images that are taken separately due to the limitation of the field-of-view of any particular microscope. After the images are taken, it is possible to stich the images correctly at overlapping areas by referencing the calibration elements of two different images and determination that these are the same.

[0022] Fig. 3 is an example illustration 300 of a portion of the carrier chip having a plurality of wells 110 and a plurality of calibration elements 220 according to an embodiment. The plurality of calibration elements 220 are positioned in predetermined intervals between any two rows of wells (or for that matter between any two columns of wells, depending on a point of view). In the example illustration 300 a first calibration element 220-n-m (where each of ‘n’ and ‘m’ are integers equal to or greater than T) is placed between two rows of wells, for example, row i-2 and i-1 , and generally, within the line of column j-3.

[0023] In an embodiment, a next calibration element 220-n-m+1 in the row direction, is positioned at a predetermined distance of ‘X’ 310, for example, but not by way of limitation, a distance of 1 mm. In yet another embodiment, a next calibration element 220-n+1-m in the row direction, is positioned at a predetermined distance of ‘Y’ 320, for example, but not by way of limitation, a distance of 1 mm. It should be noted that ‘X’ 310 and ‘Y’ 320 do not have to be equal, however, they should be at such a distance that at least two calibration elements 220, for example, 220-n-m and 220-n-m+1 , or 220-n-m and 220-n+1-m are in a single field-of-view of an imaging microscope, for the reasons discussed herein in greater detail.

[0024] Returning to Fig. 2, in an embodiment of the positions 221 within a calibration element 220, fluorescent beads are deposited within each position 221 , but for a predetermined single position, for example, but not by way of limitation, position 221-1-1. In one embodiment, another position 221 , for example, but not by way of limitation, position 221- 1-3 always has a fluorescent bead deposited therein. Thereby ensuring that it is not possible to have a calibration element 220 that has no fluorescent beads whatsoever. In an embodiment, one or more calibration elements may be further used for the purpose as a resolution target. For example, but not by way of limitation, this can be achieved by using different bead dimensions in predetermined positions. The fluorescent beads may have dimensions of, for example, but not by way of limitation, 30 .m, 40 .m or 50 .m, which can then be used to determine the resolution of the image and further properly compensate when different resolutions are used.

[0025] In an embodiment the number of positions in a first direction (for example, but not by way of limitation, an ‘x’ direction) of the calibration elements 220 is larger than the number of positions in a second direction (for example, but not by way of limitation, a ‘y’ direction). In Fig. 2 there are 4 positions in the ‘x’ direction and there are 3 positions in the ‘y’ direction. The shape allows for determination of the orientation of the carrier chip which allows for orientation of two images captured separately of each other and establishment of their relative orientation to each other. In another embodiment, it is possible to select certain positions to create a predetermined image, for example, but not by way of limitation, a “1” sign, at a predetermined orientation (can be rotated in any desired way but has to be consistent). Other signs may be used without departing from the scope of the invention and are referred to herein as an orientation mark. [0026] In an embodiment, the calibration elements 220 may be of different types, one to identify a location within a carrier chip, another for the purpose of orientation, and yet another for the purpose of resolution, or any permissible combinations thereof. It is however preferable to make sure that in each field-of-view there will be at least the minimum required number of calibration elements to ensure the ability to perform the image processing required to achieve the normalization and standardization between different images taken. By obtaining information regarding the different calibration elements 220, image processing may provide compensation for such differences between images allowing for not only qualitative analysis but also for reliable quantitative analysis. It further allows to receive similar imaging results regardless of the fluorescent microscope used.

[0027] In an embodiment, a calibration element 220 utilizes a white colored surface which is used for the purpose of flat field light distribution calibration. The white colored surface may be utilized to calibrate between two images that may have different lighting conditions resulting from use of different light sources, different light intensity, shadows, and other artifacts that impact the image quality. By obtaining information of the known white surface calibration element 220, image processing may provide compensation to such differences between images allowing for not only qualitative analysis but also for reliable quantitative analysis. It further allows to receive similar imaging results regardless of the fluorescent microscope used.

[0028] It should be appreciated that because of the ability to compensate for differences between images taken by different equipment viewing the same type of carrier chip having the calibration elements 220 described herein, it is further possible to obtain temporal cytometric data in systems where the holder of the carrier chip is capable of maintaining cell viability over time. Furthermore, the standardization and normalization provided by the addition of the calibration elements 220, enable integration of the results of separate experiments into a unified database.

[0029] While fluorescent beads were discussed in relation to the filling of the positions 221 of a calibration element 220, this should not be viewed as a limitation of the scope of the invention. Other techniques may be used, for example, etching these positions in the predetermined positions. Imaging can differentiate between etched and unetched positions 221.

[0030] In some embodiments a plurality of carrier chips may be placed on a single holder for analysis of cells trapped in wells of each such carrier chip. In an embodiment, at least one of the plurality of carrier chips should be a carrier chip having the calibration elements 220 described herein in greater detail.

[0031] Fig. 4 is an example illustration of a holder 410 holding therein one or more first carrier chips, for example, first carrier chips 420-1-1 and 420-2-2 according to an embodiment. The carrier chips 420-1 -2 and 420-2-1 may be prior art carrier chips that are not equipped with the calibration elements 220. In an embodiment, the number of first carrier chips placed within a holder 410, and positions therein are determined based on the field-of- view characteristics of the microscope used so as to ensure that regardless of the image captured it will always have at least two calibration elements 220 within the field-of-view. The calibration elements 420 should be visible even when the carrier chips are positioned within the holder.

[0032] In one embodiment, the holder 410 further comprises calibration elements 430, for example, calibration elements 430-1 through 430-8, of at least one of the types of calibration elements 220 described herein. Such calibration elements 220 may assist in the calibration of the entire system and further support the standardization and normalization of the image data captured. The calibration elements 430 are shown in Fig. 4 as positioned in the periphery of the holder 410.

[0033] However, other positions are possible without departing from the scope of the disclosed embodiments. Furthermore, while two carrier chips and two first carrier chips are shown in Fig. 4, this should not be viewed as limiting the scope of the invention, and it is specifically noted that many other combinations are possible. It should be noted that according to the embodiments described herein a well may contain one or more captured cells. That is, a well may contain therein one, two, three or more cells and the alignment and calibration described herein provides for an effective way to provide solutions for such cases that go beyond a single-cell capture.

[0034] As described herein, but not by way of limitation, some example embodiments disclosed herein include a cell capture chip that comprises a substrate, a plurality of wells arranged in a two-dimensional matrix of rows and columns on the substrate, each well designed to capture one or more cells therein, and, a plurality of calibration elements, arranged in a two-dimensional matrix of rows and columns on the substrate, wherein the plurality of calibration elements are disposed between the rows and the columns at a distance from each other such that at least two calibration elements fit within a single field-of-view.

[0035] As further described herein, but not by way of limitation, some example embodiments disclosed herein also include a device for holding a plurality of cell capture chips that includes a holder for holding therein a plurality of single-cell capture devices. In an embodiment, each single-cell capture device comprises a plurality of wells arranged in a two-dimensional matrix of rows and columns on the substrate, each well designed to capture one or more cells therein, and, a plurality of calibration elements disposed on the holder at predetermined distances from each other such that at least two calibration elements fit within a single field-of-view. Each of the plurality of calibration elements is visible when the plurality of single-cell capture devices are placed within the holder.

[0036]As used herein, the phrase “at least one of” followed by a listing of items means that any of the listed items can be utilized individually, or any combination of two or more of the listed items can be utilized. For example, if a system is described as including “at least one of A, B, and C,” the system can include A alone; B alone; C alone; A and B in combination; B and C in combination; A and C in combination; or A, B, and C in combination.

[0037] All examples and conditional language recited herein are intended for pedagogical purposes to aid the reader in understanding the principles of the disclosed embodiment and the concepts contributed by the inventor to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosed embodiments, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure.