US 2021/0065362 A1 · LIVE CELL VISUALIZATION AND ANALYSIS

Abstract

Systems and methods are provided for automatically imaging and analyzing cell samples in an incubator. An actuated microscope operates to generate images of samples within wells of a sample container across days, weeks, or months. A plurality of images is generated for each scan of a particular well, and the images within such a scan are used to image and analysis metabolically active cells in the well. Tins analysis includes generating a “range image” by subtracting the minimum intensity value, across the scan, for each pixel from the maximum intensity value. This range image thus emphasizes cells or portions of cells that exhibit changes in activity over a scan period (e.g., neurons, myocytes, cardiomyocytes) while de-emphasizing regions that exhibit consistently high intensities when images (e.g., regions exhibiting a great deal of autofluorescence unrelated to cell activity).

Claims33

Dependent claims

A method comprising:

capturing a movie of a cell culture vessel;
and
generating a static range image from the movie, wherein the range image is composed of pixels representing the minimum fluorescence intensity subtracted from the maximum fluorescence intensity at each pixel location over a complete scan period.

A method of identifying biologically active cells during one or more periods of time, the method comprising, for each of the one or more periods of time:

a. generating a plurality of fluorescence activity images of a sample contained within a sample container, wherein the sample includes biologically active cells, and wherein each image of the plurality of fluorescence activity images comprises a respective plurality of pixel values, each pixel value corresponding to a respective pixel location within an image frame;
b. generating a fluorescence range image from the plurality of fluorescence activity images, wherein generating a fluorescence range image from a plurality of fluorescence activity images comprises determining a plurality of pixel values for the fluorescence range image, and wherein determining a given pixel value for the fluorescence range image comprises determining a range of a set of pixel values, from each of the plurality of fluorescence activity images, that have pixel locations corresponding to a pixel location of the given pixel value within the image frame;
c. based on the fluorescence range image, determining a location, relative to the image frame, of one or more active objects within the sample container during a corresponding period of time, wherein each of the one or more active objects is a portion of at least one respective biologically active cell present within the sample during the corresponding period of time;
and
d. for each active object of the one or more active objects within the sample container during the corresponding period of time, determining a respective time-varying activity level of each of the one or more active objects across the corresponding period of time.

2 more elements

A method of identifying biologically active cells during one or more periods of time, the method comprising, for each of the one or more periods of time:

a. generating a plurality of fluorescence activity images of a sample contained within a sample container, wherein the sample includes biologically active cells, and wherein each image of the plurality of fluorescence activity images comprises a respective plurality of pixel values, each pixel value corresponding to a respective pixel location within an image frame;
b. generating a fluorescence range image from the plurality of fluorescence activity images, wherein generating a fluorescence range image from a plurality of fluorescence activity images comprises determining a plurality of pixel values for the fluorescence range image, and wherein determining a given pixel value for the fluorescence range image comprises determining a range of a set of pixel values, from each of the plurality of fluorescence activity images, that have pixel locations corresponding to a pixel location of the given pixel value within the image frame;
c. based on the fluorescence range image, determining a location, relative to the image frame, of one or more active objects within the sample container during a corresponding period of time, wherein each of the one or more active objects is a portion of at least one respective biologically active cell present within the sample during the corresponding period of time;
and
d. for each active object of the one or more active objects within the sample container during the corresponding period of time, determining a respective time-varying activity level of each of the one or more active objects across the corresponding period of time, wherein each time-varying activity level is determined based on a set of pixel values, from each of the plurality of fluorescence activity images, that have pixel locations proximate to the determined location of a corresponding active object.

2 more elements

A method of identifying biologically active cells during one or more periods of time, the method comprising, for each of the one or more periods of time:

a. generating a plurality of fluorescence activity images of a sample contained within a sample container, wherein the sample includes biologically active cells, and wherein each image of the plurality of fluorescence activity images comprises a respective plurality of pixel values, each pixel value corresponding to a respective pixel location within an image frame;
b. determining a location, relative to the image frame, of one or more active objects within the sample container during a corresponding period of time, wherein each of the one or more active objects is a portion of at least one respective biologically active cell present within the sample during the corresponding period of time;
c. for each of the one or more active objects within the sample container during the corresponding period of time, determining a respective time-varying activity level across the corresponding period of time;
and
d. based on the time-varying activity levels determined for the one or more active objects, selecting a subset of active objects from the one or more active objects, wherein selecting a particular active object from the one or more active objects comprises determining that the time-varying activity level determined for the particular active object exhibits at least one burst;
or
(i) generating a plurality of fluorescence activity images of a sample contained within a sample container, wherein the sample includes biologically active cells, and wherein each image of the plurality of fluorescence activity images comprises a respective plurality of pixel values, each pixel value corresponding to a respective pixel location within an image frame;
(ii) determining a location, relative to the image frame, of one or more active objects within the sample container during a corresponding period of time, wherein each of the one or more active objects is a portion of at least one respective biologically active cell present within the sample during the corresponding period of time;
(iii) for each of the one or more active objects within the sample container during the corresponding period of time, determining a respective time-varying activity level across the corresponding period of time based on respective sets of pixel values, from each of the plurality of fluorescence activity images, that have pixel locations proximate to the respective determined locations of the one or more active objects;
and
(iv) based on the time-varying activity levels determined for the one or more active objects, selecting a subset of active objects from the one or more active objects, wherein selecting a particular active object from the one or more active objects comprises determining that the time-varying activity level determined for the particular active object exhibits at least one burst.

8 more elements

Dependencies are inferred from the claim text.

Live cell visualization and analysis

Life of this patent

Live cell visualization and analysis

Life of this patent

Abstract

Claims33