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Abstract
Cytometry of Reaction Rate Constant (CRRC) is a method for studying heterogeneity of cell populations with regards to activity of cellular reactions. It is based on time-lapse fluorescence microscopy which facilitates following reaction kinetics in individual cells. The current CRRC workflow utilizes a single fluorescence image to manually identify cell contours; these contours are then used to determine fluorescence intensity of individual cells in the entire time-stack of images. This workflow can only be used reliably if the cells maintain their positions during the time-lapse measurements; if the cells move, the results of a CRRC experiment will be inaccurate. The requirement of invariant cell positions during a prolonged imaging is impossible to satisfy for motile cells. Here we report on developing an advanced workflow that makes CRRC applicable to motile cells. The new workflow combines fluorescence microscopy with brightfield (BF) microscopy and utilizes automated processing and analysis of images. A BF image is taken right after every fluorescence image and used to determine cell contours. The contours are tracked through the time-stack of BF images to account for cell movement. A set of contours, which is unique for every image, is then used to determine fluorescence intensity of cells in the associated fluorescent image. Finally, time dependencies of intracellular fluorescence intensities are used to determine the rate constant and plot a kinetic histogram “number of cells vs rate constant”. The robustness of the new workflow to cell movement was confirmed experimentally by conducting a CRRC study of cross-membrane transport in motile cells. The new workflow makes CRRC applicable to a wide range of cell types and eliminates the influence of cell motility on the accuracy of results.
Supplementary materials
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images
Description
It contains raw images for:
- Figure 2; the folder contains three subfolders named ‘region X’. Each represents a different x-y position of the cell plate. Each ‘region X’ folder contains images obtained in BF, DIC and PC mode.
- Figure 3; raw images for the time lapse experiment (time interval =10 s).
- Figure 6; it contains the set of adjacent BF and fluorescent images. The time interval between the images in the BF folder is 1 min. (time interval = 1 min) The folder also contains an image named ‘PI’. This image reveals the position of each single cell in the original workflow.
- Figure S3; this folder contains two in-focus BF images: the raw image and the image after threshold was applied. It also contains the in-focus and out-of-focus (5 µm below, 5 µm above, and 10 µm above the in-focus position, respectively) fluorescence images.
Actions
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kinetictraces_and_fittingresults
Description
It contains two different .csv files. One is named ‘Kinetic traces_and_fittingresults_new workflow’ and the other one is named ‘Kinetic traces_and_fittingresults_original workflow’. Both files contain one sheet displaying the fluorescence intensity values as a function of time for individual cells. It also contains the equation used for fitting and the fitting results computed by OriginPro (scroll down to line 69).
Actions
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Trackingvideos
Description
It contains two videos. Each video shows the cell movement of the same single cell over the course of a time-lapse experiment of 1 hour duration, with a 1 min time interval. The ‘newworkflowvideo’ shows the ability of the new workflow to track the cell’s movements over time. The ‘oldworkflowvideo’ shows the absence of tracking, leading to an incorrect integration of fluorescence intensity over time for the same single cell (which subsequently results in the overestimation of kefflux)
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