Analysis of action potentials, transients, and ion flux in excitable cells
Inventors
Cerignoli, Fabio • Gehalot, Piyush • McDonough, Patrick M. • Price, Jeffrey H. • Whittaker, Ross J.
Assignees
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Publication Number
US-10359357-B2
Publication Date
2019-07-23
Expiration Date
Abstract
Video recordings from two or more optical channels are produced, processed, and analyzed simultaneously in order to provide quantitative analysis of action potentials, calcium transients and ionic flux in excitable cells loaded with voltage or ion sensitive dyes with distinct excitation and emission wavelengths. The specific wavelengths of fluorescent light emitted from each dye are separated and recorded. The recordings are mutually registered and cytometric analysis is performed to provide a quantitative analysis of the action potentials, calcium transient, and/or ionic flux on a cell-by-cell and well-by-well basis in microtiter plates. The cells are then fixed, labeled for other biomarkers, and scanned again. The resulting fixed cell images are registered with the live cell recordings and analyzed; missing cells that were washed off are detected relative to the live recordings, and cytometry data from live and fixed cell scans is collated cell-by-cell.
Core Innovation
The invention provides a method executable by an instrument for producing simultaneous video recordings of a magnified field of view from multiple optical channels. Fluorescent light emitted from the sample is separated into distinct wavelengths and directed to respective cameras so that two or more videos are recorded simultaneously, each corresponding to varying levels of light tied to different intracellular components.
The method includes maintaining incubation conditions in an incubation chamber that maintains temperature, carbon dioxide, and oxygen at preset levels, and automatically applying electrical stimulation using a stimulator arm and a pair of electrodes into wells of a multiwell plate. It further includes autofocus using an autofocus module that moves the microscope objective relative to the sample, collects a stack of images at different focus positions, calculates a degree of focus or a sharpness index, derives a best focus position, and sets focus to the best focus position for the live cell channels.
The recordings are processed and analyzed to provide quantitative measurements including cellular action potentials, calcium transients, and/or flow of ion across a membrane. This processing corrects mirroring, X-Y shift, rotation, and magnification differences between videos from different cameras, then segments the videos of the cells to label cell boundaries and distinct subcellular regions from two or more optical channels.
In dependent implementations, the method fixes the cells and labels fixed cells for additional cellular molecules, then rescans and records images of label colors in fixed cells. The fixed scan images are registered with images and video recordings of the live scanning, missing cells washed off during the process of fixing and labeling are detected, and cytometric measurements from live cells and fixed cells are collated into a single data set to study and compare fixed cell and live cell labels together.
Claims Coverage
The independent claim is supported by the dependent claims, covering an instrument-executable workflow with simultaneous multi-channel video recordings, incubation and electrical stimulation, multi-camera registration, segmentation with periphery and subcellular masks, and extraction of kinetics-based quantitative metrics. The dependent claims extend the workflow to include fixation, additional labeling, fixed-cell rescanning, fixed-to-live registration, detection of washed-off missing cells, and collation of live and fixed cytometry measurements into a single data set.
Simultaneous multi-channel fluorescence video recording with wavelength separation to multiple cameras
Producing simultaneous video recordings of a magnified field of view from multiple optical channels by separating fluorescent light emitted from the sample into distinct wavelengths and directing each wavelength to a respective camera.
Motorized stage and autofocus using image stack sharpness index
Using a motorized stage to position a region of the sample to be imaged above the objective and using an autofocus module that moves the microscope objective relative to the sample, collects a stack of images at different focus positions, calculates a degree of focus or sharpness index, derives a best focus position, and sets focus to the best focus position.
Incubation chamber maintains temperature, carbon dioxide, and oxygen
Maintaining temperature, carbon dioxide, and oxygen at preset levels in the incubation chamber that contains the sample.
Electrical stimulation via stimulator arm and electrodes in multiwell plate wells
Automatically moving a stimulator arm to move a pair of electrodes into wells of a multiwell plate for applying electrical stimulation.
Multi-camera control system for simultaneous recording mapped to intracellular components
Recording two or more videos simultaneously from each of the two or more cameras to record two or more varying levels of light, each of which corresponds to two or more different intracellular components.
Multi-camera registration correction across cameras recording same field of view
Correcting for mirroring, X-Y shift, rotation, and magnification differences between the video recordings captured by different cameras recording from the same field of view.
Segmentation with periphery and subcellular masks for intensity-vs-time kinetics
Segmenting the videos of the cells and labeling subcellular regions of the cells from two or more optical channels to define boundaries of individual cells and distinct subcellular regions, and generating measurements from cell periphery and subcellular masks of changes in intensity over time of the fluorescent dyes.
Kinetics-based quantitative extraction for action potentials and calcium transients and/or ion flow
Plotting changes in intensity of the fluorescent dyes versus time and extracting from plotted changes in intensities measurements that characterize shape and duration of the cellular action potentials, calcium transient, ion concentrations and/or ion flow.
Fixed-cell workflow with additional labeling, fixed rescanning, and fixed-to-live registration
Fixing the cells and labeling fixed cells for additional cellular molecules, rescanning and recording images of the label colors in fixed cells, and registering images from fixed scanning with images and video recordings of the live scanning.
Detection of washed-off missing cells during fixing and labeling
Segmenting fixed-scanning images to detect fixed cells and subcellular regions and detecting missing cells washed off during the process of fixing and labeling.
Collating live and fixed cytometry measurements into a single data set
Collating cytometric measurements from live cells and fixed cells into a single data set to study and compare fixed cell and live cell labels together.
Overall, the claim set covers simultaneous multi-camera, wavelength-separated fluorescence video recording tied to intracellular components, with autofocus, incubation maintenance, electrical stimulation, multi-camera registration, and segmentation into periphery and subcellular masks to derive quantitative intensity-vs-time kinetics and characterize shape and duration of action potentials, calcium transients, and/or ion concentrations or ion flow. The dependent claims add a fixed-cell workflow including additional labeling, fixed rescanning, fixed-to-live registration, detection of washed-off missing cells, and collation of live and fixed cytometry measurements into a single data set for comparison.
Stated Advantages
Provides quantitative measurements including one or more of cellular action potentials, calcium transients, and/or flow of ion across a membrane.
Enables characterization of shape and duration of cellular action potentials, calcium transient, ion concentrations and/or ion flow from changes in fluorescent dye intensity versus time.
Enables correction for mirroring, X-Y shift, rotation, and magnification differences between recordings from different cameras recording the same field of view.
Enables extraction of measurements using cell periphery and subcellular masks derived from changes in intensity over time.
Enables combining fixed-cell and live-cell cytometry measurements by collating into a single data set for study and comparison of fixed cell and live cell labels together.
Documented Applications
Cardiomyocyte recordings and fixed-vs-live data merging are illustrated in examples and figures.
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