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-9939372-B2
Publication Date
2018-04-10
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 computer-executed method for processing recordings made by an instrument to produce simultaneous video recordings from multiple optical channels of a magnified field of view. Cells are loaded with two or more fluorescent dyes designed to respond to action potentials, calcium transients, or the flow of ions across a membrane, and the instrument separates fluorescent light emitted from the sample into distinct wavelengths directed to respective cameras.
The instrument includes an optical path with a light source, microscope objective, mirrors, optical filters, and two or more cameras arranged to separate wavelengths. The system further includes a motorized stage to position a region of the sample above the objective, and an autofocus module that moves focus in relation to the sample to focus the image using one of the cameras to collect a stack of images at different focus positions and derive a best focus position.
After autofocus, the method enables live cell channel collection by using an automatic filter changer to change the filter after autofocus to collect a corresponding live cell channel, or by collecting the stack on the same fluorescent channel as the corresponding live cell channel. The recordings are processed and analyzed to provide quantitative measurements including cellular action potentials, calcium transients, and ion flow across a membrane, and the method corrects mirroring, X-Y shift, rotation, and magnification differences between video recordings from different cameras recording from the same field of view.
The method further segments the videos of the cells and labels subcellular regions from two or more optical channels to define boundaries of individual cells and distinct subcellular regions. It generates measurements from the cell periphery and subcellular masks of change in intensity over time of fluorescent dyes, plots change in intensity versus time, and extracts measurements characterizing the shape and duration of the action potential, calcium transients, or ion concentrations.
Claims Coverage
The partial content contains one independent claim, and its inventive coverage is concentrated in a multi-camera, multi-optical-channel computer-executed processing method with autofocus-based focus selection and subsequent multi-channel correction, segmentation, and kinetic feature extraction for action potentials, calcium transients, and ion flow.
Simultaneous multi-optical-channel recordings for action potentials, calcium transients, and ion flow
A computer-executed method that produces simultaneous video recordings from multiple optical channels of a magnified field of view for cells loaded with two or more fluorescent dyes that respond to action potentials, calcium transients, or flow of ions across a membrane, using two or more cameras to separate fluorescent light into distinct wavelengths and direct each wavelength to a respective camera
Camera-driven autofocus with focus-stack sharpness index and best-focus selection
An autofocus module that, using one of the two or more cameras, collects a stack of images at different focus positions, calculates a degree of focus or sharpness index, derives a best focus position based on the degree of focus data, and sets focus to the best focus position, including the option that the images in the stack are either of a different fluorescent color followed by filter change or are collected on the same fluorescent channel as the corresponding live cell channel
Multi-camera video correction, cell/subcellular segmentation, and periphery/subcellular kinetic measurements
Processing of recordings including correcting for mirroring, X-Y shift, rotation, and magnification differences between video recordings captured by different cameras recording from the same field of view; segmenting videos of cells and labeling subcellular regions from two or more optical channels to define cell boundaries and subcellular regions; generating measurements from cell periphery and subcellular masks of change in intensity over time; and plotting change in intensity versus time
Extraction of action potential and calcium/ion concentration shape and duration from intensity-vs-time kinetics
Extracting measurements that characterize the shape and duration of the action potential, calcium transients, or ion concentrations from the intensity-over-time kinetics of the fluorescent dyes
Across the independent claim, the inventive coverage combines simultaneous multi-wavelength/multi-camera channel acquisition from fluorescently labeled excitable cells, camera-based focus-stack sharpness index autofocus with best-focus selection and live-channel capture, and processing that corrects inter-camera geometric differences, segments and labels cells and subcellular regions, and extracts quantitative kinetic measurements characterizing action potential, calcium, and ion dynamics.
Stated Advantages
Documented Applications
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