研究目的
To automate the intracellular calcium profiles extraction from fluorescence image sequences of endothelial cells under different stimuli.
研究成果
The proposed automated system for intracellular calcium profiles extraction from fluorescence image sequences effectively tracks endothelial cells under different stimuli, providing accurate and efficient data extraction. The system's ability to automatically relocate regions of interest and handle large image sets offers significant advantages over manual methods.
研究不足
The study acknowledges the complexity of finding an exact mathematical model to describe the motion of cells and the potential for cells to temporarily disappear during some frames due to movement artefacts, which may affect tracking accuracy.
1:Experimental Design and Method Selection:
The study combined digital image processing techniques with a multi-target tracking approach supported by Kalman estimation to automate the extraction of intracellular calcium profiles from fluorescence image sequences.
2:Sample Selection and Data Sources:
Image sequences from two different stimuli (chemical stimulus with ATP and mechanical stimulus delivered by a glass microelectrode) were used.
3:List of Experimental Equipment and Materials:
Equipment included an upright epifluorescence Axiolab microscope, a Zeiss 40× Achroplan objective, a filter wheel, and a camera. Materials included Fura-2/AM for calcium measurements.
4:Experimental Procedures and Operational Workflow:
The procedure involved loading fluorescence images, generating ratio image sequences, applying noise reduction and adaptive thresholding, and tracking cells using a Kalman filter.
5:Data Analysis Methods:
The mean intensity within each region of interest (ROI) for every frame throughout a ratio image sequence was collected to generate calcium profiles.
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Filter wheel
Lambda 10
Sutter Instrument
Positions alternately along the optical path the two filters that allow the passage of light at 340 and 380 nm.
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Fura-2/AM
Molecular Probes
Calcium-sensitive fluorophore for measuring intracellular Ca2+ signals.
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Axiolab microscope
Carl Zeiss
Upright epifluorescence microscope for visualizing endothelial cells.
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Zeiss 40× Achroplan objective
Carl Zeiss
Water-immersion objective for microscopy.
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Camera
Extended-ISIS Camera
Photonic Science
Used to capture fluorescence images.
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