Automated Image Analysis
Image analysis pipeline (workflow) for automated quantification of whole-cell, near-membrane, and perinuclear FRET signals. This pipeline was used to measure localized subcellular cAMP signals as they evolve over time, using a cAMP FRET reporter and hyperspectral imaging microscopy.
Subcellular regions are identified using automated image analysis. Time-dependent FRET traces may then be measured.
LC Pro Analysis Software
Intracellular calcium signals occur in virtually all cells, and mediate a myriad of functions such as proliferation, secretion, fertilization, contraction, and memory. To investigate these phenomena, calcium signals are commonly measured using fluorescent microscopy preparations loaded with calcium indicator dyes, which fluoresce when bound to free cytosolic calcium. However, intracellular calcium signals encode specific responses via complex spatial and temporal patterns including localized oscillations and cell-wide waves, which can be difficult to decipher manually.
Therefore we developed LC_Pro, an algorithmic solution to the analysis of calcium signal dynamics. LC_Pro performs automated region of interest (ROI) analysis on image sequences of calcium signals in both cell culture and tissue preparations by identifying the location of signal events and measuring their magnitude as a function of time. These measurements are then used to calculate relevant signal parameters such as the maximum magnitude, duration, and spatial spread of signal events, which are processed into graphical output to facilitate the decoding of calcium signal patterns.
LC_Pro is implemented as a freely available, open source plugin for the National Institutes of Health (NIH) image analysis software package ImageJ. Since the development of LC_Pro, we have published multiple validations of its efficacy as well as the results of its application in physiological systems. We continue to employ LC_Pro to decipher intracellular calcium dynamics, and are currently developing more sophisticated algorithms for cell signal analysis.