Abstract
Calcium signals act as a ubiquitous secondary messenger in regulating many body functions. The detection of calcium microdomain signals is greatly facilitated by the existence of biomarker-targeted fluorescent probes. In this study, SRRF (super-resolution radial fluctuations) algorithm were used to compare the loci and the intensity of fluorescent probes before and after SRRF analysis. The implementation of SRRF algorithm was aimed for automatically resolving delicate and small calcium signals (to avoid the overlapped loci) on original images. For assessing the spatial accuracy of image intensity, immunofluorescence staining of retina cryostat slice for connexin 36 (Cx36) was microscopically imaged with or without the successive SRRF reconstruction. For characterizing the temporal association between SRRF and non-SRRF images, the changes of Cx36-GCaMP calcium indicator were recorded from transfected HeLa cells in response to the transient puffing of ionomycin. Image processing and analyses were conducted with Image J and Matlab. Through this study, SRRF reconstruction was found to confer an accurate measure for the identification of subcellular molecules, such as gap junctions. Compared with the conventional imaging, SRRF reconstruction generated better image resolution for the precise registration of individual signals. Temporally, the ratios of change in fluorescence intensity between SRRF and non-SRRF images were significantly correlated in the presence or absence of the subtraction of high background intensity. Quantitatively, the ratios of change in fluorescence intensity between SRRF and non-SRRF images with or without background subtraction were also significantly correlated. The merit of SRRF application on calcium live imaging was validated with the reporter gene system we worked on.