Epsilon Open Archive

Abstract

Background
Calcium imaging is based on the detection of minute signal changes in an image time-series encompassing pre- and post-stimuli. Depending on the function of the elicited response, change may be pronounced, as in the case of a genetically encoded calcium-reporter protein, or subtle, as is the case in a bath-applied dye system. Large datasets are thus often acquired and appraised only during post-processing where specific Regions of Interest (ROIs) are examined.

New method 
The scintillate software provides a platform allowing for near instantaneous viewing of time-sequenced tiffs within a discrete GUI environment. Whole sequences may be evaluated. In its simplest form scintillate provides change in florescence (ΔF) across the entiretiffimage matrix. Evaluating image intensity level differences across the whole image allows the user to rapidly establish the value of the preparation, withouta prioriROI-selection. Additionally, an implementation of Independent Component Analysis (ICA) provides additional rapid insights into areas of signal change.
 
Results 
We imaged transgenic flies expressing Calcium-sensitive reporter proteins within projection neurons and moth mushroom bodies stained with a Ca2+sensitive bath-applied dye. Instantaneous pre-stimulation background subtraction allowed us to appraise strong genetically encoded neuronal Ca2+responses in flies and weaker, less apparent, responses within moth mushroom bodies. Comparison with existing methods At the time of acquisition, whole matrix ΔF analysis alongside ICA is ordinarily not performed. We found it invaluable, minimising time spent with unresponsive samples, and assisting in optimisation of subsequent acquisitions.
 
Conclusions 
We provide a multi-platform open-source system to evaluate time-series images. Abbreviations ΔF,change of intensity between frames; ICA,independent component analysis