ABSTRACT
Cameleons are genetically encoded fluorescence resonance energy transfer (FRET)-based Ca(2+) indicators. Attempts to use cameleons to detect neural activity in vertebrate systems have been largely frustrated by the small FRET signal, in contradistinction to the higher signals seen in Drosophila and Caenorhabditis elegans. We have developed a statistical optimization method capable of detecting small ratiometric signals in noisy imaging data, called statistical optimization for the analysis of ratiometric signals. Using this method, we can detect and estimate anticorrelated ratiometric signals with subcellular resolution in cultured, dissociated zebrafish spinal neurons expressing cameleon or loaded with fluo-4 and fura-red. This method may make it possible to use yellow cameleons for measuring neural activity at high resolution in transgenic animals.
Subject(s)
Calcium-Binding Proteins , Calcium/metabolism , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes , Neurons/metabolism , Recombinant Fusion Proteins , Zebrafish/metabolism , Algorithms , Animals , Calcium Signaling/physiology , Cells, Cultured , Data Interpretation, Statistical , Neurons/cytology , Zebrafish/anatomy & histologyABSTRACT
Performing reflection-mode (backscatter-mode) confocal microscopy on cells growing on reflective substrates gives images that have improved contrast and are more easily interpreted than standard reflection-mode confocal micrographs (Keith et al., 1998). However, a number of factors degrade the quality of images taken with the highest-resolution microscope objectives in this technique. We here describe modifications to reflection-enhanced backscatter confocal microscopy that (partially) overcome these factors. With these modifications of the technique, it is possible to visualize structures the size-and refractility-of individual microtubules in intact cells. Additionally, we demonstrate that this technique, in common with fluorescence techniques such as standing wave widefield fluorescence microscopy and 4-Pi confocal microscopy, offers improved resolution in the Z-direction.
Subject(s)
Microscopy, Confocal/methods , Microtubules/ultrastructure , Animals , Cell Line , Kidney , Microtubules/physiology , Sensitivity and Specificity , TelophaseABSTRACT
When PC12 cells are grown on substrates showing a gradient of nonspecific adhesion, they preferentially initiate neurites in directions of changing adhesivity, whether that change is in the direction of increasing or decreasing adhesivity. This preference for changing adhesivity is ablated both by C. difficile toxin A, which inhibits all Rho-family GTPases, and by C. botulinum C3 exoenzyme, which specifically inhibits Rho.