Digital analysis of light microscope immunofluorescence: high-resolution co-localization of synaptic proteins in...

A protocol is presented for determining the subcellular distribution of fluorescently labeled proteins in neurons using deconvolved images gathered with a wide-field microscope. The protocol includes optimal settings for the numerical algorithm used to deconvolve the images and an objective method for thresholding the deconvolved images to retain only high-intensity, specific labeling. The effectiveness of the protocol is demonstrated using a fluorescent antibody stain directed towards the alpha1 subunit of the GABA(A) receptor in cultured neurons. We also show, using an antibody against the presynaptic vesicular protein synaptophysin, that the technique can detect presumptive regions of synaptic contact between neurons. Double-labeling with the anti-alpha1 and anti-synaptophysin antibodies in a cultured neuron reveals regions of both synaptic and non-synaptic alpha1 labeling. Thus, numerical postprocessing of wide-field images can be used to efficiently locate receptor proteins in neurons in relation to functionally important structures. This confocal-like functionality is attained without the excessive bleaching and phototoxicity associated with the intense laser excitation light used in confocal techniques.