Objective quantification of acetylcholine receptor aggregation using fast Fourier transforms

A new approach for objectively analyzing the aggregation of acetylcholine receptors (AChRs) through power spectrum analysis derived from fast Fourier transform (FFT) of images has been developed. Presently, detection of AChR aggregates at neuromuscular junctions is not easily accomplished. Though the formation of AChR clusters results in periodic gray-level variations that differ with time, no study reporting their correlation with frequency information in the Fourier domain for aggregates’ detection purposes exists. To this end, we processed time-lapse images of AChR aggregates’ formation on murine myotubes to extract peak values of power spectra. To validate interpretation of the Fourier spectra analysis, a computer routine was developed to semi-automatically count AChR aggregates. We found: (1) logarithmic maxima of Fourier spectra correlated significantly with experimentation time; (2) cluster count correlated significantly with time only after clusters were discernable from images, signifying that this method heavily depended on definitive growth data and thresholding values; (3) exponents of Fourier maxima versus time and cluster count versus time profiles during this phase compared favorably, indicating that both methods were analyzing identical cluster growth rates. Our observations suggest that analysis via FFT power spectrum is sensitive and robust enough to automatically quantify AChR aggregates.