Cell Image Quality and Point Spread Function Depends on Photomultiplier Sensitivity in a Fast Frame-Rate Multi-color Laser-Scanning Confocal Fluorescence Microscope

Abstract

A typical parameter used for describing the resolving capability of a fluorescence microscope is the full width at half maximum (FWHM) obtained by fitting a Gaussian curve to the point spread function (PSF) generated by a fluorescent bead.In this work we used a custom-built video-rate confocal microscope at the diffraction limit to measure variations of PSF FWHM as a function of PSF intensities at different photomultiplier (PMT) sensitivities. Images were obtained by summation (128-256) of pixels with 62.5 ns dwell time. We used fluorescence beads and fluorescence-conjugated IgGs excited at 488 and 561 nm with emission recorded centered at 536 nm and 607 nm, respectively. In general, FWHM as a function of PSF peak intensity remains invariant using maximum photomultiplier (PMT) sensitivity. On the other hand, when using lower PMT sensitivities, FWHM values were correlated with PSF intensities, being at the diffraction limit.In agreement with this finding, we found that at a lower PMT sensitivity the image quality of cells (e.g. isolated heart myocytes labeled with anti-L-type Ca2+ channel and anti-ryanodine receptors) is greatly improved. These proteins accumulate along the tubular transversal structure. It was remarkable that when using high pixel sensitivity (25-16 nm/pixel) we could discriminate discrete molecular clusters near the diffraction limit (∼250 nm). In summary, proper usage of PMT sensitivity helps revealing more subcellular structures using a multi-color laser-scanning fluorescence microscope.