Behavior of the point-spread function in photon-limited confocal microscopy.

Abstract

We study the behavior of the point-spread function (PSF) of the confocal scanning optical microscope (CSOM) when the available optical energy density from the sample plane is low (<7.5 microJoule/micrometers2). The PSF profile is analyzed under three photon-limited imaging conditions: (1) reflection-type CSOM with a weak source and a perfectly reflecting sample, (2) reflection-type CSOM with a strong illumination source and a weak sample, and (3) fluorescence CSOM with a weak fluorescent sample. Linfoot's image quality criteria of fidelity, structural content, and correlation quality are used to assess the reproducibility of the PSF profile as a function of the photon number. Low photon numbers yield a PSF profile that is difficult to maintain from one location in the sample plane to another. The optical sectioning capability of the CSOM was found to deteriorate more quickly against light power reduction than its transverse resolving power. The signal-to-noise ratio of the scanned CSOM image improves exponentially with the photon number from the sample plane. The noise that is generated by an unstable PSF has an average amplitude that decreases exponentially with the photon number and is significant only at low photon numbers. The CSOM image quality deteriorates because of spurious high-frequency components, degradation in the PSF dynamic range, and varying resolving power.