Spatial resolution of weakly reflecting objects in confocal optical microscopy

Abstract

Imaging with a confocal scanning optical microscope (CSOM) allows for high contrast and high spatial resolution, depth discrimination or optical sectioning and rejection of scattered light. Such optical systems can be characterized by its point spread function (PSF) and the resolving power can be related to the full width at half maximum (FWHM) of the effective PSF. For a CSOM, the PSF is obtained from the product of the PSF of the two imaging lenses in a confocal arrangement. These classical resolution criteria can be achieved only for very high signal-to-noise ratio (SNR) images. The image resolution, even for a well-corrected lens, is ultimately limited not by diffraction but by the SNR. A good survey of resolution can be found in J. Opt. Soc. Am. A 14, 547 (1997).

The SNR, and consequently the image resolution, is determined by random and systematic errors in the detected image. In a CSOM, image resolution is further by the loss in sensitivity and photon count due to the size of the pinhole at the detector and source. The pinhole leads to the rejection of out of focus rays in the system and this property results in the unique optical sectioning capability of a CSOM. The enhanced image resolution gained from the confocal configuration of the microscope is offset by the reduced photon count at the detector plane. The quantum efficiency of the photo-detector will also affect the number of photons contributing to the detected image. For our investigation, we shall consider a very fine array of photodetectors with quantum efficiency η at the detector plane. In practice, η is lower than unity and contributes further in lowering of the intensity of the detected light. In addition, a weakly reflecting object, or similarly, a highly absorbing one, will limit the photons that will arrive at the detector pinhole.

In this paper, we show the behavior of spatial resolution of a weakly reflecting object in a confocal setup. We discuss the numerical model we used and the instrumentation geometry of the confocal system. The results of the simulation are presented, and a summary and discussion are given, including some possible applications of non-classical light in photon-limited imaging.