Confocal scanning light microscopy with high aperture immersion lenses

TLDR

The scanning approach makes all possibilities of electronic image processing available in light microscopy. The study investigates the imaging characteristics of a confocal scanning light microscope with a high‑aperture, immersion lens (NA = 1.3). In the confocal arrangement, illumination and detector pinholes are aligned at a common point through which the object is mechanically scanned. Results show a 1.4‑fold improvement in response over standard microscopy, low side‑lobe intensity and no glare enable high‑resolution imaging of weak details near strong features, and apodization with an annular aperture yields an additional 1.25‑fold improvement in point resolution.

Abstract

SUMMARY The imaging characteristics of a confocal scanning light microscope (CSLM) with high aperture, immersion type, lenses (N.A. = 1·3) are investigated. In the confocal arrangement the images of the illumination and detector pinholes are made to coincide in a common point, through which the object is scanned mechanically. Results show that for point objects the theoretically expected improved response by a factor of 1·4 in comparison with standard microscopy can indeed be realized. Low side lobe intensity and absence of glare permits the imaging at high resolution of weak details close to strong features. A further improvement by a factor of 1·25 in point resolution in CSLM is found after apodization with an annular aperture. Due to the scanning approach all possibilities of electronic image processing become available in light microscopy.