Optimizing the performance of confocal point scanning laser microscopes over the full field of view

Abstract

Summary To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.

Dendritic cells from CML patients have altered actin organization, reduced antigen processing, and impaired migration

“…14 The specimens were illuminated with nonsaturating levels of the 543 nm line from a HeNe laser. The fluorescent emissions were filtered out with a 560 nm long-pass filter where the 12-bit gray scale values in the final image data sets were proportional to the number of photons that emanated from the specimen.…”

RhoE Regulates Actin Cytoskeleton Organization and Cell Migration

“…Images of cells were visualized with an MRC 500 confocal visualization system (Bio-Rad) or with an LSM 310 apparatus (Zeiss, Welwyn Garden City, United Kingdom) mounted over an infinity-corrected Axioplan microscope by using a ϫ10 eyepiece and a ϫ63 NA 1.4 oil immersion objective (Zeiss). The MRC 500 was configured and used as described previously (15). Image files were collected with the LSM 310 as a matrix of 1,024 by 1,024 pixels describing the average of eight frames scanned at 0.062 Hz, where FITC and TRITC were excited at 488 nm and 543 nm and visualized with bandpass filters of 540 Ϯ 25 and 608 Ϯ 32 nm, respectively, where the levels of interchannel cross talk were insignificant.…”

Application of a laser scalpel to sectioning fragile tissues prior to optical sectioning microscopy and 3D reconstruction

“…giving final power densities of 3 × 10 7 to 9 × 10 7 W m −2 . These slices, which were not further treated or stained in any manner, were then visualized with an LSM 510 confocal microscope and visualization system fitted with a ×10 NA 0.3 Neofluar air objective (Zeiss, Germany) aligned as described previously (Entwistle & Noble, 1994). The specimens were alternately illuminated with non‐saturating levels of the 488 nm line of an argon ion laser and the 543 nm line of a HeNe laser and the autofluorescent emissions from the tissue were collected through a 505–550 nm band pass filter or a 560 nm long pass filter, respectively, such that the grey scale values in the final image data sets were proportional to the number of photons that emanated from the specimen (Bögler et al ., 1993).…”