Three-dimensional microscopy using a confocal laser scanning microscope

Abstract

In a scanning laser microscope detecting fluorescent light from the specimen, the depth-discriminating property of confocal scanning has been used to carry out optical slicing of a thick specimen. The recorded digital images constitute a three-dimensional raster covering a volume of the specimen. The specimen has been visualized in stereo and rotation by making look-through projections of the digital data in different directions. The contrast of the pictures has been enhanced by generating the gradient volume. This permits display of the border surfaces between regions instead of the regions themselves.

Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets

“…The fine details of protein storage and interaction can therefore not be fully resolved in conventional optical imaging techniques such as wide-field or confocal microscopy, where the resolution is limited to a few hundreds of nanometers. 1 Recent developments in fluorescence nanoscopy, with techniques such as STED 2,3 , PALM 4 and STORM 5 offering an order of magnitude higher resolution, now drastically improve the means of overcoming these limitations. However, these super resolution techniques put higher and more specific demands on the optical system as well as on the fluorescent probes.…”

Automated Motile Cell Capture and Analysis with Optical Traps

“…Later, stage scanning confocal microscopes using laser light sources and nonimaging (photomultiplier tube, PMT) detectors were developed (Wilson, 1980;Wilson et al, 1980) for scanning electronic devices. In the 1980s, laser scanning confocal microscopes (LSCMs) (Carlsson et al, 1985(Carlsson et al, , 1987 came into the forefront in which confocal fluorescent images of biological specimens were acquired by steering the laser across a stationary specimen. It was apparent that the out-of-focus light rejection and optical-sectioning capability of LSCM over conventional image collection permitted the visualization of individual biological structures never before visualized in fluorescence (White et al, 1987).…”

“…As a result, fluorescence is emitted and subsequently collected by the objective lens from the true focal plane, as well as from out-of-focus objects. In LSCM, laser light is focused into the specimen and excites fluorescence, which passes through a dichroic filter and is reimaged onto the detection pinhole which is placed ''confocal'' to the laser focus spot (Carlsson and Aslund, 1987;Carlsson et al, 1985). As a result, fluorescence originating from the laser focus passes through the detector pinhole, while that originating from out-of-focus regions does not.…”

How the Confocal Laser Scanning Microscope entered Biological Research

“…Although we were unaware of it until quite late, a parallel development had been going on in the Institute of Physics of the University of Stockholm (Carlsson et al 1985). This resulted in a commercial system, launched almost simultaneously with ours.…”