Laser Scanning Confocal Microscopy (LSCM)

Laser scanning confocal microscopy (LSCM) is a technique for obtaining high resolution optical images by scanning laser light across a sample surface and collecting either the reflected light or, more importantly, the fluorescence emission point-by-point with a photomultiplier tube. The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the plane of interest, a whole image is obtained point-by- point and line-by-line. The brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The key principle in LSCM is the use of spatial filtering techniques to remove out-of-focus light or glare in samples whose thickness exceeds the immediate plane of focus. This results in a large improvement over widefield optical microscopy in terms of image quality (both laterally and in the z-axis direction), and in the capability to collect serial optical sections (z-stacks) allowing for 3-dimensional reconstructions of topologically complex specimens.