Confocale fluorescentiemicroscopie

A confocal laser scanning microscope or CLSM uses a laser and a series of pinholes to section a sample into thin optical slices. These slices can be compiled to reconstruct a sharp, detailed image.

Unlike traditional fluorescence microscopes where the entire field is illuminated, CLSM uses a laser beam to restrict the excitation of fluorophores to a single point at a given time in a thin section of the sample. This results in a slower loss of fluorescence by photobleaching.

An illumination pinhole focuses the light, and an emission pinhole allows light only from the focused focal plane to pass through to the detector, reducing background fluorescence and blurriness in the images.

The laser scans the entire sample repeatedly at different focal planes along the Z-axis, capturing the images to generate a series of optical sections.

These optical sections can be used as a two-dimensional image or compiled together as a Z-stack using image-processing software for reconstructing a high-resolution, three-dimensional image.