Basic Science Imaging Platform

Confocal microscopy allows one to capture images with as little out-of-focus blur as possible, thereby generating optical sections through specimen stained with fluorescent dyes. In principle, this is achieved by light routed through pinholes and at these apertures the out-of-focus light (having slightly different focal lengths than in-focus light) is blocked. An image is generated by scanning the point of illumination through an optical plane and stacks consisting of consecutive optical sections in z-direction can be used to digitally generate volume data.
Confocal microscopy is used by several platform members to study cell monolayers, tissue sections, whole or dissected chicken and mouse embryos.


Brief technical description

Most fluorescently labelled specimen can be recorded by confocal microscopes. Besides fixed specimen, live cells can also be recorded when expressing fluorescent proteins. Molecular dynamics can be measured using methods like fluorescence recovery after photo bleaching (FRET) or förster/fluorescence resonance energy transfer (FRET). Volume data can be easily generated using confocal microscopy for qualitative evaluation. Absolute quantifications however, rely on proper image restoration algorithms (deconvolution) to compensate for the unequal image resolutions in x-y and z direction inherent to confocal microscopy. (C Schöfer, Jan 2012)