Instrument: Nikon Eclipse upright microscope with A1-Si Confocal Microscope
In this technique an object is scanned using a laser beam to build up the image a line at a time.
The microscope works in two basic modes; epifluorescence and reflection. In epifluorescent mode, the laser excites fluorescence within the sample, this is either natural fluorescence (autofluorescence) or by means of a fluorescent dye which has been attached to a specific site on the sample using an antibody label. We can also use fluorescent dyes which are non-specific. Using different dye and laser combinations, we can simultaneously label different parts of the sample to differentiate specific features in 3D.
In reflection mode, the laser is simply reflected off surfaces within the sample. The microscope can also combine epifluorescent and reflection mode images.
Using either method, the returning light rays are fed through a pinhole which excludes out of focus information. This means that only the focused image plane is viewed.
Sequential image planes can be scanned and built up into a stack of images. These can be manipulated to provide extended focus images and 3D images, or the image stack can be rotated to provide views from tilted viewpoints or at right angles to the plane of section.
The system includes 4 lasers at 405nm, 488nm, 561nm and 640nm with 4 PMT detectors, a 32-channel spectral detector with 2.5nm, 6nm or 10 nm diffraction grating and a transmitted light detector.
Image Output: Up to 4k x 4k pixels TIFF images with up to 4 fluorescence channels and one transmitted light channel simultaneously.