Zeiss LSM510 META

	Fluorescent Emission Fingerprinting on Zeiss META Transients of calcium ions in the salivary glands of the blow fly. Video clip reproduced with the kind permission from Carl Zeiss, Inc. Conventional fluorescent microscopes rely on combination of filters to allow only the desired wavelengths to reach the camera or PMTs. The increasing number of fluorophores used by cell biologists inevitably narrows the gap between emission peaks of various fluorescent molecules. This means that many commonly used fluorophores have overlapping spectra, contributing to crosstalk between channels used in regular fluorescent microscope. Investigators who routinely examine thick tissue samples using fluorescent microscopy will notice the tremendous background autofluorescence from tissues. These problems pose great challenge to the successful and accurate interpretation of fluorescent imaging data. The recent breakthrough in the technology of fluorescent emission fingerprinting provides a solution to these problems. The capability of the META module, fitted on the LSM510 laser scanning confocal microscope, to detect the full spectra of multiple fluorophores simultaneously means that users can generate a library of reference spectra, such as those shown below. These spectra will include the distinct spectral signature from tissue background autofluorescence. Please consult the page on technical tips for dealing with tissue sample autofluorescence. In addition to Z-stack, LSM510 META is also capable of generating a lambda stack with spectral distributions of fluorescent emissions. In the example above, the lambda stack displays four different color in a mixture of fluorescent beads. The users can then make and store reference spectral library by first identifying the region of interest (ROI) in the pictures, the spectra of which will be extracted in a spectral read-out as shown below: Finally, by performing a function called linear unmixing, users can separate these highly overlapping spectra (even GFP and FITC) into different channels. The ability to spectrally establish emission characteristics is a critically important breakthorugh for researchers who need to use immunofluorescence in tissue with severe auto-fluorescence background. The autofluorescence can now be extracted from the rest of the useful signal, thus allowing accurate quantification of fluorescent intensity. An example of the practical use of META is shown below:
		Total fluorescence from tissue biopsy sample. Sample was stained with TOTO (green nuclear stain), and Cy-5 labeled anti-Ki67.
		Combination of all fluorescence after linear unmixing of every emission fingerprints by Zeiss META system.
		Pure TOTO stain in the sample, separated from all tissue autofluorescence.
		Ki-67-positive nuclei, separated from the TOTO signal, as well as tissue autofluorescence.
		Left-over autofluorescence from tissue sample, which is now separated into another channel.