Spectral imaging uses a computational approach to visually separate overlapping fluorescent spectra.  This is accomplished in several ways by utilizing"spectral fingerprinting" and/or "linear unmixing".  In most methods, a "lamda series" is collected by scaning the sample with one or more lasers, but the emission light from every pixel is spread into its component wavelengths using an optical grating or prism.  With the Zeiss Meta system, every 10.7 nm of wavelength is sent to a separate PMT.  A maximum of 32 channels, over a range of 342 nm can be displayed.  Up to eight channels can be acquired simultaneously. 

In the illustration to the left, each space between the red lines represents an image collected at a specific 10 nm-wide wavelength band.  Four of the five "lamda" images would result from the contribution of two separate fluorescent dye spectra, while the last would be from only one dye. 

Image intensity is a linear combination (mixing) of all contributing fluorescent components.  Linear unmixing uses algebraic algorithms to separate the contribution of each component.   The algorithm uses weighting based on spectral characteristics of each dye involved.  Ideally, those characteristics are extracted from landa scans of samples known to contain just one fluorophore. This routine enables the system to identify how much each source contributes to the overall signal at each pixel.

Spectral fingerprinting means that the spectral profile of a dye has been defined from a sample using the Meta system.  Control samples with a single stain, as well as an unstained sample, are examined to define the spectra of the individual dyes and autofluorescence.  These profiles reflect the influences on the spectra contributed by the conditions existing within the specimen.  Once these profiles are saved, they can be retrieved to identify and separate spectra in multi-stained samples.  In addition, autofluorescence can be either removed or included as an additional channel.  All this is done using Linear Unmixing, the most accurate method of spectral imaging.

Alternatively, spectra can be captured after a lamda scan of a multi-stained sample if the dyes are known to be spatially segregated.   This can be accomplished manually, using theACE routine, or the Multichannel Unmixing option.  In the case of ACE, the system automatically identifies the most likely structures emitting a single dye spectrum, constructs profiles, then applies those profiles to the entire image.  Multichannel Unmixing can use a lamda scan or a single image, and is the least accurate method, especially when autofluorescene is significant.

Finally, the Meta detector can be used as a customizable emission filter tool.  The user can set the band width of up to 8 channels.  When used in the Meta Tracking in Frame mode, each channel has its own gain and offset control.