Resources

Education in Microscopy and Digital Imaging

Spectral Imaging

Spectral imaging coupled to linear unmixing is becoming an important staple in the microscopist's toolbox, particularly when applied to the elimination of autofluorescence and for FRET investigations. Instruments equipped for spectral imaging are becoming increasingly popular and many confocal microscopes now offer this capability. Widefield fluorescence and brightfield microscopy are also being used more frequently for resolving complex fluorophore and absorbing dye mixtures, a trend that should continue into the future.

Spectral Imaging

Introduction to Spectral Imaging and Linear Unmixing

Spectral imaging combined with linear unmixing is a highly useful technique that can be used in combination with other advanced imaging modalities to untangle fluorescence spectral overlap artifacts in cells and tissues labeled with synthetic fluorophores that would be otherwise difficult to separate.

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Spectral Imaging

FRET Microscopy with Spectral Imaging

In FRET applications, spectral imaging relies on excitation of the donor alone, followed by acquisition of the entire emission spectrum of both the donor and acceptor fluorescence. Spectral imaging is very useful in the examination of fluorescent protein FRET biosensors.

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Spectral Imaging

Practical Aspects of Spectral Imaging

Among the most important considerations for spectral imaging and linear unmixing is to note that the success of this technique depends upon a number of factors that are within the control of the microscopist. This section outlines critical aspects and artifacts with spectral imaging and linear unmixing.

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