Spectral Unmixing

Spectral unmixing is a confocal microscope application that can be used to separate dyes or fluorescent proteins with overlapping spectra.



Above image showing spectral bleed through  with DAPI and Alexa 488              Image after spectral unmixing using  Nikon A1R.

DAPI and Alexa 488 emission spectra overlap. Simultaneous excitation of these two dyes will lead to spectral bleed through.

Over the last past decade a variety of new fluorescent dyes and proteins have become available for microscopy applications. This  make it much easier to find appropriate dyes that  minimize spectral crosstalk. However situations do arise where dye or protein do overlap  and in these cases spectral unmixing is a powerful application that can be used with on our confocal microscope systems. It can also be applied to samples where auto fluorescence is causing problems.( tissue samples)
The new Zeiss 710 confocal microscope is equipped with a QUASAR detection unit. This unit can acquire an entire lambda stack with a single scan. The benefits are fast lambda stack acquisition and minimal laser exposure of the sample. Using this detector unit overlapping dyes can be easily unmixed.


Above QUASAR detection L.Boland                                     Lambda stack  acquired on the Zeiss 710                                          Unmixed Image


Introduction to Spectral Imaging and linear Unmixing(Zeiss)













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Copyright 2009 Beatson Advanced Imaging Reaource
Last modified: 02/25/16