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
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
Introduction to Spectral
Imaging and linear Unmixing(Zeiss)