Nuance
Spectral Unmixing System:
The
Nuance spectral unmixing system is a generous gift of Dr. Charles
V. Clevenger, Diana Princess of Wales Professor of Cancer Research
and Pathology
his Conventional
fluorescent microscopes rely on combinations of filters to allow
only the desired wavelengths to reach the camera or PMTs. The
increasing number of fluorophores and tissue stains used by
cell biologists inevitably narrows the gap between emission
peaks of various fluorescent molecules. This means that many
commonly used fluorophores/stains have overlapping spectra,
contributing to crosstalk between channels used in regular fluorescent
microscopes. 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 Nuance multispectral imaging system, like the META module
on the LSM510 laser scanning microscope (link to META sub),
is capable of simultaneously detecting the overlapping spectra
of multiple fluorophores or tissue stains in a thick section
sample. It can generate a library of component spectra, including
spectra of tissue background autofluorescence, then “unmix”
or separate each spectrum into an individual channel. In contrast
to the LSM 510 META, the Nuance system is ideal for brightfield
imaging of tissues treated with clinical stains, such as hematoxylin
and eosin, and also for fluorescence imaging of tissues stained
with various fluorophores.
The Nuance camera is mounted on a Zeiss Axioskop 50, and is
equipped with white light and mercury arc illumination. Filter
sets are suitable for the excitation and visualization of DAPI/Hoechst,
FITC/ GFP, rhodamine/dsRED, and Cy5 fluorophores.
How it works:
1. A background-
corrected RGB image is gathered.
