Spectral cytometry is a new approach to
classical cytometry.
Where a detector used to correspond to a color, the spectral
cytometer collects the fluorescence of small spectral bands for
each of the lasers present in the instrument.
The spectral deconvolution allows to decompose the spectrum of
light emitted by each cell into each of the spectral components
from each fluorochrome present on the cell, whatever the number of
fluorescent molecules (Figure 1).
Figure 1 : For each laser, the spectrum is split into small
spectral bands allowing to calculate a specific spectrum for
each analyzed fluorochrome: on the left, the spectrum is split
for the blue laser; on the right, the spectrum is split
globally on 5 lasers.
(Front. Mol. Biosci., DL Bonilla, G Reinin, E
Chua)
Before any analysis by
spectral cytometry, we need to extract the spectra of the
fluorochromes used one by one.
For this purpose, we need to analyze:
1) Totally unlabeled cells. The autofluorescence of the
cells is indeed considered as a fluorochrome. It is
therefore possible to evaluate the autofluorescence spectrum
and subtract it from the fluorescence signals.
2) All individual monolabeling. The preparation of the
monolabel must be identical to the sample (dissociation,
washes, permeabilization, fixation...). In case of
uncertainty on the presence of a marker on your cells (or
lack of cells for controls) and in order to make the
extraction of the spectrum, it is necessary to provide beads
that bind your antibodies. The beads must undergo the same
treatments as the cells. There are also specific beads for
viability markers.
Once the spectral deconvolution is done and specific spectra
of your fluorochromes are obtained (Figure 2), the
cytometric analysis can finally start in a classical way
with parameters reduced to the number of fluorochromes to
analyze.
Figure 2 : Example of spectrum calculated after spectral
deconvolution of PE on a Cytek Aurora 5-laser spectral
cytometer
This
technology allows for a finer analysis of fluorescence and
makes it possible, for example, to detect in the same tube
molecules that are totally indistinguishable with conventional
cytometry. The technique allows the simultaneous measurement
of fluorochromes that have up to 98% similarity (Figure 3
and 4).
Figure 3 : left: GFP and YFP spectra (indistinguishable
on a conventional cytometer); middle: GFP and YFP spectra
after spectral deconvolution on the Cytek Aurora (74%
similarity); right: simultaneous GFP and YFP analysis on
the Aurora
Figure 4 : Left: APC and Alexa 647
spectra (90% similarity); right: comparison of a double
APC, Alexa 647 labeling with the same APC, PE labeling.
The percentages of detected populations are similar.
The other interest of spectral cytometry is to be able to
subtract the background noise of the analyzed cells on each of
the measured channels allowing to detect specific
fluorescences which would be drowned in the background noise
on a traditional cytometer (Figure 5)
Figure 5 : Analysis of specific mCherry labeling in a
population with high 'autofluorescence'.
