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mouse. Alternatively, it is possible to zoom directly to a specific area within
the image by scrolling the mouse wheel over the area of interest.
The Tool task pane also includes a measurement tool. Use Measurement
Mode to make linear measurements within the image. To turn
Measurement Mode on or off, click on the ruler icon
.
Spectral Unmixing
Spectral unmixing may be required to remove fluorophores that produce
signal in both color channels. These properties should be saved prior to
adding images to an image collection or performing an analysis.
NOTE
: It is important to perform the spectral unmixing in small increments to
determine the appropriate value. Figure 15 below illustrates spectral
unmixing.
Top Row:
Appropriate removal of the red fluorophore from the green channel using the spectral unmixing tool
.
Bottom Row:
We lowered the green fluorescent channel to show the difference between optimized and
overcorrected spectral unmixing. Using the NucLight-Red A549 cell circled in yellow as a guide, the red
fluorophore is visible in the green channel with an average Green Calibrated Unit (GCU) of 7.5 and an average
Red Calibrated Unit (RCU) of 11.2. Optimization of the red fluorophore in the IncuCyte™ ZOOM showed that an
8% spectral unmixing of the red removed from green was required [GCU - (RCU x 0.08) = amount of red signal
removed from the green channel]. If too much of the red signal is removed from the green channel,
overcorrected pixels will develop, giving the appearance of “holes” within the image. Overcorrection of spectral
unmixing may affect assay metrics.
Figure 15.
Spectral unmixing of a mixed population of A549 cells expressing NucLight Green or Red
