USER GUIDE
IncuCyte ZOOM Live-Cell Analysis System
®
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IncuCyte™ Coincident Object Analysis User Guide
Coincident Object Analysis is a new software feature in
v2016A enabling the user to quantify the extent of the
overlap between green and red fluorescent objects
within each image. The tool calculates both the
number and area of the overlapping objects, enabling
discrimination between single or dual labeling.
Coincident Object Analysis is useful for a wide range
of biological applications, including measurements
of co-expression of different genes or fluorescent
biomarkers, cellular localization of proteins, and
internalization assays.
Figure 1. Selectively quantify red, green or co-labelled
(yellow) fluorescent objects for labeled cells. Total
fluorescence and the level of coincidence (overlap) are
represented by the graph.
TOTAL
GREEN
TOTAL
RED
22
19
Total cells: 30
(11 green only + 8 red only + 11 overlap)
Coincident Object Analysis Workflow
Green-labeled only: 11
RED & GREEN OVERLAP: 11
Red-labeled only: 8
1) Create a suitable image mask for
the GREEN fluorescent objects –
filter out objects that you do not
wish to include in the Coincident
Object Analysis.
2) Create a suitable image mask for
the RED fluorescent objects –
filter out objects that you do not
wish to include in the Coincident
Object Analysis.
3) Click the Overlap command tab.
Check the ‘Analyze’ box. (“Unused” changes to “Analyze”)
4) The software will create a 3rd mask
(Overlap) based on the overlap of
the red and green image masks.
This overlap mask can also be
filtered for size (to eliminate small
or large objects) if required.
5) Run the analysis job; the mask and
overlap metrics can be visualized
and plotted in the IncuCyte™
software in the same ways as other
channel metrics.
A
B
C
Figure 2. Discriminate labeled populations
with different levels of coincident fluorescence
using Coincident Object Analysis. Each example
yields identical values from the green and red
fluorescent channels, however the coincidence
varies greatly.
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USER GUIDE
IncuCyte™ Coincident Object Analysis
essenbioscience.com/IncuCyte
Figure 3. Worked example: BHK-21 cells were simultaneously cotransfected with two foot-and-mouth disease virus (FMDV) replicons
containing either a GFP (green) or mCherry (red) reporter cassette in
place of the structural proteins (as described previously, Herod et al,
JGV, 96:3507-6518). Coincident object analysis was used to quantify the
proportion (area metric) of cells expressing one (green or red) or both
(yellow) of the replicons over time. This approach can be used to study the
interactions of cells containing two replicons.
Data courtesy of Dr. Morgan Herod (University of Leeds).
A
Panel A
Both green and red FMDV replicons
were genetically identical (except
for the reporter). Here, a subset
of red cells also contained green
replicons (yellow).
48%
33%
19%
Panel B
Both replicons are genetically
distinct with expression of the
red replicon dependant on the
presence of the green replicon.
at t=6h
B
48%
33%
19%
90%
10%
Frequently Asked Questions
Q: What metrics does COA return?
A: The coincident objects are quantified by count (number of
objects) and area (µm2). As with other object metrics, count
data can be displayed as units per image, per well or per
mm2. Area metrics can be displayed as units per image or
units per well.
Q: I cannot see any intensity metrics in the Overlap channel.
Why is that?
A: The Overlap mask does not take into account the intensities
of the green or red fluorescent channels, but simply quantifies
the overlay of the masked areas (area or count). The individual
red or green fluorescent masks can be adjusted for object
intensity thresholds – this will change the masked area in that
channel as well as the related COA mask.
Q: Can I export the COA metrics into other programs, e.g.
Microsoft Excel?
A: Yes, the COA (Overlap) metrics can be exported using the
same method as for all other metrics.
Q: Can I use COA to calculate the percentage of green objects
that are red, or vice-versa?
A: Yes, the overlap metrics can be normalized to the individual
fluorescent channels to calculate a % value. This feature is
not available within the IncuCyte software - the data must first
be exported to an analysis package such as Microsoft Excel to
make this calculation.
Q: Can I reference either of the fluorescent channels to the
phase channel and measure COA for phase contrast/green
or phase contrast/red?
A: Not currently. While the phase contrast channel is not available
for the overlay analysis, the phase metrics may prove useful for
normalization (cf Q4 above).
Q: Is there anything I need to be aware of when comparing
the time-courses of green and red fluorescent objects?
A: The COA metrics allow quantification of the relationship
between the time-courses of the green and red fluorescent
channels (e.g. when did red objects appear and coincide with
green objects).
However, the user should be aware that differences in
the masks of the individual channels can create apparent
differences in the time-courses – the impact of this is
amplified when two masks are compared to return the COA.
Care should be taken (1) in ensuring that the fluorescent
masks faithfully describe the fluorescent objects, and (2)
when interpreting small differences in time-courses across
fluorescence channels.
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