Automatically quantify red and green fluorescence overlap. Selectively analyze co-labelled objects and measure changes in fluorescence coincidence over time.
The IncuCyte® Coincident Object Analysis tool enables calculation of the total number, and area, of overlapping objects. Example applications include:
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).
Q: What metrics does COA return?
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 normalised 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. Whilst the phase contrast channel is not available for the overlay analysis, the phase metrics may prove useful for normalisation (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
New fluorescence analysis tools for co-culture scratch wound assays
Improved scheduling flexibility using the new Restore feature
|Features and Functionalities||Version|
|IncuStore™ Expansion Data Storage Compatibility|
|Whole Well Imaging|
|Points of Interest (POIs) - Digital Monitoring Tools|
|Automated vessel marking - IncuCyte ZOOM® Marking Tool|
|35mm Dishes, 6-, 12-, 24-, 96-Well Enabled|
|New Movie Formats (MPEG-4, AVI uncompressed)|
|Improved Scheduling Flexibility|
|Fluorescence Analysis Tools for Co-culture Scratch Wound Assays|
|Enhanced IncuCyte® NeuroTrack Fluorescence Tools|
|Enhanced autofocus and additional plate support for single spheroid applications in round bottom ULA plates|
|Background Fluorescence Subtraction|
|Fluorescent NeuroTrack™ Analysis|
|Import/Export Image Processing Definitions|
|Coincident Object Analysis|
|IncuCyte ZOOM® Rack System|
|Space Saving Rack - Holds 2 instruments and 2 IncuStore™ Units|
|Uninterruptable Power Supply|
|'In The Lab' Access to User Interface|
|Cell Culture QC|
|Immune Cell Killing|
|Immune Cell Proliferation & Clustering|
|Neurite Dynamics - Label-free*|
|Neuronal Co-Culture - Fluorescence*|
|Proliferation - Cell Count|
|Proliferation - Confluence|
|Scratch Wound Migration*|
|Stem Cell Monitoring & Reprogramming|
* additional software module required