Invitrogen EVOS M5000 - Measure Confluence; Confluence Tool

5.
To delete a tag, right-click on the tag you wish to delete
6.
To delete all tags for a label, check the
Delete box for the label, then click the Trash button.
7.
To delete all tags for all labels, check the Delete
boxes for each label, then click the Trash button.
8.
When
finished with the count, save your count results (see “Save analysis results” on page92).
Measure confluence
Confluence
tool
Confluence is a measure of how densely cells are distributed in culture. When all available growth area
is utilized in a culture vessel and the cells make close contact with one another, the culture is at 100%
confluence.
The Confluence tool measures the percent area covered by cells in the image, which is required to
calculate transfection eciency.
Guidelines for confluence measurements
•
We recommend that you visualize your cells using transmitted light and a phase objective with 4x
to 10x magnification. Set the phase ring to Oly 4x (for Olympus
™
4x phase objective) or 4x/10x
(for
EVOS
™
phase objectives) using the phase ring selector (“Front view” on page13).
•
If you plan to calculate the transfection eciency after measuring the confluence, capture the
image in both the tr
ansmitted light channel and the fluorescence channel for the marker that your
cells are expressing.
•
For analysis, only use 16-bit image files (TIFF or PNG), which contain the full dynamic range and
metadata needed for quantitative analysis.
•
Increasing the number of targets and background areas improves accuracy. You can select up to 5
target areas and 5 background areas.
•
The Confluence tool uses a texture and intensity-based algorithm. The sensitivity slider adjusts
the algorithm sensitivity to pixel intensity (higher intensity = more pixels included). Decreasing the
sensitivity reduces the confluence value.
•
Dierent cell types have dierent confluence “patterns”, and variability in morphology and contrast
can influence the absolute confluence measurement between dierent cell types. However, within
a given cell type, you can optimize the reproducibility of your measurements. Reproducibility in
confluence measurements is more important than absolute percentages.
Measure confluence
1.
In the transmitted light channel, adjust lighting and focus on the sample, then click Capture to
acquire an image of your culture.
Note:
If you plan to calculate the transfection eciency after measuring the confluence, capture
the image in both the tr
ansmitted light channel and the fluorescence channel for the marker that
your cells are expressing.
Chapter5Measure, annotate, and analyze captured images
Measure confluence
5
54
EVOS
™
M5000 Imaging System User Guide