Countess
™
II Automated Cell Counter User Guide
37
Set nominal focus
Overview
Nominal focus is the Z-point (i.e., depth) around which the auto focus function
searches to provide fine focus to the sample.
The auto focus algorithm of the Countess
™
II FL Automated Cell Counter is
designed to highlight the differences between live and dead cells in the brightfield
channel. The optimal focus level is where the “live” cells have a light colored center
and the “dead” cells are dark throughout (see examples below).
Focus is not optimal
Focus is optimal
To enable optimal auto focus functionality, you may need to initially refine the
brightfield focus by adjusting it manually and then setting the nominal focus. This
allows the auto focus function to have a set point from which to focus on the cells
in subsequent samples.
Set nominal focus
1.
Prepare the sample by adding 10 μL of cell suspension to 10 μL of 0.4% trypan
blue stain. Mix the sample mixture well by pipetting up and down a few times.
2.
Load 10 μL of the sample mixture into the Countess
™
Cell Counting Chamber
Slide (page 15). Let the sample mixture settle for 30 seconds to ensure a uniform
focal plane.
3.
Insert the sample slide into the slide port of the instrument, making sure that
the side containing the sample is inserted completely.
4.
When the slide is inserted, the instrument automatically illuminates the sample,
sets the intensity of the brightfield light source, and auto focuses on the cells.