Symptom
Possible Causes
Recomended Solutions
Fluorescence
imagery appears
too dim
Image display settings
are set too low
Increase the image display gain and/or change to log in the appropriate
camera channel.
Sample did not label well
Look at the sample with a fluorescent microscope.
Insufficient illumination
Turn the appropriate lasers on. Set the laser powers to maximum and
decrease them to prevent pixel saturation.
If the probing protocol results in dim staining, sensitivity of the instrument
can be increased by changing the fluidics speed to Lo / Hi sensitivity mode.
Core stream position is
grossly off-center within
the flow cell due to air or
clog in the fluidics
Run the purge bubbles script from the instrument drop-down menu.
more information, see Unstable fluidics (Air or clog in system)
Excitation laser is mis-
aligned
Run calibration particles on the FlowSight
®
Instrument. Load the default
template. Open ASSIST, re-run the laser alignment calibration for the
appropriate laser line, and verify it passes.
Fluorescence is
too bright,
images have a
contrasting color
or appear flat
Image display settings
are set too high
Decrease the image display gain and change to linear in the appropriate
camera channel.
Instrument sensitivity is
set too high
Decrease the excitation laser power to prevent pixel saturation. Saturation
is indicated in the image gallery by pixels colored in a contrasting color
(generally red or white).
Set the brightfield intensity to 800 counts by pressing “Set Intensity”.
The sheath syringe is
empty
Load sheath, then go to the instrument drop down and run prime.
There is a clog or air
bubble in the system
Run the Purge Bubbles script from the instrument drop-down menu.
more information, see Unstable fluidics (Air or clog in system)
One channel sat-
urates while the
others do not
Instrument sensitivity is
not optimized
The best instrument setup maximizes the dynamic range of fluorescence
signal, while at the same time avoiding image pixel saturation (which can-
not be compensated). In general, decreasing the laser powers until no
pixels saturate.
Probing protocol
requires better stain bal-
ance
Reduce the concentration of the stain that produces the saturating signal
so that all probes can be simultaneously imaged without excessive sat-
uration.
Excessive fluorescent
dye is left in the sample
buffer.
Some DNA dyes are required to run with the sample to stain properly, how-
ever if too much dye is in solution it can cause the core stream to fluoresce.
It’s important to balance the concentration of these dyes so that the cells
can be imaged properly. Typically the concentrations in “Current Protocols
in Cytometry” should work.
For Research Use Only. Not for use in diagnostic procedures.
50
Amnis
®
FlowSight
®
Imaging Flow Cytometer User Manual
