Molecular Devices ImageXpress Confocal HT.ai, User Manual

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Chapter 5: Configuring Plate Acquisition Options
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Option Description Notes
FL
Subtraction
Only Background subtraction is performed
on the images. No shading correction
is performed. Background subtraction
removes stray light that is unrelated
to the sample. The system collects a
reference image for each wavelength
at the start of the plate acquisition and
then subtracts this image from the
acquired images. Not recommended for transmitted
light images.
Not recommended for time lapse
assays of dim samples.
Not recommended for Z-stack
assays where 3D reconstruction is
used.
Not recommended for use with oil-
immersion objectives.
Low magnification objectives may
produce an artifact as it may catch
the edge of the plate in the
reference image.
Incorrect instrument configuration
could produce a stage error.
You should use the Auto Expose
tool when you optimize exposure
times or temporarily disable the
shading correction if you use snaps.
Turn back on to run Plate
Acquisition.
Subtraction is not available for
water immersion objectives.
FL Shading
Only Images are corrected for shading
automatically. The shading correction
is tuned for fluorescence and it
adjusts for uneven illumination of the
sample. An in-focus reference image
for each wavelength is collected at
the beginning of the plate acquisition.
This reference image is used to
calculate the shading pattern per
wavelength and then applied over the
plate. The same reference image is
used for all time points in a time
series. Recommended for most fluorescent
assays.
Not recommended for transmitted
light images.
Use if Auto Correction for FL is
producing an artifact from catching
the edge of the plate in the
reference image.
Table 5-10: W# Tab: Shading Correction Options (continued)