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Chapter 5: Configuring Plate Acquisition Options
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Option Description
Laser and
Image Displays for the first acquisition wavelength (W1) and only if you select Enable
Laser-Based Focusing and Enable Image-Based Focusing on the Acquisition
tab. If selected, the laser autofocuses first, and then image-based focus fine
tunes the focus. You must specify values for the following:
Image-Based Range +/- µm specifies the search range for the image-based
portions of autofocus.
Max. Step specifies the maximum step size of a single Z move in µm that is
required to get the correct focus position. This setting depends on the
objective. Because the focus peak is narrower, use a smaller step size with
higher NA objectives.
If you select Custom Exposure Time on the Autofocus tab, with Laser and Image
selected, specify a value for the following:
Exposure (ms) specifies the exposure time in milliseconds for the acquisition
wavelength when autofocusing.
Laser with
Image
Recovery Displays for the first acquisition (W1) wavelength and only if you select Enable
Laser-Based Focusing and Enable Image-Based Focusing on the Acquisition tab
and you select Allow Image-Based Focusing for Recovery from Laser-Based Well
Bottom Failures on the Autofocus tab.
When selected, specify the following options:
Image-Based Range +/- µm specifies the range to use for the image-based
portions of autofocusing.
Note: Adjust the range based on the sample variability. A larger range
requires a longer time to focus.
Max. Step specifies the maximum step size in µm of a single Z move to use to
get the correct focus position. This setting is dependent on the objective.
Because the focus peak is narrower, use a smaller step size with higher NA
objectives.
Note: Smaller step sizes require more steps to arrive at the final focus
position. Increasing the number of image autofocus steps increases the
chances of photobleaching or phototoxicity.
If you select Custom Exposure Time on the Autofocus tab, and you select Laser
with Image Recovery, specify a value for the following:
Exposure (ms) specifies the exposure time in milliseconds to use for the
acquisition wavelength when autofocusing.
Z-Offset from
W1 Available for all acquisition wavelengths other than W1. The offset for W1 must be
calculated before you can set this value. Select this option to move the W1 focus
position to the offset. Specify the post-laser offset in µm in the Offset field or
click Calculate Offset to calculate the offset for all subsequent wavelengths.
Table 5-8: W# Tab: Autofocus Options (options vary based on Acquisition tab selections)
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