To adjust the Autofocus Plate Scanning parameters:
From the Resolve3D window,
click the Experiment
icon to view the Design/Run Experiment window.
Select the Design tab and then select the Plate tab.
Activate the Do Microtiter Scanning check box and click the More Settings button next to the Autofocus Frequency field. The Plate Autofocus Settings window is displayed.
The fields in the Plate Autofocus Settings window are used as follows:
Channel for Autofocus: Select the desired channel for autofocus. You are selecting the EX Filter. The parameters for EM and ND are taken from the last image acquired with that EX filter in the Resolve3D window.
Autofocus Exposure: Set the autofocus exposure time. You will want to use the same or a shorter exposure time than what will be used during the acquisition time in your experiment. In order to make sure that your Autofocus Exposure time is not longer than necessary, test the exposure time using the same binning value in the Resolve3D Window.
Note: You may have to adjust the Resolve3D Autofocus settings using the Autofocus icon in order to find the best focal plane. Remember that the Plate Autofocus Settings are not the same as the Resolve3D Autofocus settings.
Plate Scan Focus Binning: The default is set to a bin of 2x2. It is recommended to use binning for the autofocus for most samples in order to reduce the amount of light exposure to the cells. Please enter a single digit for this value. A Plate Scan Focus Binning of 2 is similar to a 2x2 in the Resolve3D window.
Note: The software is set up so that you can autofocus with a different bin than acquisition. For example, you can autofocus with a binning of 2x2 and acquire data with a binning of 1x1.
Plate
Scan Long Focus Range: This value is the Maximum Z Test Range
for the first image in every well. If autofocusing Once
Per Well, this is the only Focus Range that is used for the
experiment. If autofocusing Each
Panel, this Focus Range is used only on the first panel in
each well. The Plate Scan Long Focus
Range is combined with the Lens’ Depth of Focus in order to
find the best focal plane during the scan. It is imperative that the
correct lens has been selected in the Resolve3D window and that the
Lens has a valid Lens ID.
You cannot change the Z Step Size for the Plate
Scan Long Focus Range. The system will take the same sized
steps no matter the value of the Plate Scan Long Focus Range. Therefore,
a scan of 100 microns will take twice the amount of time when compared
to a scan of 50 microns.
When you are imaging multiple wells, the Plate Scan Autofocus starts
from the best plane of focus from the previously scanned well. It
then searches for the best signal-to-noise ratio in the distance ½
of the value of the Plate Scan Long
Focus Range above that focal plane and ½ of the value of the
Plate Scan Long Focus Range
below that focal plane.
Plate
Scan Short Focus Range: This is the Maximum Z Test Range between
panels in a plate scan. If imaging more than one panel in each well,
this value will be used for each panel after the first one. The Plate Scan Short Focus Range is
combined with the Lens’ Depth of Focus in order to find the best focal
plane within panels during the scan. It is imperative that the correct
lens has been selected in the Resolve3D window and that the lens has
a valid Lens ID.
You cannot change the Z Step Test Size for the Plate
Scan Short Focus Range, which is the same as with the Plate Scan Long Focus Range.
To determine if autofocusing within wells is necessary or to determine
the value for the Plate Scan Short
Focus Range, measure the change in Z throughout one well. Find
the best focal planes in different fields of view within close proximity
in a well and use the Z coordinates to find out the distance between
focal planes. Double the largest difference in Z to get the Plate Scan Short Focus Range.
Note: If the distance between Z planes is not significant, you may want to consider autofocusing Once Per Well only. Running the autofocus once per well instead of per panel will save time for your experiment and will save your sample from the effects of photobleaching and/or phototoxicity.
Design an Acquisition for a Plate