Molecular Devices ImageXpress Confocal HT.ai, Руководство пользователя

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ImageXpress Confocal HT.ai High-Content Imaging System User Guide
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Plate Acquisition Settings
You make most plate acquisition settings in the Plate Acquisition Setup dialog in the
MetaXpress software. The settings are dependent on the content and distribution of the
samples, as well as the requirements of the experiment.
To help you determine the settings, use the following sections as a checklist.
Magnification, see below
Correction Collars, see below
Binning, see below
Widefield vs. Confocal Imaging Mode, see page 59
Site Selection, see page 59
Shading Correction, see page 59
Autofocus, see page 59
Illumination Settings, see page 60
Exposure Time, see page 60
Z Series Acquisition, see page 61
Magnification
The magnification setting depends on the type of information you want to obtain. Generally,
higher magnification gives higher resolution of the objects of interest, but it produces a smaller
field of view, which requires more sites to give the equivalent number of cells. To count cells, a
4x objective is often suitable. A 10X PA objective is often used to acquire spheroid images for
morphology analysis. For translocation, a 20x or 40x objective can be appropriate. Counting or
localizing small organelles might require objectives above 40x.
Other considerations include the numerical aperture (NA) of the objective, the working
distance, and the optical corrections built into the objective. With the magnification constant,
brightness is proportional to the square of NA. Higher NA objectives also produce a sharper
picture when you match the plate-bottom thickness to the objective correction thickness,
typically 170 µm. Unfortunately, higher NA objectives, such as a 40X Plan Apo, fail to reach the
outer rows and columns of some multi-well plates because of the plate skirt height. Molecular
Devices furnishes Plan Apo Lambda objectives that allow acquisition of more of the plate.
Correction Collars
If you use an objective with a correction collar, ensure that the correction collar is set
appropriately for the plate you use. See
Adjusting Objective Correction Collars on page 213 .
Binning
Another method to increase the signal-to-noise ratio (S/N) is to bin the pixels from the camera.
Binning combines the electrons from adjacent pixels to create the effect of a single, larger
pixel. Binning increases the S/N at the expense of decreased resolution. Use binning to
decrease the exposure time dramatically while maintaining the same signal intensity. Another
positive feature of binning is that it produces smaller images that require less storage space
and are analyzed more quickly. Some assays benefit greatly from binning, while others require
unbinned images.