Molecular Devices ImageXpress Confocal HT.ai, Руководство пользователя

Страница: 59 / 256

Chapter 4: Preparing for Acquisition
5084773 A 59
Widefield vs. Confocal Imaging Mode
Widefield imaging includes fluorescence from parts of the sample that are above or below the
focal plane. Confocal imaging excludes fluorescence from parts of the sample that are out of
focus. The depth of focus depends on the optics, particularly the objective lens, and the
degree of confocality depends on the size and shape of the hole that is used. Small pinholes
achieve higher confocality and better sectioning capability than widefield, at the expense of
reduced light intensities.
Therefore, if it is important to measure the total intensity of a particular fluorophore in cells, it
might be preferable to use widefield imaging. Widefield imaging also tends to perform better
with cells that are flat. If it is important to measure colocalization between two markers,
particularly in a thick sample, then confocal imaging is recommended. In some cases,
collecting a Z-series of multiple focal planes might also be needed to obtain suitable images
of thick samples.
Site Selection
Use multiple sites in a single well to acquire images from a greater area of the well. If you
select the Single Site option on the Sites to Visit tab, the software acquires an image for only a
single site located in the center of the well. Use the Fixed Number of Sites option to acquire
separate images within a given well, and those images can be of contiguous or distributed
areas.
Use the stitch command to assemble the smaller separate images into a single large image.
Use this to retain image resolution while increasing the image area of coverage. Unless you
use a journal to change settings during the experiment, the sites you select are used during the
entire experiment.
Sites can be used to include specific areas of the wells in the plate, while at the same time
excluding other areas of the well. For example, the center of the well might exhibit a pipetting
artifact from an automated pipettor, or the cells might clump more at the edges of the wells.
Shading Correction
All microscopes exhibit some degree of illumination variation, or shading, across the field of
view. Shading is an artifact that can come from the objective, optics, light source, or
background light from the room. The ImageXpress Confocal HT.ai system is designed to
minimize shading effects and provides a shading correction feature within the software. This is
recommended for assays where comparison of individual cell intensities is critical, such as a
cell cycle assay. See
Table 5-10: W# Tab: Shading Correction Options on page 102 for details
on shading correction options.
Autofocus
The ImageXpress Confocal HT.ai system includes an advanced laser autofocus system. Laser
autofocus is fast, reliable, does not photo bleach the sample, and is not dependent on the
quality of the staining. Most experiments require only laser autofocus with a defined Z-offset
for each wavelength. Some assays, such as those that use whole organisms or suspension
cells, might require some image-based autofocus in addition to the laser autofocus. You define
laser autofocus settings for each plate type and objective. The Laser Autofocus Wizard helps
optimize these settings. If you see frequent focus failures, contact your system administrator or
Molecular Devices representative.