In the Resolve3D window ND field, set the neutral density filter to 100%.
In the Exposure field, enter a low value (e.g., 0.1 sec) for exposure time.
In the dZ field, enter the desired value for the Z step.
Move through the area of interest
by pressing 
and
to move the
stage in the Z direction.
Click the Acquire 
icon. If performing
a 2D experiment, go to Step 11.
Continue performing Steps 4 and 5 to obtain a sampling of Z sections. Observe the value of the Max field in the Resolve3D window to find the maximum intensity in these sample images. The focal plane with the highest maximum intensity value is the plane of optimal focus.
Note: There could be a higher intensity value in the image data that you did not sample. Therefore, do not set the exposure time to the limits of the sampled data because a higher intensity, unsampled plane may saturate the camera. |
Move to the plane of optimal focus. This is the Z section with the highest intensity value.
From the Resolve3D menu, choose View | Points List.
Click Mark Point. The Z value is the third coordinate listed for the point.
Document the Z value with the brightest intensity.
In the Exposure field, increase the exposure time (consider the amount of exposure the specimen can tolerate). Intensity values increase in direct proportion to exposure time. This is only true for fixed cell experiments. The signal-to-background ratio is much lower (approximately 2) for live cells.
Repeat for all desired wavelengths.