Confocal Settings

These are the basic settings we recommend when using the Bio-Rad confocal microscopes at the QEII campus and Crawley campuses.  They should be used as a guide, but as samples differ you may need to contact Paul or Tracey if you have special requirements.

Laser Power

Always use the lowest laser power required to get an acceptable image to reduce bleaching your sample.  The lasers vary in strength and should be used at different powers:

Pinhole Iris

The pinhole controls the thickness of the optical section.  Do not use it to control the brightness of an image unless absolutely necessary.

Gain

This affects the brightness of the image; it is the amount of power the detector uses to see the light.  The higher the value the higher the signal.

Black level

The black level affects the dark areas of an image. 

Kalman Averaging

Multiple scans of the same image reduces the noise in the final image.  Values below are given for slow scan collection.  If using normal scan speed then increase the number of Kalmans by 2.5 times.

Zoom

The amount of zoom you can use depends on the NA of the objective.  Check the side of the objective for the NA.

Low NA (<0.9) use a maximum of approximately 2x zoom.

High NA (1.2-1.4) max zoom depends on the magnification of the objective. 

Lookup Tables (LUTs)

LUTs are an overlay on the display used to represent the intensity of the pixels, they do not affect the image collected.  Use the SETCOL.LUT table.  Red represents the brightest pixels (250-255) and Green the darkest (0-5).

If you have solid red areas your image is saturated and you will lose data in the brightest parts.  Reduce laser power or gain settings.  If there are solid green areas the dark regions of the image are too dark, increase black level.

You should aim for some small areas of red and mottled green background, but not solid areas.