One golden rule: “Use the lowest laser power to give an acceptable image.” When an acceptable image can not be obtained and all other parameters have been adjusted to the point they are causing problems with the image, only then is it usual to increase the laser power. Remember, excessive laser light can alter your specimen and cause photodamage and photobleaching. Using the lowest possible laser power for the shortest possible time will best preserve the fluorescence or biological activity in your samples.
Laser powers are given using quite different scales on microscopes by different manufacturers. Sometimes percentage of maximum power is used, sometimes values from 0 to 255 are used, and sometimes actual laser power is measured at some point in the optical path (which is not necessarily the same as at the specimen focal plane). Additionally, different lasers are used with quite different power outputs. So, it is nearly impossible to compare laser power from one confocal microscope to another confocal microscope, even if manufactured by the same company.
So how much power should one use? “Use the lowest laser power to give an acceptable image”. (Do you think someone is trying to tell you something here?)
Adjusting the Image and Detector Controls
Gain or HV
Gain (also called HV or high voltage) is a relative measure of the amplification one applies to the photon detection system. Usually, higher gain settings result in brighter images (increased signal). However, as one increases the gain, increasing noise becomes evident in the image. (This is visible as a “speckle” overlying the image data.) So, at some point as the gain is increased (and the overall image gets brighter), the signal/noise ratio (S/N) starts to decrease. The overall aim is to get the highest S/N, NOT the brightest image.
Offset or Black Level
Offset (also called black level) is the dark current setting on the amplifiers of a PMT detector. In effect, this control sets the black values in an image or display.
By increasing the offset, the darkest areas of the image get brighter.
By decreasing the offset, the dark areas of the image get darker.
Caution: by decreasing the offset too far, very faint data will actually be lost - it is critical that this is not allowed to happen. New users are often tempted to decrease the offset value to try to eliminate background or non-specific signal. This should NEVER be done.
In order to collect the very best images, it is essential to capture both the very darkest values in our data as well as the brightest values. The difference between the brightest and darkest values is termed the dynamic range.
Correct adjustment of the gain and offset values is ESSENTIAL to obtain the highest dynamic range (and, therefore, the best looking image).
Look Up Tables (LUTs)
Look up tables are simply coloured overlays that sit over your display to indicate the brightness of individual pixels.
Many microscope manufacturers use specific LUTs to indicate when pixels in an image are saturated – where brightness (laser power or gain) is set too high, or undersaturated where offset is set too low.
It is critical, especially for newer confocal microscopists, to use LUTs to correctly set the gain and offset, thereby collecting the very best range of values (dynamic range) and therefore the very best image. Remember, the data in the image that you collect on the microscope is the very best it will ever be. Later image processing to improve the appearance of your image usually throws some data away.
To explore the effect of photomultiplier settings, consider the three images and photomultiplier outputs above. In Figures a) and b) the offset on the PMT is raised or lower to make the image brighter or darker. However, the images are both somewhat washed out with a) being overall too bright and b) being overall too dark. In Figure c) the PMT gain has been increased, which results in an image with better contrast, more dynamic range and is overall a much clearer image. PMT offset and gain are analogous to brightness and contrast in photography. Both have to be set correctly to get the optimal result.