Download Adjusting the PMT signal gain

Adjusting the PMT signal gain
A photo-multiplier tube (PMT) photo-multiplies the weak signal emitted by the fluorophore, so that it can be detected.
Photo-multiplier tubes are extremely sensitive and very rapid detectors of light and can multiply the fluorescent signal by
a factor as much as 108. For further information on how a PMT works, see: http://en.wikipedia.org/wiki/Photomultiplier_tube
and/or
http://micro.magnet.fsu.edu/primer/digitalimaging/digitalimagingdetectors.html
Because they are fast, speed in detection by a PMT comes at the price of efficiency: a PMT has a QE of 12-15% at best,
and so only detects 1 in every 7 or 8 photons of fluorescent signal.
As the voltage gain applied to the PMT increases, so the detected signal displayed on the monitor gets brighter until it
saturates, contrast is lost, and no more detail can be discriminated against the background.
There are two rules that apply to image collection with the confocal microscope above all others:
1) Do not bleach your sample with the laser – keep the power to a minimum
Otherwise there is no signal to detect in the first place!
2) Do not oversaturate the emitted signal into the display
Otherwise no detail can be seen in the image, and 3-D reconstruction is impossible.
A photomultiplier will only see shades, or tones, of gray, not colour. The colour applied to the display is a false pseudocolour to help us recognize each fluorophore. The photomultipliers can operate in either 8-bit or 12-bit depth. The bit
depth defines the number of gray levels discriminated by the PMT: 8-bit depth = 28 = 256 grey levels from zero (black)
to 255 (white). 12-bit = 212 = 4,096 grey levels. CCD cameras are usually 12-bit; by comparison, colour images are 24-bit
depth (3x 28; one 8-bit depth per red, green and blue channel).
Computers have on-off switches to encode information. They therefore store data in binary (base 2) form, unlike humans
who have ten digits and so count in base 10. Hence the bit-depth for encoding increasing tones, or shades, of gray,
depends upon the exponential power of 2.
For most applications use 8-bit detection unless you are collecting quantitative data from which you
must extract numerical measurements, in which case 12-bit depth is better.
Our eyes only see information either as colour (change in wavelength) or intensity (change in amplitude of the wave). We
are better at discriminating colour than tones of gray, and our eye will therefore only see the central part of the 8-bit gray
scale. At best we can only see up to 60 tones of gray.
In order to contrast stretch the emitted signal from the sample into the PMT, and not over- or under-saturate it, we must
use a false-colour look-up table (LUT) to pseudo-colour the display so that we can accurately set the voltage gain on the
emitted signal so that it fills the entire PMT range without under-saturation and (much more important) without oversaturation.
1. On the Leica confocal go to the Q-LUT button in the image window. Toggle through pseudocolour, grey scale and LUT functions.
2. On the Nikon confocal the LUT toolbar is always open in an image window. Toggle the
LUT button on/off to switch between the raw image and that with activated LUT. The
Show LUT window button allows adjustment of the LUT parameters"
3. On the Olympus confocal go to the LUT settings in the image window to bring up a separate
dialogue box. Set the Hi-Lo range with the button in the lower right-hand corner.
3. On the Zeiss confocal go to the Palette button in the image window. Select Range Indicator.
A. Set the black level of the PMT first. This is controlled by the Amplifier Offset.
(It is usually a more sensitive control than the Detector, or HV, gain).
B. Once the Amplifier offset has been used to set the background at just pure black, increase the detector gain (you are
increasing the voltage of the PMT by doing this) until the image just shows 1-2 pixels saturated.
Here is an example taken from the Zeiss LSM 510 confocal. In both the Zeiss and Olympus confocals, the LUT sets
under-saturated pixels as blue, and over-saturated pixels as red. In the Nikon confocal you can set the pixels to a colour of
your choice, which is really good. In the Leica SP-series confocals, the under-saturated pixels are set as green and the
over-saturated pixels as blue. You can usually get away with more under-saturated blue pixels than shown here (the
ideal), but too many over-saturated red pixels will mean a loss of contrast, loss of fine detail and the inability to get good
3-D reconstructions.
Our eyes perceive colour with differing sensitivity, and can see green better than red or blue. We pseudo-colour the PMT
signal in order to relate quickly and efficiently to the fluorophores we are detecting. If it is important to pick up fine detail
in the image, consider using white as your pseudo-colour to give the highest contrast against the black background.
Words and Images © Copyright JB Sanderson, Sheffield, April 2008