LSM 710 and LSM 780
MULTIPHOTON LASER SCANNING MICROSCOPY …
Systems
Non Descanned Detection (NDD)
Carl Zeiss
02/2010 M60-1-0025
e
17
How to Use the GaAsP nosepiece NDD Detector:
A. General Instructions for Use
Before using this detector for image acquisition, proceed as follows:
•
If standard non-descanned detectors (NDDs) are provided on the microscope,
first
use these to set the
optimum acquisition parameters such as laser power, PMT high voltage, filter selection, and the
positions of specimen and focus. If no standard NDDs are provided on your system, please use the
detectors provided in the scan head for setting these parameters. You should also consider the
spectral properties of the filters whether you use NDDs or the scan head detectors. Always start at the
surface of the specimen.
•
As soon as you remove the GaAsP nosepiece NDD detector from the microscope, please store it in the
light-tight box supplied.
•
If you follow these instructions, the GaAsP nosepiece NDD detector can be safely used and will give
optimum performance.
B. Proper Use of the GaAsP nosepiece NDD Detector for Image Acquisition
It is assumed that you have first used at least the internal detectors for identifying the optimum
acquisition parameters (laser power, PMT high voltage, and filter selection, focus and specimen position).
1.
Best switch off the room light completely or at least dim it to the least possible level, or screen the
specimen space against all external light.
2.
Switch off the halogen, X-Cite or HBO lamp, or make sure that the shutters are closed.
3.
Deactivate any other laser sources. Diode lasers have to be turned off via the software; all other
lasers, when not in use, are switched off or blocked by a shutter in the laser module.
4.
Set the power of the multiphoton laser to a minimum level which is smaller than or, at most,
equal to the level you used for image acquisition with the scan head detectors or standard NDDs.
5.
Activate the GaAsP nosepiece NDD detector via the software (tick the check box). Make sure that
the detector high voltage is set to zero.
6.
Start image acquisition. Slowly increase the high voltage (detector gain) of the detector, and
watch the intensity in the image. With very high voltage, you should essentially see only dark
noise. If a lot of noise is visible in the image already with lower voltage, this is probably due to
room light or light from other sources. These light sources may not only cause damage to the
detector but also greatly impair the quality of the image. Switch these light sources off, or
improve the screening of the specimen area. After this, reset the high voltage to zero.
7.
If there is nothing but dark noise, manually tilt the color beamsplitter provided right above the
objective into the beam path, and slowly increase the high voltage of the detector again, while
watching the image. The optimum range of high voltage is about 500 – 800 V. If the signal is too
strong, reduce the laser power. If the signal is too weak in the optimum high voltage range,
slowly increase the laser power until the image is well balanced.
If too much light (e.g., room light) hits the GaAsP nosepiece NDD detector, the light is directed
onto a light trap, and no signal or a faint signal only, is detected. Stop the scan. Reduce light
incidence by screening off ambient light, switching off light sources, or reduce the laser power.
Then restart scanning.
