The Laser and Detector window (below) is used to set the lasers and detectors you want to use. A black button indicates that the laser or detector is OFF, while a colored button indicates that the laser or detector is ON. The pinhole can also be changed in the Laser and Detector window. Generally, use the small or medium setting (above). Use the drop-down menu on the top right of the screen to select the objective you are using. In the Laser Control Bar and control panel: Set the power of the laser(s) you are using. A good idea is to start at 5-10% power and increase if needed. The amount of power needed depends on the power of the laser. For example, the 543 nm laser is not very powerful, and needs to start around 20-30%, rather than 5-10%. Set the detector gain at about half way (~6 B). The detector gain amplifies the signal transmitted through the photomultiplier tubes. The general idea is to use the minimum amount of laser power to get a signal and then increase the gain, without getting high background noise. Hit to scan the specimen with the initial settings once, or to begin “Single” “Live” continuous scanning. Adjust the gain and laser power to optimize your image. If you do not see anything in one or more channels, click the or “Best” “Bright” button under the Color tab near the bottom of the screen. Usually, this will adjust the settings so that your sample will be visible. You can focus your sample by adjusting the fine focus with the remote fine focus controller box. Under the Color tab, be sure that the Color Mode is set to Multi Channel Pseudo Color, and check the Add saturation indicator box. This will display saturated pixels in a contrasting color. Then, adjust the detector gain such that just a few saturated pixels are visible in your image. If your signal is still weak, you can adjust the pixel dwell, which sets the laser irradiation time for each pixel. The default setting is around 1.68 µs, and this can be increased to 2 or 3 µs safely. This setting is located in the XY Basic panel of scan settings.
The Laser and Detector window (below) is used to set the lasers and detectors you want to use. A black button indicates that the laser or detector is OFF, while a colored button indicates that the laser or detector is ON. The pinhole can also be changed in the Laser and Detector window. Generally, use the small or medium setting (above). Use the drop-down menu on the top right of the screen to select the objective you are using. In the Laser Control Bar and control panel: Set the power of the laser(s) you are using. A good idea is to start at 5-10% power and increase if needed. The amount of power needed depends on the power of the laser. For example, the 543 nm laser is not very powerful, and needs to start around 20-30%, rather than 5-10%. Set the detector gain at about half way (~6 B). The detector gain amplifies the signal transmitted through the photomultiplier tubes. The general idea is to use the minimum amount of laser power to get a signal and then increase the gain, without getting high background noise. Hit “Single” to scan the specimen with the initial settings once, or “Live” to begin continuous scanning. Adjust the gain and laser power to optimize your image. If you do not see anything in one or more channels, click the “Best” or “Bright” button under the Color tab near the bottom of the screen. Usually, this will adjust the settings so that your sample will be visible. You can focus your sample by adjusting the fine focus with the remote fine focus controller box. Under the Color tab, be sure that the Color Mode is set to Multi Channel Pseudo Color, and check the Add saturation indicator box. This will display saturated pixels in a contrasting color. Then, adjust the detector gain such that just a few saturated pixels are visible in your image. If your signal is still weak, you can adjust the pixel dwell, which sets the laser irradiation time for each pixel. The default setting is around 1.68 µs, and this can be increased to 2 or 3 µs safely. This setting is located in the XY Basic panel of scan settings.
nikon-d-eclipse-c1-user-guide.docx