Table 1. Mirror Buttons
1.5 Finding the sample - confocal
1. Select the dyes from the Dye List Window (Section 2.1);
2. Set collection parameters for minimal photobleaching – See “Optimizing Image Collection”;
a. Minimal laser intensity, Acquisition Setting Window>Lasers, Fig. 7, start with 1%;
b. set detector HV fairly high ~1000 V, Image Acquisition Control Window (See section
2.7.1 regarding Auto HV)
3. Set the Live View to tile view, each channel is displayed in a separate window;
4. Set to gray scale (control-h);
5. Set scanning to simultaneous (Sequential checkbox, Fig. 4.9, is cleared);
6. Initiate scanning: Image Acquisition Control Window>Focus X2 or Focus X4, Fig. 4.5;
7. Adjust focus and position of sample;
8. Roughly adjust laser power and HV to reduce saturation;
9. Stop scanning, Image Acquisition Control Window>Stop Fig. 4.8.
10. Refer to Section 2 for more information on settings.
11. Select Sequential, Frame, Fig. 4.9;
12. Select each channel and adjust to minimal laser power and HV less than 750 to avoid saturation
(red masked pixels);
13. Set the Offset to see only a few or no pixels of intensity = 0 (blue masked pixels)
14. Set the dwell time, 2 or 4 µs are good starting points
a. Acquisition Setting Window>Fast-Slow slider, Fig. 6.2;
15. Scan XY Repeat, Image Acquisition Control Window, Fig. 4.6 to preview full resolution;
Olympus Fluoview-1000 User’s Guide
V.M. Bloedel Hearing Research Center, Core for Communication Research
Center on Human Development and Disability, Digital Microscopy Center
May 11, 2011 10
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