Olympus Fluoview-1000 Benutzerhandbuch - Seite 22

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V.M. Bloedel Hearing Research Center, Core for Communication Research

Center on Human Development and Disability, Digital Microscopy Center

3.2.3 Scan mode and sequential capture

1. 'Scan X2', 'Scan X4' or 'XY Repeat' - click on the desired channel for Frame scanning. That

channel will be scanned until the user clicks on 'Stop' or selects a different channel.

2. 'XY' scan button will acquire all active channels in the selected sequential manner, and display

the result.

3.3

A group number is visible in the title bar for each detection channel, Figures 5.7, 9.2, 9.2A. Channels

given the same group number will be scanned simultaneously during sequential capture. Any channels

that do not display significant bleedthrough, e.g. Alexa 488 and Alexa 660, may be scanned together

during sequential capture as shown in Figs. 9.2A, 9.3A. These 2 detectors are set to the same group

number and scanned simultaneously while the other channels would be scanned sequentially. The

advantage to channel groups is that acquisition time can be reduced during sequential image acquisition.

When adjusting detectors using Frame Sequential, selecting a group will scan both channels, Fig. 9.3A.

3.4

Bleedthrough is commonly encountered with many fluorophore combinations. Channel adjustment is

best accomplished by using sequential imaging.

1. Live Window is set to gray scale (control-h) in order to observe saturated pixels;

2. Set Live Window for tile view (each detector is viewed in its own pane);

3. Click on 'Sequential';

4. Select 'Frame';

5. Click on 'Group 1', Figure 9.2;

a. the channel(s) in this group will be scanned

6. Scan with 'Scan X2' or 'Scan X4';

7. Adjust PMT settings and laser power for image quality

a. try to keep 'HV' below 750 to minimize detector noise

b. minimize laser power levels to reduce bleedthrough and photobleaching

8. Click on the next channel group and repeat the process;

9. Acquire images using 'Line' sequential.

3.5

Bleedthrough for some fluorophore combinations can be minimized during simultaneous imaging solely

with the laser power and detector settings. In general, strive to minimize the strength of the signals from

shorter wavelength fluorophores by reducing the laser power for those channels. This reduces the red

side of their emission spectra to reduce their contributions to the signals passing the filters for the longer

wavelength dyes.

Conversely, the HV setting for each channel controls sensitivity to any photons transmitted through the

barrier filter, regardless of which fluorophore from which they originated.

May 11, 2011

Olympus Fluoview-1000 User's Guide