Olympus Fluoview-1000 Manual del usuario - Página 10

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Olympus Fluoview-1000 Manual del usuario

1. Add a Scale Bar to Your File

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

Center on Human Development and Disability, Digital Microscopy Center

Mirror Button

LSM

DAPI

FITC

TRITC

Cy5

DICT

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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;

May 11, 2011

Olympus Fluoview-1000 User's Guide

Function

Confocal Imaging

UV excitation

Blue excitation

Green excitation

Red excitation

DIC/ Brightfield

Brightfield

Mode

Confocal

Epi-fluorescence

Transmitted light

Visualization

Confocal only, automatic

Eyepieces only

Eyepieces or Camera

Confocal or Eyepieces