1. Live Window is set to gray scale (control-h);
2. Set Live Window for tile view (each detector is viewed in its own pane);
3. Turn off ‘Sequential’ imaging (clear the check box);
4. Click on ‘Scan X2’ or ‘Scan X4’;
5. Choose the channel with the lowest excitation wavelength;
6. Reduce the laser power to the lowest possible level while adjusting ‘HV’, keeping below 750, until
an acceptable image is obtained;
7. Select the next channel and adjust the appropriate laser power as low as possible but, take care in
adjusting the HV so that the shorter wavelength channel does not contribute significantly to the
image;
8. The channel with the longest excitation wavelength can be adjusted to a low HV and a relatively
higher laser intensity to reduce amplification of bleedthrough from the other fluorophores;
9. Acquire your images.
See Section 6, ‘Optimizing your images’ for details on setting the detectors.
4. Collecting a Z-Series.
A z-series is a set of images collected with an increment of focus between each image plane. This is an
automated process where the user sets the upper and lower limits of the focus travel with an interval for
incrementing the focus between captures.
4.1 Determine the depth of the optical volume.
Focus through the sample with Scan X2 or Scan X4 and note either the Current Focus settings (Figure
10.3) or structural landmarks. The focus increment (Step Size) should be approximately one-half the
size of the smallest object to be axially resolved, or one-half of the objective lens axial resolution. Each
lens has a limit of axial resolution, Table 2. See Optimizing Your Image.
4.1.1 Which way does the focus move?
The objective lens moves in the direction of the back side of the focus knob. To focus “upwards”,
through the coverslip and into the sample, turn the knob so the top moves towards you.
Focus “downwards”, towards the coverslip, by turning the knob so the top moves away from you.
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 23
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