4.2
Settings for z-series collection.
1. Determine the axial extent of volume needed (how far you need to focus through the sample);
2. Focus to lower extent of volume by manual focus or by the large yellow arrowhead pointing
downwards, Figure 11.6, (increments focus by the Step Size);
3. [Optional] Use the small downwards arrowhead to finely establish the volume limits, Figure 11.6
(increments focus by 1/2 Step Size);
4. Click on the ‘Set’ for the “End” (lower) limit, 11.1;
5. Establish the upper extent of the volume and click ‘Set’ for the “Start” limit, 11.1;
6. Set the StepSize, Figure 11.3, by one of 3 manners:
a. enter a value (
µ
m) and press the ‘Return’ key
b. click on the small black arrowheads
c. Click on ‘Op.’ to select an “optimal” value calculated from the objective lens and
wavelength, Figure 11.2
7. The number of optical slices will appear in box 11.4;
8. Move to the XY acquire button, Figure 4.7;
9. Select ‘Depth’, Figure 4.7;
10. Click on the XY acquire button to collect the z-series, Figure 4.7;
11. When the series is completed, you will be prompted with 2 options:
a. “Series Done” – click here if you are content with the results
b. “Append” – click on this to add sections to the end of the z-series, the number of sections
to append is controlled by the box next to the button
The red number below the lens icon in this window displays the current axial position. You can simplify
keeping track of focus distance by clicking on the ‘Set 0’ button before beginning to scan or set focus
limits. This will set the current axial position to “0” and subsequent focus positions will be displayed
relative to this position.
4.2.1 What is axial resolution?
Axial resolution describes the smallest depth interval that can be resolved by the lens, it depends on the
lens NA, emission wavelength and quality of the specimen preparation. Air lenses have roughly 3-5
times lower axial resolution than lateral resolution while immersion objectives have 2 times lower axial
resolution. The optimal setting button sets the z-step to 1/2 of the calculated axial resolution. The 20X/
NA 0.75 air lens has calculated axial resolution of 2.32
µ
m, for a calculated optimal z-step of 1.16
µ
m.
In practice, smaller z-steps, down to 0.4
µ
m result in higher quality images for this lens. Images
collected for deconvolution, benefit from “over-sampling” in the z-axis by a factor of 2, or using a z-step
that is at least 4 times smaller than the calculated axial 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
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