3. Set the Live Window to ‘Tile Display’;
4. Press ‘control-h’ to set the look-up tables (LUT) to gray scale;
5. Select zoom setting and box size (zoom = 1 and box = 512 are good starting points);
6. Raise PMT HV to about 1000 V;
7. Set to Simultaneous capture;
8. Collect image with Scanx2 or ScanX4;
a.
bring specimen into focus and frame the field
9. Choose Sequential capture, Frame (see below);
10. Select each channel group in turn and scan at ScanX2:
a. minimize laser intensity while adjusting PMTs to less than 750 V to minimize saturated
pixels (red)
b. set Offset to eliminate pixels with an intensity of zero (blue)
c. leave Gain set to 1
11. Select pixel dwell time for image collection, 2
µ
s is a good starting point;
12. [Optional] choose Kalman averaging (see below);
13. Capture image with XY button.
6.3
Labeling Controls.
Labeling controls are essential in determining acquisition settings and avoiding artifactual images.
Control specimens also can save hours of work by helping to pinpoint the source of problems.
6.3.1 Negative controls
Negative controls, lacking primary antibody, are essential to determine degree of autofluorescence,
background labeling or bleedthrough. Samples with no exposure to secondary antibodies or using
antibodies subjected to immuno-absorbtion are also useful.
6.3.2 Positive controls
Known positive controls are also important. Positive controls labeled for each single label are crucial
when imaging specimens with multiple labels. They can verify that the labeling protocol is working,
and avoid wasting hours on the confocal looking for non-existent labeling. Single label controls also
help in tracking sources of bleedthrough.
6.4
The confocal aperture
The confocal aperture (also called “pinhole” or “iris”) controls the amount of light rejected from out of
focus regions. It should be set at the lowest setting consistent with the nature of the sample and your
image requirements. Smaller iris diameters provide improved contrast and thinner optical sections. A
wider iris increases the amount of out-of-focus light collected, the thickness of the optical section and
reduces contrast. Diameters less than 1 Airy disk have no meaningful effect with air objectives having
NA less than 0.9.
6.4.1 Optimal Iris Diameter
The optimal iris diameter matches the diameter of the outer edge of the first dark ring of the Airy disk, at
the detector (refer to textbooks for more discussion). This is generally considered the diameter of 1 Airy
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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