1. Quick Guide to Getting an Image
Examples of the software control windows are on the following pages. The text contains numbers that
refer to specific numbered controls in figures.
Your overall goal with fluorescent microscopy is to image the specimen with the least amount of
photobleaching. This requires limiting specimen exposure to damaging levels of excitation light.
1. Find the specimen using the eyepieces and epi-fluorecence. This is faster than scanning.
2. Low magnification objectives allow rapidly searching over large areas, but usually dimmer.
3. Low intensity transmitted light is safest for finding and positioning the sample.
4. DIC is usually better than brightfield for low contrast samples, such as monolayers or picking out
structures in complex specimens.
5. Epi-fluorescence is usually faster than confocal to find the fluorescent sample.
6. Epi-fluorescence allows you to see a blurry glow from out of focus objects, unlike confocal.
7. Use the least amount of light necessary to find the sample to reduce photobleaching and toxicity.
8. Begin confocal imaging using low laser intensity with high PMT HV while surveying the
sample.
9. Focusing on the edge of the coverslip by transmitted light can help to find initial focus.
Figure 1. Main Fluoview window.
Drag this corner to resize the window
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 6
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