10. No Image!
10.1 Start-up problems.
•
Fluoview starts to load but stalls with the Fluoview workspace open - Is the microscope
controller turned on? See Start-up protocol.
•
Fluoview starts to load but stalls with a driver error – Is the Scan Control Unit turned on? See
Start-up protocol.
10.2 Scans, but no image.
•
Is the Mirror in microscope control window set to “LSM”?
•
Is the selected laser turned on? Check the power strip, power supply key and rocker switch.
•
The 405 nm laser has a toggle switch to open its internal shutter, did someone flip it and close
the shutter?
•
The 561 nm laser has a green button adjacent to its key that must be pushed to turn it on.
•
Check the fluorophore selection (Dye List) – is it appropriate for your sample?
•
Is the sample in focus? The depth of field for epifluorescence is much greater than for the
confocal image.
•
Is the manual shutter below the turret open or closed?
•
Is the DIC analyzer partially in place and blocking the optical path?
•
Use tungsten or mercury lamp to make sure that the sample is in position and focused.
•
Is the laser intensity sufficient? If you can see the sample by epi-fluorescence, no more than 3
mW of laser power are usually required to get an image.
•
Can the detector HV be increased?
•
Has the detector Offset been increased? – This should be 5-7.
•
Are you adjusting to the correct PMT and laser?
•
Does the Confocal Aperture need to be opened up?
•
Do you need to use special imaging methods for extremely low signal levels?
•
Are you using the correct objective lens? non-immersion lens?
•
Is there oil, glycerol, water, fingerprints, etc. on a dry lens?
•
Do you have the correct immersion medium for your immersion lens?
•
Can you observe any laser illumination coming through the objective lens?
•
Is the Scan Control Unit on?
•
Is the Scan Control Unit key turned to the on position – it controls a scanhead safety shutter.
•
Has there been an earthquake recently? Components can be shaken out of position.
10.3 The confocal image appears unevenly illuminated:
•
If focusing on an uneven surface or thin specimen, only part of the field will be in focus.
•
If the slide is setting unevenly on the stage, sample surfaces will not be uniformly in focus.
10.4 The epifluorescent or tungsten images appear unevenly illuminated:
•
The objective is dirty or there is an inappropriate immersion medium present.
•
Unusually dim image - the mercury lamp aperture is closed.
•
The mercury lamp arc may have shifted, facility staff will need to re-align the bulb.
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 50
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