Lines in Images---what could cause this?

[Andorin]

Hi, I am cutting mouse brain sections to 10um on a cryostat (perfused in paraformaldehyde and frozen into a block of OCT), staining them with various antibodies, and visualising using a Zeiss 510 confocal microscope.

I have been finding that some of my images have black lines running across them; an example of this is shown in the image at srcf.ucam.org/~ns452/lines.JPG (address needs those three letters put before it... but I can't put them there due to spam filter).

If I move in the image, then the lines stay where they are, so I take that to mean that it is actually something present on the slide.... but it is not visible using bright-field mode.

Any suggestions on what this could be? Could it be caused by my cryostat technique?

Thanks

[5µl-pipette_death_punch]

Sorry I can't open the image, but as the ZEISS 510 is a old confocal (we are using the same equipment as you do), I suppose that there is a problem with the scanning unit. I would try first to switch off the bidirectional scanning (if it is activated) and then decrease the scan speed. Maybe it is not on the slide...

[haisond]

I've experienced similar lines while making adjustments for the autofocus mode. The lines are generated from the laser burning or photobleaching while you are setting the autofocus parameters. This effect can be eliminated by reducing your laser power, and number of scans.