Hello World,
After some beautiful pictures on this blog here is a little gory one. I was trying to take some images of certain type of nuclei in my cell sample. The procedure I followed was – take an image at minimum scan zoom, identify the region of interest (ROI), select that ROI and let the laser scan only that region – effectively zooming in to the ROI. Every time I would select an ROI and apply the scan zoom, I would see mysterious looking distortions – as if there was a water bubble on top of the region being scanned. I was using a water immersion lens at that time, so thought something was really wrong in the way I was putting water drop on the coverslip. Even after repeated attempts at draining and wetting the coverslip, I couldn’t get rid of distortion. Then it occurred to me that I should take an image at lower zoom after I produce such distortion.
That image revealed that I was frying the cells at high scan zoom – I was putting too much laser power in very small physical area!! See a circular ‘burn mark’ in following image. Also notice how the burn mark causes warping of signal coming from fluorescent probes. Effectively, burn mark acts as a lens. I am still not sure if the cells were ablated by local heating or the mounting medium evaporated.
When we scan the laser over area zoomed, by say, factor of 50 – the average laser intensity in the scanned area also goes up by 50. And it proved too much for cells to handle. So it is good idea to reduce the laser power when you use the scan zoom in your imaging experiments as you focus on smaller and smaller areas.
But I wonder why local heating was prominent only in the circular path and not throughout the scanned area.
Can anyone throw some light? 
Posted by Shalin Mehta 
