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? 
November 13, 2007 at 4:57 pm |
Hi.
It is really good idea to set up laser power in the zoom factor you are going to use. Aproximately. So if you want to scan for example one nucleus per image, first zoom up one nucleus and than balance laser power and gain on photomultiplier. During scanning at lower zoom, intensity can be easily increased just by increased sensitivity. Laser power should be enought for lower magnification. Ok. In the case you are not switching image pixel size. Usually for zoomed nucleus is 512×512 sufficient resolution, for zoom 1 you should use 1024×1024 for the approximately same pixel size. So the balancing is necessary anyway.
IMHO the circular shape of you distorsion originated from evaporated medium which made a buble and destroyed a cell little bit. Just my opinion. I have never seen something like this before.
Sincerley – Ondrej.
November 13, 2007 at 10:39 pm |
I have seen this many times in our facility. In our case it is due to heating by the pulsed IR laser during DAPI imaging in 2P mode (well, really ANY dye in 2P mode). If we raise the laser output above 3% we seem to run a risk of creating bubbles or “burning” our samples. I believe we saw more of this phenomenon with cells mounted in Vectashield but I was also successful in frying a Molecular Probes test slide on multiple occasions. Interestingly we don’t see problems when imaging cells in glass bottom dishes surrounded by media – perhaps due to more efficient heat dissipation?
November 20, 2007 at 12:28 am |
I would second David’s reasoning. Peter So mentioned during today’s class of Optics course, that cells sometimes gobble up debris that absorb IR (this absorption is ‘one photon’ and depending on the fluorescent properties of the debry may or may not be accompanied by subsequent emission). They seem to have seen ‘unclean’ cells getting fried. He also mentioned that melanin leads to heating of skin samples when imaged under 2P because of its high absorption in IR. These IR absorbants can heat up very quickly under focused IR and cause the burning of samples.
November 26, 2007 at 3:17 pm |
Its very common for live cell imaging with one photon microscope, because of high laser power (also depends upon which wave length you are using). the important thing is that, it never create any circular line , its just an illision. when you are zooming you are using more power compair to the full cell. Some times , due to high laser power cell die and comes out from glass surface. generally it happens when you are using DMSO or other cmecicals. its always good to use 2 photon for your experiment. its also happens in case of two photon also.
December 18, 2007 at 6:02 am |
Hi
I have read couple of your posts on the confocal listserv and thats where I found the link to your blog. We have seen exactly the same thing as David has reported on the Molecular Probes slide.
Just out of curiosity, why are you using Zoom 50 and which lens ?
Abhi