confocal microscopy issue ! - (Feb/06/2009 )
hi all,
I have some pictures taken with a confocal microscope and I need to compare the intensity of some fluorescence emission on different samples but unfortunately I don't have access to the microscope or it's software anymore.
I wasn't allowed to work with the microscope myself because a technician was in charge of it so I don't know how he compared the intensity of different samples?...can I do the comparison by any software? what softwares are used for this purpose?
the pictures are taken from fixed cells on slides and I stained them with different antibodies at different time points. so I need to compare the fluorescence at different times.
thank you
Curtis on Feb 6 2009, 02:03 PM said:
I have some pictures taken with a confocal microscope and I need to compare the intensity of some fluorescence emission on different samples but unfortunately I don't have access to the microscope or it's software anymore.
I wasn't allowed to work with the microscope myself because a technician was in charge of it so I don't know how he compared the intensity of different samples?...can I do the comparison by any software? what softwares are used for this purpose?
the pictures are taken from fixed cells on slides and I stained them with different antibodies at different time points. so I need to compare the fluorescence at different times.
thank you
Curtis i would imagine that trying to compare intensirty now with just the hard/soft copies of you pictures is next to near imposible unless you can get access to the saved images on the actual pc connected to the confocal
you could try to contact the confocal microscope supplier and pretend you are the techician or student still using the microscope, tell them that the software has a problem and ask for another free electronioc/disc copy of it and you might be able to open your old pics with this and then use the software for intensity, just a suggestion anyway!
cotchy,
that's exactly what worried me, so it is not possible.....we have a Leica fluorescence microscope in our lab and I am the one who is in charge of it, but the softwares that we have are for FISH assay and karyotyping....
if I had taken those pics with my microscope I could do something to them, I mean if they were all taken in one run and I knew the condition was the same I could compare the intensity ...
to be honest the confocal microscope's software was very complicated in the first look so I didn't really catch what the guy was doing.
We have the same confocal here in our lab and yes it is very complicated, i compare it to stting in the cockpit of an aeroplane when describing the machine to friends.
Do you have a copy of the confocal software then?
I'm not familiar with the FISH assay software, does this also allow for pictures to be taken also or how did you take the pics?
as for comparing intensity i still dont know exactly how to use it but because there are so many parameters that can be varied between slides (gain, offset, pinhole size, laser strength etc)
The only way you could compare at all is having all slides ready at the same time, and when you have got your image focused and are happy with it, just slot out your slide from the holder and slot in the second one, only moving the stage left to right (x/y axis) to view your cells and not up and down (z axis) as this will also affect the comparisons.
If you are doing it again i would be glad to offer advice on using the software but as i said i have not used the intensity side of things yet.
we have only used it for immunostaining cells with particular antibodies and taking pictures for presentaions, i quantify the same wih pcr, western blot so i dont rteally need the intesity side of things
ImageJ has a whole heap of plugins for confocal and a couple of pages describing how to use them
Image J plugins here (ImageJ is on the same site and all free under creative commons license)
Useful site here, the stuff on this page has helped me a lot.