Hi, i am working on fluorescence microscopy..i am trying to build a small microscope to study fluorescence (eGFP) ..I would like to know if there is any limitation on the excitation time and imaging time. I mean, how long can my excitation source be ON? and for what time period can I capture fluorescence? Is there any delay required between excitation and fluorecence image capture? or, can the excitation light be ON throughout the process?
The theory is important, of course. That tells you that the absorption, fluorescence, and decay happen in tiny fractions of a second. But you also need to see how fluorescence imaging is accomplished in practical terms. Fortunately, this equipment is common in biomedical research. A web search reveals that the Department of Biotechnology at your institute has fluorescence and confocal microscopes. It may be helpful for you to visit so you can see fluorescence microscopy first-hand, and understand the essential components.
https://www.iitm.ac.in/btresearch
It would have been much more helpful if you would have helped me to gain the required knowledge, instead. Anyways, thanks for your comment.
The theory is important, of course. That tells you that the absorption, fluorescence, and decay happen in tiny fractions of a second. But you also need to see how fluorescence imaging is accomplished in practical terms. Fortunately, this equipment is common in biomedical research. A web search reveals that the Department of Biotechnology at your institute has fluorescence and confocal microscopes. It may be helpful for you to visit so you can see fluorescence microscopy first-hand, and understand the essential components.
https://www.iitm.ac.in/btresearch
Theresa
Why didn't his supervisor give him directions, when it is in the same university or even the same department?! Does not sound good for a research-start
the project of Mr. Rudra Chaudhary is rally appreciable, and i think he would have been gone through the basics, and the question put up by him is part of research, for which he would have been thought Researchgate as best platform....so instead of going off the track, we should have a healthy discussion .....
Yours,
.......skumar
@Rudra,
As stated above: start first from gaining the fundamental knowledge about fluorescence and microscopy. There are plenty of books and articles available that discuss those topics. Then, if you face a specific problem or you will need a clarification, come back here and ask the question.
@Suresh,
I have a doubt if Rudra went through the fundamental aspects of fluorescence and microscopy if he asks those kind of questions. Other possibility is that his background is completely different from the research project he is undertaking.
Dear Rudra,
To actually be slightly more constructive, I would suggest you start with this free ressource from Nikon:
http://www.microscopyu.com/
It goes through all the basics of fluorescence microscopy. Building a fluorescence microscope is not especially difficult, but you will need dichroic mirrors, filters and light source, which make it more expensive than "a simple" bench microscope.
To answer your question:
Yes, usually the source is always ON, because the time difference between excitation and emission is typically in the order of tenths to a few nanoseconds.
Dear Rudra,
I agree with the above mentioned worries on your start in this project.
But to your question on the technical aspect Pierre is right. But keep in mind that your eGFP will bleach while it is excited. So this on/ off topic is not so much of a problem if you just look at your sample but it becomes a problem if you want to follow it with life cell imaging and if you want to quantify changes over time.. . There is not just the technical challenge but also some knowlegde and maybe experience needed on the sample side.
Anyway best luck with your project.
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