Re: CONFOCALMICROSCOPY Digest - 6 Nov 2009 to 8 Nov 2009 (#2009-253)
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Joost Willemse |
Re: CONFOCALMICROSCOPY Digest - 6 Nov 2009 to 8 Nov 2009 (#2009-253)
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Hi Serra, As your previous questions were answered more or less I am only going to give my opinion on the third question you raise. The out of focus drift in my experience happens very often in living cells. Although I have never found theoritical proof for this in my experience cell tend to move away from the intense laser power. I have been doing bleaching experiments on arabidopsis nuclei, upon bleaching with 100% laser intensity for 3 itterations (also lsm 510) the nucleus moves away from the position where you bleached. The only way I have been able to adapt to this problem is by reducing laser intensity and increasing the ammount of itterations. Don't know if this will help in your case as well, but I do know that living cells tend to move away from things that could potentially harm them, so I guess it is worth a try. Good luck Joost Willemse Ontdek nu Windows phone. De smartphone van dit moment Hi all! Currently i try to establish FRAP with my ECFP tagged protein which is distributed in cytoplasm of neurons and i use LSM 510. However i have some problems and i would love to get your valuable opinions. Here are my questions: 1. How should i find the correct "laser iteration" to bleach the region that i want? Is there a calculation or a formula to find out the iteration of the laser when the laser is %100? 2. Even though my second question seems to be the same with the first one actually it is a bit different. I don't always get the same transfection efficiency in my cells. So how can i manage the iteration of my
laser? Should i have always the same iteration value? But then i dont have efficient bleaching in cells where ECFP is highly expressed. (For example,last week i remember that in one cell i was able to do bleaching with 10 iterations but in another cell i had to use 50 iteration). On the other hand the more i increase the iteration, bleaching time increases too. What is the standardization? 3. After the laser finishes the bleaching, cells always go out of focus and i lose the view. I checked many times that the cells had enough medium in the dish and they didn't get dry. But still i couldn't get rid of this problem. I think this happens because of the temperature change after bleaching. Any other suggestions?I need to overcome this... 4. I am also open to hear any "good FRAP paper" where i can learn more about the protocol. Thanks so much in advance and i hope
someone in this email group can help me. Warmest regards, Serra Dear Serra, concerning your questions: 1)
There is no formula available
to calculate the best laser intensity for bleaching. As the correct intensity
pretty much depends on the sample and the result you want obtain this is not
really surprising. In general: the more fluorophores you bleach the higher the
probability of inducing unwanted reactions like crosslinking or even
photodamage. If you are looking at fast diffusion you want to bleach as fast as
possible. Thus you want to keep the number of iterations as low as possible. 2)
Changing the number of
iterations will affect your results. If you want to compare your results you
will have to keep the number of iterations constant. 3)
Diffusion is temperature
dependant. Thus you want to do your experiments preferably at a constant temperature.
If bleaching is heating up your sample you have to decrease the number of
iterations or the laser power used for bleaching. Additionally you should make
sure that the temperature is kept constant (for mammalian cells usually 37deg). 4)
At the following link you can
find a nice tutorial for FRAP experiments. Best regards Arne Seitz --------------------------------------------------------------- Dr. Arne Seitz Head of Bioimaging and Optics Platform (BIOP) Swiss Institute of Technology (EPFL) http://biop.epfl.ch/ From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Serra Akinturk Hi all! Currently i try to establish FRAP
with my ECFP tagged protein which is distributed in cytoplasm of neurons
and i use LSM 510. However i have some problems and i
would love to get your valuable opinions. Here are my questions: 1. How should i find the correct
"laser iteration" to bleach the region that i want? Is there a
calculation or a formula to find out the iteration of the laser when the laser
is %100? 2. Even though my second question
seems to be the same with the first one actually it is a bit different. I
don't always get the same transfection efficiency in my cells.
So how can i manage the iteration of my laser? Should i have always the same
iteration value? But then i dont have efficient bleaching in cells where ECFP
is highly expressed. (For example,last week i remember that in one
cell i was able to do bleaching with 10 iterations but in another cell i
had to use 50 iteration). On the other hand the more i increase the iteration,
bleaching time increases too. What is the standardization? 3. After the laser finishes the
bleaching, cells always go out of focus and i lose the view. I checked many
times that the cells had enough medium in the dish and they didn't get dry. But
still i couldn't get rid of this problem. I think this happens because of the
temperature change after bleaching. Any other suggestions?I need to overcome
this... 4. I am also open to hear any
"good FRAP paper" where i can learn more about the protocol. Thanks so much in advance and i
hope someone in this email group can help me. Warmest regards, Serra |
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