Is it possible to selectively "burn" some regions with a laser pulse from a two-photon microscopy? I would like to selectively damage some brain regions in insects in order to study neuronal connectivity and plasticity.
I don´t think so. The multiphoton is not excited by continues high energy photons, but by a pulse of two or three low energy photons.
Indeed, but i know that you can induced a lot of photodamage when you focus the laser in a certain spot of your sample..i just do not know if it was ever done live as "microsurgery" tool to study recovery or plasticity..
It is possible indeed. Davalos et al. did a great paper on brain injury (ATP mediates rapid microglial response to local brain injury in vivo. Dimitrios Davalos, Jaime Grutzendler, Guang Yang, Jiyun V Kim, Yi Zuo, Steffen Jung, Dan R Littman, Michael L Dustin & Wen-Biao Gan. Nature Neuroscience). The technic is pretty simple and I have done it myself a few times: I usually use a 800 nm wavelength on a specific region. I change the scanning time to about 100 µs/pixel and use a laser power of about 80% (Ti:Saphire laser, Mai Tai). About 90% of the time, the lesion is "autofluorescent" in the GFP, RFP and far red channels. Hope this help!
Yes, it is possible. Depends only on the laser power you are using. Usually powers from 10 to 50mW at the sample are enough for imaging and this is very low relative to what is available from the IR laser. A Ti-Saph laser at 800 nm, as suggested above, delivers more than 2.5 watts at the exit of the laser. It is not uncommon that MPE users damage their samples because of excessive laser power at the sample level. Then, if you have a MP confocal microscope, just point the laser where you want damage to be done and increase the power. If your equipment allows to scan lines, circles or different directions, play around. and be careful not to cause a too extensive damage by using excessive power or excessive exposure to IR laser.
The atofluorescence may be due to NADH and oxidation of lipids, products that will be released from the cells and are excited around 780 nm. Actually, most of the cells autofluorescence is caused by these molecules.