I used DiI and I traced very well some fibers, but a disadvantage is that when it's exposed to UV light in a microscope, loses fluorescence very quickly. I need a tracer in fixed tissue because it's easier to administrate the tracer in the zone that I'm studying than in stereotaxic. Thanks.
Greetings,
From what I understand of your question, DiI is working for you, but you are having issues with photobleaching. Unfortunately, DiI is actually one of the more photo-stable dyes out there. You may want to try optimizing your imaging technique. I am not entirely sure why you are seeing bleaching form from UV since DiI is excited primarily by blue/green light. To minimize bleaching you can try some of the following:
1: Reduce imaging time (try performing your analysis on pre-imaged sections or virtual slices)
2: Minimize excitation energy (photo-bleaching of DiI follows 1st order kinetics and is dependent on excitation energy (see link). Also, at some point more excitation does not equal more fluorescence instead you just get more background and bleaching, and is very easy to do with laser excitation.)
3: Using a mounting agent with an anti-fading agent. These work by preventing oxidation of dyes. They do reduce the amount of fluorescence, but they also increase their longevity.
4: Double check your filters and light source. Mercury HBOs tend to have a lot of energy in the UV emission spectra. If your UV excitation filter lets the 405nm peak through this may have a strong impact on bleaching.
Best of luck.
http://www.ncbi.nlm.nih.gov/pubmed/3920227
In addition to Justin's excellent comments above, there's a sampler kit for lipophilic tracers which might prove useful for testing DiI derivatives and analogs (including DiR and DiD) against each other for your application:
https://www.lifetechnologies.com/order/catalog/product/L7781
Some variants may perform better than others for you, but try Justin's suggestions first, since it does indeed sound like the tracer is working as intended but that the imaging needs troubleshooting.
In my case I have used DiI (Red) and DiO (green). I performed axonal tracing experiments after fixing the brain slices in PFA and this worked very well. I could perform confocal imaging for a long time without any problems. if you need more details let me know
Justin's answer pretty much covers everything. The only thing I would add is to maybe think about using multiphoton excitation if you continue to have issues. Dil can be excited at ~ 700 nm and this would significantly reduce photobleaching, as well as increase axial/lateral resolution due to the two-photon effect.
Thank you very much everyone, especially Justin, valuable information, now I'm performing my experiments and I am incorporating your advice.
Dear Alejandra,
I also agree with Justin's comments. However, you don't mention how the DiI is being exposed to UV light--are you also using another stain, like DAPI/Hoechst for nuclei, or Fluorogold? I did find out the hard way that one should image the UV channel last when viewing DiI labeled axons in a tissue that has also had Fluorogold, but then I label a very small number of fibers in the tissue (on purpose). If you are also labeling with a second dye that is excited in the UV, be sure to acquire that channel last.
In addition to the lipophylic dyes available from Molecular Probes, there are also the NeuroVue dyes (available from Polysciences here in the US). A publication from Bernd Fritzsch is posted here on ResearchGate:
https://www.researchgate.net/profile/Bernd_Fritzsch/publication/5687008_Long-distance_three-color_neuronal_tracing_in_fixed_tissue_using_NeuroVue_dyes/links/0912f50b4fe62a321d000000.pdf
I also have a question for David--have you had good success with MP of DiI? We have been underwhelmed, to say the least, with the signal (it could be the thickness of our tissue, though). If you have any hints, I would greatly appreciate hearing them.
Best of Luck,
Jill
Article Long-Distance Three-Color Neuronal Tracing in Fixed Tissue U...
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