Hi. I want to use caged ligands in cell-attached configuration, as an alternative to perfusing the electrode, i.e. patch clamp -> measuring single channel w.o. ligand -> uncaging ligands in the electrode -> measure single channel with ligand. Has this ever been done before? What kind of UV illumination do I need (flash lamp/LED, intensity, radiant flux etc.)? As I don't need a focal uncaging, but rather uncaging the entire electrode, I don't want to use UV laser, but is there any other source with sufficient power to penetrate the glass of the electrode?
Perhaps a similar approach to the one in this paper would work for you - maybe then you could use an inexpensive fiber-coupled UV LED (e.g. Thorlabs M365L2 or M375L3) and obtain adequate intensity within the pipette to uncage.
Katz, Y., O. Yizhar, J. Staiger, and I. Lampl (2013, March). Optopatcher-an electrode holder for simultaneous intracellular patch-clamp recording and optical manipulation. Journal of neuroscience methods 214 (1), 113-117.
http://www.ncbi.nlm.nih.gov/pubmed/23370312
Alternatively, a high-NA immersion lens focused onto the tip of the pipette might work with UV through the standard epi-fluorescence illumination path.
We used to use a Rapp UV flash lamp for caged calcium photolysis, however, I had a chance to see the Optopatcher system at the recent Biophysical Society meeting, and I think it would work for your application. I believe that ALA Scientific Instruments markets a commercial version. With the Rapp system we illuminated via the epi-illumination pathway and we used an aperture in the field diaphragm plane to restrict illumination to the cell plus only in the tip of the patch pipette. This way, we could photolyze the caged compound localized to cell and the pipette tip, after which nonphotolyzed compound in the rest of the pipette would replace the photolyzed compound in the cell and pipette tip. This allowed multiple uncagings, with a few-minute pause between flashes.
I was uncaging with a "classical" light source (do not remember exactly but a typical mercury lamp based one) couple to a filter and send to the sample via the epiflorescence path. I had some problems diffusing the dye in the cell and I remember that the uncaging was going on very well in the pipette. Rapp systems and others will be good if you need a good time resolution and/or high power and/or short pulses.
The optopatcher is a product from AM sysytems, but developed at Weizmann Institute. The light guide goes straight into the pipette. Now, I don't know if you'll get enough energy to uncage your compound, that will depend in the photochemestry of your sample. Surely is enough excite light activated ion channels. You should talk with Drs. Lampl and Katz of the Weizmann, they developed the optopatcher.
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