*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Artur,
I worked a bit on this during my PhD. I published a protocol for making such substrates in an appendix of a paper:
Combined scanning probe and total internal reflection fluorescence microscopy
In the journal Methods (2008)
I never used these for SPT, but I would think the key is to cut the mica as thin as possible. This took a lot of practice to get good at. You really want the thickness to be such that the mica sheet when pinched between your fingers at the edge flops downwards or flaps a bit when you wave it in the air. Start by cleaving a single mica piece with a fresh razor blade. Poke the edge and work it around the edges a bit. Eventually you'll be able to easily pull the two pieces apart. Repeat this process on the cleaved pieces until you get a good one with the thickness properties I described above.
Use only a very small amount of optical adhesive to join this piece to a glass coverslip. Let it spread before curing.
If you can, try rotating the TIRF polarization to the "p" position, you might get a bit more signal that way too.
If you need more details on this, contact me offline. Maybe I can make a YouTube video showing my method sometime when I get a chance.
John Oreopoulos
On 2012-04-09, at 2:51 AM, Artur M <
[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Dear all,
>
> do any of you have an experience with mounting mica on coverslips for TIRF-SPT
> (SPT of labeled lipids in supported lipid membranes)? So far I was doing it by
> gluing thin slices of mica to coverslips with NOA63, but I think thickness is still to
> high and I'm loosing a lot of resolution (and signal). It will be fine for 'normal'
> fluorescence studies, but I can forget about SPT. Any hints?
> Thanks,
> AM.
Locked
