Posted by
Satoru Uzawa on
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Hi,
When I imaged 15 nm gold beads on Leica SP2, I had to use hardening mounting media (I tried both ProLong Gold and Vectashield and ended up using ProLong Gold) to “fix” the gold beads. Glycerol based mounting media are useless here. Also, agarose will not make individual beads immobile. Many beads got stuck to either the coverslip or to the slide glass, but I could find some that were in the middle. Gold beads stuck on the coverslip are useless, since the reflection form the glass surface will dominate the signal.
I used 12 uL of mounting media with series of dilution of the gold beads to mount with 18x18 mm coverslips. Vortex hard before you mount them. If you can sonicate them, that’s better. I hanged the slide upside down while they were curing to increase the distance from coverslip to slide (to be honest, I don’t know how much it helped, though). As far as I can tell, the slide lasted quite long time, more than a month.
I would start with low dose of laser with PMT, but not with HyD or APD. To find beads, you can start at the surface of the coverslip and move slightly into the mounting media where you will find some beads on a slide with high concentration of beads. The faintest stable signal there would be from a single bead. Once you know the intensity from the single beads, you can search one further inside the mounting media on a slide with lower concentration of beads. In your case, it should be easier to find beads as they are substantially larger than what I used. I noticed that you may not need to identify any single gold bead, so you could just go with high concentration of beads. I don’t think the distributor/manufacturer of the gold beads makes any difference for imaging.
Good luck!
Best,
Satoru
______________
Satoru Uzawa
Meyer lab, HHMI/U.C. Berkeley
Dept. of MCB
131 Koshland
Berkeley, CA 94720-3204
> On Jul 17, 2017, at 10:55 AM, Arvydas Matiukas <
[hidden email]> wrote:
>
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>
> Dear microscopists,
>
> We are building STED microscope, and now are at the step of
> aligning 488nm excitation and 580nm depletion beams in the focal plane
> by imaging gold 100nm beads (from Sigma). Surprisingly it was difficult
> to see the reflected signal off the beads either visually, using CCD camera,
> or APD detector (while 83nm green fluorescent beads produced a nice image).
> So far I was able to see the beads in a 1uL drop of stock solution squeezed
> between coverslip and slide (visually or with CCD camera at slightly defocussed 580 laser
> beam) . However they were violently moving due to Brownian
> motion. Diluting and immobilizing beads in agarose made them "dissappear".
>
> Please share your experience of imaging gold nanobeads (~100nm in diameter).
> Specifically I have three questions:
> 1. Is there a preference for a specific manufacturer ? Do the beads degrade with time
> (my are not fresh but the bead stock still has pale red color).
> 2. Please share a bead slide preparation protocol that is working.
> 3. Any advise on illumination and detection settings? How bright the bead reflection
> signal is expected to be?
>
>
> Thanks,
> Arvydas
>
>
>
>
>
>
>
>
> *******************
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> SUNY Upstate Medical University
> Neuroscience & Physiology Dept
>
> Room 4607 IHP
> 505 Irving Ave
> Syracuse, NY 13210