Posted by
Arvydas Matiukas on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Trying-to-image-Cy7-5-AxioObserver-Z1-blocking-IR-tp7586976p7587000.html
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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