Re: PSF with DIC
Posted by
Bruno Afonso on
URL: http://confocal-microscopy-list.275.s1.nabble.com/PSF-with-DIC-tp3781952p3785576.html
hey,
We had to remove our prism in our 100x DIC objective (in a nikon te2000) when doing sub-difraction fluorescence microscopy. We were getting the same you saw with the beads. So, imaging beads will affect also widefield microscopy when using the prism.
b
On Wed, Oct 7, 2009 at 09:42, MODEL, MICHAEL
<[hidden email]> wrote:
While I'm here...has anyone properly investigated the effect of the DIC objective prism in confocal fluorescence imaging? I had always assumed (rightly or wrongly) that it's presence didn't influence the PSF, but last week I was imaging some subresolution
beads and found that, particularly on our IX81-based FV1000 confocals, the DIC objective prism had quite a pronounced effect on the psf. Specifically the psf was distorted along a diagonal axis and at the point of focus, the bead appeared significantly larger
with the prism in place. The implication of this is that for confocal fluorescence imaging, the resolution of the microscope is reduced when the DIC objective prism is in place. I've also looked on our Zeiss Axiovert 200 and Nikon TE-2000 based systems which
employ a slightly different method of DIC and there the effect is much less pronounced although noticeable.
Simon
Simon - thanks for your input on the noise issue - and as for the DIC prism, I haven't looked at the PSF but have noticed slight deterioration in resolution of fluorescent details, and try to remember to take it out when not needed.
(Besides, the handle gets on the way of our Prior stage and has been broken several times).
Mike