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I put the gel with the beads inside on the coverslip and I also squirt some
fluorescent liquid on the one corner of the coverslip ,once I find the
focused image of the fluorescent liquid I set the position on zero and then
I go through the agarose gel,by this way and by repeating the experiment for
at least 7 times,I am able to go deep up to 640um (the lens is Olympus
uplapo 1.2 60x)!

Sarah

On Feb 5, 2008 7:11 PM, Julio Vazquez <[log in to unmask]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> - Sarah,
>
> To follow up on Jim's, the only 60x/1.2 objectives I know of are PlanApo
> water immersion lenses, and they tend to have working distances of about
> 200-240 microns...  unlikely you could focus on a sample at a depth of 600
> microns, even without coverslip (which you should be using)...
>
> If you want to get deeper (and have enough resolution and brightness to
> see your beads), you should consider a 10x water immersion lens, or a long
> working distance water dipping lens. Don't know about Nikon, but Zeiss has a
> 10x/0.45 C-Apo water immersion, and a 40x and/or 60s/0.8 water dipping
> lenses, all with about 1.5 -2.00 mm working distance.
>
> What do you mean by penetration depth anyway? If you are trying to measure
> the working distance of the lens, you can get that from the objective's
> specs on the vendor's catalog...
>
> If you are trying to determine the location (depth) of a a bead inside a
> gel, you should have a look at these pages (and possibly others on the same
> site):
>
>
> http://www.microscopyu.com/articles/formulas/formulascoverslipcorrection.html
>
> http://www.microscopyu.com/articles/optics/waterimmersionobjectives.html
>
>
>
>
>  --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA 98109-1024
>
> http://www.fhcrc.org/
>
> =
>
>
>  On Feb 5, 2008, at 3:18 PM, Sarah Kefayati wrote:
>
>  Hello all!
>
> I have used the latex beads inside the agarose gel and I have measured the
> penetration depth of NA: 1.2 60X lens about 600um,could it be wrong?
>
> Thanks
> Sarah
>
>
>