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Search the CONFOCAL archive at
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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
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >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 Dear Sara, You don't say, but I believe that the (Nikon?) objective you describe is supposed to be used with a coverslip. If you just use it just with agarose, two things will happen: You will have VERY high spherical aberration (and therefore z-direction measurements are not worth much) and, because the coverslip is missing, the working distance will appear to be more than the (about ) 200 µm that the lens was designed for. Add a 170 µm coverslip on top of the agarose, and you should be able to see about 200 µm below the agarose/coverslip interface. Cheers, Jim P. -- ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2008 "If it ain't diffraction, it must be statistics." Anon. |
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In reply to this post by Sarah Kefayati
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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): -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 = On Feb 5, 2008, at 3:18 PM, Sarah Kefayati wrote:
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Search the CONFOCAL archive at
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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 <[hidden email]> wrote:
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal That lens definitely cannot focus so deeply. How firm is your agarose? You may be bringing the lens into contact with the coverslip and flattening the gel. Glen On Feb 5, 2008, at 4:15 PM, Sarah Kefayati wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- > bin/wa?S1=confocal > 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 <[hidden email]> 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 > > |
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
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Sarah,
Olympus has two 60x/1.2 water lenses (check their online catalog): their working distance is 250 and 280 microns, respectively. Therefore, it is not possible to focus 640 microns past the coverslip. The most you can go until you touch the coverslip is 250/280 microns. Water lenses were designed so that people could see deeper into acqueous samples, which could not be achieved with oil immersion lenses due to mismatch of refractive index between oil (about 1.52) and water (about 1.34). Agarose, however, is not water, and the refractive index of your gel could be as high as 1.4. Therefore, even with a water lens, you will get spherical aberrations that will distort your image and result in false perceptions of depth. The other possibility is that your stage or focus drive is way out of calibration, and that you are actually not focussing 600 microns when you think you are... Julio. On Feb 5, 2008, at 4:15 PM, Sarah Kefayati wrote:
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
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Dear Sarah,
I didn't find a "uplapo 1.2, 60x" but I did find a
"UPLSAPO 60XW, 1.2 NA. The latter lens is listed at
http://www.olympusamerica.com/seg_section/files/bro_objectives.pdf
as having a working distance (max distance that one can focus into a
specimen mounted on the far side of a coverslip of specified
thickness, of 280 µm (This lens has a collar that corrects for
coverslips between 0.15 and 0.2 mm thick).
So I doubt that you are really looking into the specimen 640
µm. Is this really the name on your lens? How thick is your coverslip?
Where is the collar set? How are you measuring distance? Is the
z-motion perhaps miscalibrated (move it 1000 µm, and set if it is a
mm)?
Cheers,
Jim P.
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 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 <[hidden email]> wrote: --
**********************************************
Prof. James B. Pawley, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 14-26, 2008, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ |
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
The exact name of the lens wich is mentioned on the lens is: UPlanApo/IR 60X/1.2 W !
Regards
sarah
On Feb 6, 2008 10:08 AM, James Pawley <[hidden email]> wrote:
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