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Hi Andrea,
This discussion is something I've not found a definitive answer for yet.
From Inoué, S. (1986) Video Microscopy. Plenum Press, New York, p.114, the
lateral resolution is calculated using x=(1.22 lamda)/2NA. If your numbers
are plugged into this, it yields 225nm. I'm particularly concerned about
this because I have a web page on this topic
http://www.mcb.arizona.edu/IPC/zoom_Page.htm, and I'm still not comfortable
with what users might take as the gospel. Another burning question is
z-resolution, and how the pinhole size effects this, if at all. Finally,
why is there uncertanty about how to determine these values?
Regards,
Carl
Carl A. Boswell
Dept. of Mol. Cell. Biology
Univ. of Arizona
(520) 626-8469
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----- Original Message -----
From: "andrea manazza" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Monday, December 02, 2002 8:39 AM
Subject: Re: confocal resolution
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Kathy,
the maximum resolution for optic microscopy should be 200 nm, according
to standard physics laws: that means that any higher resolution will
give you no more information on the sample.
As for useful formulas:
- the Nyquist theorem for lateral resolution should be similar to
X= 0,4*lambda/NA, where NA is numerical aperture of your objective and
lambda the wavelength. It means that, if you’re working with 63x
objective and 1,32 as NA, at 488nm, the lateral resolution is 148nm;
dividing this number by 3, you obtain almost 49 nm. 49 is the raster
distance you should set in your scan, in order to avoid loss of info
from the specimen —> it does NOT mean that you will obtain resolution
higher than 200nm, but only that you’ll obtain good data by scanning
thrice the 148nm distance obtained before; in order to modify the
raster distance you should change the electronic zoom and the scan
format.
At least, this is what I've found; if anyone else has nice data, I'll
be interested as you in reading more :)
Thank you and have a nice time.
AM
dott. Andrea Manazza
Dipartimento di Oncologia, Università di Torino
CeRMS, ospedale Molinette
Via Santena, 7 - 10126 Torino, Italia
tel: +39-11-6706522
lab: +39-11-6336859
fax: +39-11-6336887
----- Original Message -----
From: Kathy Spencer <[log in to unmask]>
Date: Wednesday, November 27, 2002 9:32 pm
Subject: confocal resolution
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello All!
> Basic generic question....what is the limit of resolution
> on a
> confocal? I'm using an Olympus Fluoview confocal, 60x 1.4NA
> objective, oil
> (1.516), pinhole automatically set for 1 Airy disk. For 488nm
> laser, I
> calculate 174nm using resolution=wavelength/2NA. But can the confocal
> resolve more than that? What is a good formula?
> Thanks!
> Kathy
>
>
>
>
>
>
>
>
> Kathy Spencer
> The Scripps Research Institute
> 10550 N. Torrey Pines Road
> ICND 202
> La Jolla, CA 92037
> 858-784-8437
>
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