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Okay, when hitting the send button I just realized my mistake. The notch
filter in the epifluo cube will not work on the confocal since the
excitation path coincides with the emission path at that point. Sorry for
that. It is different on our widefield setup.
On the confocal you would have to put the notch filter on the emission side
of the first dichroic. Generally you won't have access to that very easily.
I guess you will have to wait for the manufacturers after all ...
Best regards,
Kevin
Kevin Braeckmans, Ph.D.
Lab. General Biochemistry & Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89
E-mail: [log in to unmask]
> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List
> [mailto:[log in to unmask]] Namens Reece, Jeff
> (NIH/NIEHS) [E]
> Verzonden: dinsdag 5 september 2006 22:38
> Aan: [log in to unmask]
> Onderwerp: Re: High NA objectives for confoal microscopy
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I'll start with a minor point. The RI of cytoplasm has been
> reported in the range 1.35-1.37. So if you use RI=1.36
> instead of 1.33, the critical angle is now 63 degrees. On a
> confocal microscope, the intensity of excitation light within
> the solid angle between 63 and 67 degrees is much less than
> that within 0-63 degrees. I would guess the proportion
> varies wildly from manufacturer to manufacturer, microscope
> to microscope within a manufacturer, and possibly even laser
> to laser on a single microscope. I wouldn't trust any
> theoretical derivations of this.
>
> On the other hand, when you are talking about excitation at
> z=0, the coverglass surface, from angles close to the
> critical angle, the TIRF excitation is about 4-5x more
> efficient than the non-TIRF excitation (according to
> Axelrod's 1984 paper; TIRF users might have noticed this
> non-intuitive effect). So, it is a good question, especially
> if you are trying to justify the purchase of a $8-10k water
> immersion lens: how much of the confocal signal at z=0 is
> actually due to TIRF? You should be able to get a decent
> measure of this if you have NA=1.4 and NA=1.3 lenses, looking
> at two focal planes: z=0 and a couple of microns further into
> the sample. The sample ideally would have sparse, punctate
> fluorescence in both focal planes.
>
> A related question: for most microscope users with fixed
> samples, does the RI mismatch problem go away? I know that
> the subject of mounting media has been discussed before on
> this listserv, but I haven't found from that discussion or
> anywhere else a mountant that prevents photobleaching, has
> RI=1.52, solidifies, and does not require dehydration of the
> sample. Please everyone, let me know if such a mountant exists.
>
> Now to one of my current pet peeves, but I think it's an
> important tangent. I think the biggest problem for most
> confocal users using
> NA=1.4 and a sample of unmatched RI is not spherical
> aberration or TIRF signal. It is the sometimes significant
> reflection that gets back to the detector when near the cover
> glass, especially with low fluorescence signal. This
> situation does not go away if everyone uses the perfect
> mounting medium on fixed cells. Many confocal users with
> live cells are interested only in the region just above the
> coverglass, and of course would rather use the NA=1.4 lens
> because of its better resolution and more efficient light
> collection. Having TIRF going on at the same time could
> actually be advantageous. In such cases, the filters used by
> the manufacturers do not provide enough blocking of the
> excitation light.
> My hope is, for the practical benefit of this significant
> group of users, that the microscope designers have already
> started addressing this issue, taking advantage of recent
> advances in filter technology. I propose that, with NA=1.4
> and detector gain at its maximum, one should not be able to
> detect the glass-water interface with an XZ scan.
> Accomplishing this should require a relatively inexpensive
> upgrade to most existing CLSM systems, as well as adding
> little to the production cost of new systems. If I'm wrong
> about the cost, let it be an option.
>
> Cheers,
> Jeff
>
> Jeff M. Reece
> Confocal Microscopy Center
> National Institute of Environmental Health Sciences (NIEHS)
> National Institutes of Health (NIH) P.O. Box 12233, MD F2-02
> Research Triangle Park, NC 27709
> (919) 541-0311
> [log in to unmask]
>
>
>
> > -----Original Message-----
> > From: Haridas Pudavar [mailto:[log in to unmask]]
> > Sent: Sunday, September 03, 2006 5:24 PM
> > To: [log in to unmask]
> > Subject: High NA objectives for confoal microscopy
> >
> > ---------------------- Information from the mail header
> > -----------------------
> > Sender: Confocal Microscopy List
> <[log in to unmask]>
> > Poster: Haridas Pudavar <[log in to unmask]>
> > Subject: High NA objectives for confoal microscopy
> > --------------------------------------------------------------
> > -----------------
> >
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> > Content-Transfer-Encoding: 7bit
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Hi,
> > I was wondering what is the highest NA objective one can use for
> > confocal without getting into total internal reflection at
> > coverslip/media interface. It seems to me that with NA 1.4,
> the angle
> > of incidence (~67 degree) is already higher than critical
> angle. (~ 61
> > degree assuming a RI of 1.52 for oil and 1.33 for cell/media.)
> > Therefore, NA above 1.3 (~ 57 degree for 1.3, which is
> smaller than
> > critical angle)will give huge TIR from coverslip/media interface.
> > But all the microscopy manufacturers are selling 1.4 NA objectives
> > for high resolution confocal microscopy.
> > Am I making any fundamental mistake in the calculation ?
> > Can somebody help me?
> >
> > Haridas
> >
> >
> >
> > --
> > Haridas Pudavar, Ph.D.
> > Research Asst. Professor,
> > Inst. for Lasers, Photonics & BioPhotonics,
> > 458, NSC, Dept. of Chemistry,
> > SUNY at Buffalo, Buffalo, NY-14260
> > Ph: (716) 645 6800 x 2220
> > Fax: (716) 645 6945
> >
> >
> > --------------020807090008000002070803
> > Content-Type: text/html; charset=ISO-8859-1
> > Content-Transfer-Encoding: 7bit
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > <!DOCTYPE html PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
> > <html>
> > <head>
> > <meta content="text/html;charset=ISO-8859-1"
> > http-equiv="Content-Type">
> > </head>
> > <body bgcolor="#ffffff" text="#000000">
> > <p class="MsoNormal">Hi,<br>
> > I was wondering what is the highest NA objective one can use
> > for confocal without getting into total internal reflection at
> > coverslip/media
> > interface. It seems to me that with NA 1.4,<span style="">
> > </span>the angle of incidence (~67 degree) is already higher than
> > critical
> > angle. (~ 61 degree assuming a RI of 1.52 for oil and 1.33 for
> > cell/media.)<span style=""> </span>Therefore, NA
> above 1.3
> > (~ 57 degree for 1.3, which is smaller than critical
> > angle)will give
> > huge TIR from coverslip/media interface. <span style=""><br>
> > </span>But all the microscopy manufacturers are
> > selling 1.4 NA objectives for high resolution confocal
> microscopy.<br>
> > <span style=""></span>Am I making any fundamental mistake in the
> > calculation
> > ?<span style=""> </span><br>
> > Can somebody help me?</p>
> > <p class="MsoNormal"><o:p> Haridas<br>
> > <br>
> > </o:p></p>
> > <br>
> > <br>
> > <pre class="moz-signature" cols="72">--
> > Haridas Pudavar, Ph.D.
> > Research Asst. Professor,
> > Inst. for Lasers, Photonics & BioPhotonics,
> > 458, NSC, Dept. of Chemistry,
> > SUNY at Buffalo, Buffalo, NY-14260
> > Ph: (716) 645 6800 x 2220
> > Fax: (716) 645 6945</pre>
> > </body>
> > </html>
> >
> > --------------020807090008000002070803--
> >
>
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