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Kevin,
I'll admit, I have no idea what a notch filter is. Would you
have a moment to explain the basis of its function? I replied to you
on the list in case there are few others also curious.
Thanks,
Tobias
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>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
>> > --------------------------------------------------------------
>> > -----------------
>> >
>> > This is a multi-part message in MIME format.
>> > --------------020807090008000002070803
>> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>> > 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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