Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Parfocality for confocal should be a fair bit better with one
of the new violet-corrected objectives that they all make now.
But with TIRF you don't have that option.  We use a multiline
argon for CFP/YFP FRET on our Leica but on our new Nikon C1 we
have a pulsed 440nm diode (not DPSS) laser.   This works for
both conventional imaging and FLIM.  Very preliminary tests
(we are still setting up the system) seem to show reasonable
parfocality through 407nm, 440nm, 488 and 561.  But I'll
do some serious testing once it's all fully comissioned.
One thing with these diode lasers is that they actually vary
a bit in wavelength, and we specifically asked for a 'short'
440nm laser.  But I haven't yet checked exactly what it's
giving.

                                                      Guy

> Both TIRF and parfocality will depend on correction for chromatic
> aberrations (at least a tiny bit for the latter and maybe more for
> TIRF). For TIRF, the "critical" angle is a function of the lens and
> medium RI. If your objective is not perfectly corrected chromatically
> over all wavelengths and/or the medium RI is sensitive to wavelength
> (which it is), then the critical angle for different lines will be
> different and require adjustment of the laser angle for each line to
> get TIRF to function. As we all know, not all 1.45 or 1.6 NA
> objectives perform alike.
>
> As Michael points out, at 440 nm he gets good performance but must
> change the angle when he excites at 488 nm and longer. For a
> different objective, different angles and performance are to be
> expected at different wavelengths.
>
> Mario
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>At 12:25 PM 02/09/05 -0500, you wrote:
>>>Search the CONFOCAL archive at
>>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>Thanks for the info George.  You bring up a good point with the
>>>parfocality issue.  I'm actually more interested in TIRF applications,
>>> so
>>>it makes me wonder if parfocality will be more or less of an issue for
>>> TIRF?
>>>Sarah
>>
>>
>>The angle for TIRF varies with wavelength (at least with the Olympus N.A.
>>1.45 objective).  We have a 440nm diode laser which works great for CFP,
>>but when we want to use excitation at 488 nm we need to change the
>>angle.  Often we can get away with one angle for 488 and 543, but even
>> here
>>tweaking at 543 improves the TIRF.
>>
>>
>>
>>____________________________________________________________________________
>>Michael Cammer   Analytical Imaging Facility   Albert Einstein Coll. of
>> Med.
>>Jack & Pearl Resnick Campus      1300 Morris Park Ave.     Bronx, NY
>> 10461
>>(718) 430-2890       Fax:  430-8996      URL:
>> http://www.aecom.yu.edu/aif/
>>   **This electronic transmission contains information that is
>> privileged.**
>
>
> --
> ________________________________________________________________________________
> Mario M. Moronne, Ph.D.
> NanoMed Technologies LLC
> President and CTO
> ph (510) 528-2400
> FAX (510) 528-8076
> 1561 Posen Ave
> Berkeley, CA
> 94706
>
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--
Associate Professor Guy Cox
Electron Microscope Unit,
University of Sydney,
NSW 2006, Australia

Phone:+61 2 9351 3176    Fax:+61 2 9351 7682
http://www.guycox.net