Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Peng (and George),
We currently run an Enterprise II that produces 60mW of 351/364nm UV on
an old Biorad MRC1024 UV system. When imaging probes like Hoechst or
DAPI, we routinely use 10mW 351nm output on the laser with a 10%
transmission ND filter in the excitation path. This should lead to
approx 1mW minus any losses in the optics of the scanhead and
microscope. While I haven't measured the power directly, I suspect we
are only getting, at most, 100 - 200uW at the sample.
Given that our second Enterprise laser is almost at the end of its life
(currently over 5000 hours), I was also thinking of purchasing a 20mW
375nm laser for imaging our probes. I would have thought that 20mW would
be sufficient power to image your autofluorescence, provided there are
not significant losses in the optical path. I would be very interested
to hear of your experience with this laser should you decide to purchase
it. Please keep us informed of your progress.
Regards
Paul
Dr Paul Rigby
Senior Lecturer
Centre for Microscopy, Characterisation and Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley WA 6009
Ph (61-8) 9346 2819
Fx (61-8) 9346 3469
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Peng Xi
Sent: Wednesday, 6 February 2008 11:50 AM
To: [log in to unmask]
Subject: Re: 350nm laser delivered into a microscope
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi George,
Nice to hear from you. Do you seriously need 80mW -- when I did
autofluorescence spectroscopy in Hong Kong I used no more than 1mW on
sample (355/457nm), otherwise the photobleaching is very huge. I am
planning to buy a 20mW 375nm laser, it will be bad if I end up with no
signal. Please help me to double check the power on your 364nm line
(it is not surprise for an argon laser to have 80mW output on
488nm/514nm).
It will be nice if I can use DAPI, but I plan to image the
metabolic state with autofluorescence of NADH/FAD, through in vivo
imaging. :) Thank you for reminding me with GFP! Long time ago we have
an transgenic mouse that have GFP, but I need to know more about it
since I am not a biologist.. :)
Today is the Chinese New year, and I wish all of you a very happy
2008!
Peng
Associate Professor
Institute for Laser Medicine and Biophotonics
Shanghai Jiao Tong University
800 Dongchuan Rd.
Shanghai 200240, China
Tel: (86) 21-3420-4076
http://biophotonics.sjtu.edu.cn/
On 2/5/08, George McNamara <[log in to unmask]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Peng,
>
> I estimate between 5 and 10% of confocal systems have a 351/364 nm
> laser like the 80 mW Coherent Enterprise on the Zeiss LSM 510 in our
> core (I suspect not a whole lot of the 80 ends up at the specimen for
> most objective lenses. Our 63x/1.4 NA lens is downright dim). We have
> an Axiovert 200M with the scanhead on the side port. Inside the scan
> head's two fiber input ports are collimator lenses to adjust the
> focus for UV and visible light respectively (or vis and IR for the
> 510 multiphoton systems). Keep an eye on
> http://www.zeiss.com/sensitivity for the next generation.
>
> My experience has been that most autofluorescence is pretty similar
> from UV through green excitation (elastin being a partial exception).
> In the past I found that a CFP filter set worked nicely to estimate
> the autofluorescence component of a GFP set (both off the shelf
> Chroma filters).
>
> I recommend you focus more on the 364 nm laser line, if you are using
> a UV Argon ion laser that has both. I also recommend using UV to
> image moderate to high concentration DAPI, rather than low SNR dim
> autofluorescence.
>
> If you are using a solid state laser for 350 nm, please let me know
> what kind so I can consider replacing my Enterprise.
>
> George
>
>
> At 07:51 AM 2/4/2008, you wrote:
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Hi,
> > Has anyone have the experience of delivering 350nm laser in to a
> >microscope? I'm planning to use this line for autofluorescence
> >excitation with a Nikon microscope, I'll use the side-port to deliver
> >the excitation to construct a custom-made confocal setup, but I don't
> >know the absorption ratio. Currently I'm worrying about the
absorption
> >from the reflective prism; if the absorption is very large I'll
> >consider to use the back port.
> > Thank you for any input!
> >
> >Best,
> >Peng Xi
> >Associate Professor
> >Institute for Laser Medicine and Biophotonics
> >Shanghai Jiao Tong University
> >800 Dongchuan Rd.
> >Shanghai 200240, China
> >Tel: (86) 21-3420-4076
> >http://biophotonics.sjtu.edu.cn/
>
>
>
>
>
>
> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [log in to unmask]
> [log in to unmask]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc
> (Analytical Imaging Core Facility)
>
|
