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350nm laser delivered into a microscope

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Peng Xi-2

350nm laser delivered into a microscope

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/

Barbara Foster

Re: 350nm laser delivered into a microscope

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

Hi, Peng Xi

The big issue is going to be the transmission of any of the optics,
from the fiber optic through the microscope (including prisms and
objectives). Most glassware drops off abruptly somewhere between 360
and 380. My best advice: have a long chat with Nikon tech support.

Hope this was helpful,

Best regards,
Barbara Foster, President

Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: (972)924-5310
Skype: fostermme
W: www.MicroscopyEducation.com

MME is now scheduling customized, on-site courses through July
2008. Call us today for details.

We are sorry to report that Optimizing Light Microscopy for the
Biological and Clinical Laboratory is no longer available.

At 04:53 PM 2/3/2008, Peng Xi 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

Re: 350nm laser delivered into a microscope

In reply to this post by Peng Xi-2

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:

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
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)

Michael Weber-4

Re: 350nm laser delivered into a microscope

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

Hey Peng,

another way of bringing the laser to the object plane would be not
through the microscope ports, but directly below the objective via a
dichromatic mirror for UV. For example people in Czech Republic (Brno)
build a little laser coupling device that one can screw between
objective and turret. This way you can get around the spectral response
issues with the microscope prisms.

Direct coupling of the laser or scanning system to the microscope would
allow you to work around fiber coupling issues. Then you probably need
to use existing ports - maybe one can change the prisms to good
dichromatic mirrors?

---

Beside that, I would also be interested in news about 350nm diode
lasers. We have a setup with a (nearly dead) Enterprise laser and where
looking for diode lasers to get rid of the heavy and hot beast. But the
information we got so far from companies and independent people was that
the current generation of UV diode lasers has too low quality (long-term
stability, beam shape etc.) for setups like confocals.

cheers,
Michael

George McNamara 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
> [hidden email]
> [hidden email]
> 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)

Peng Xi-2

Re: 350nm laser delivered into a microscope

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

Hi Michael,
Thank you for your reply! I think if it is heavily absorbed when
using the side port, I'll use the mercury lamp port. I have
successfullly modified a Nikon TE200 to a two-photon microscope with
the mercury lamp port, to minimize the dispersion from the reflective
prism. But still, I think it is much convenient to use the side-port
for my confocal construction, if the absorption is not an issue... :)
Thank you!

Best regards,
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/

On 2/5/08, Michael Weber <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hey Peng,
>
> another way of bringing the laser to the object plane would be not
> through the microscope ports, but directly below the objective via a
> dichromatic mirror for UV. For example people in Czech Republic (Brno)
> build a little laser coupling device that one can screw between
> objective and turret. This way you can get around the spectral response
> issues with the microscope prisms.
>
> Direct coupling of the laser or scanning system to the microscope would
> allow you to work around fiber coupling issues. Then you probably need
> to use existing ports - maybe one can change the prisms to good
> dichromatic mirrors?
>
> ---
>
> Beside that, I would also be interested in news about 350nm diode
> lasers. We have a setup with a (nearly dead) Enterprise laser and where
> looking for diode lasers to get rid of the heavy and hot beast. But the
> information we got so far from companies and independent people was that
> the current generation of UV diode lasers has too low quality (long-term
> stability, beam shape etc.) for setups like confocals.
>
> cheers,
> Michael
>
>
> George McNamara 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
> > [hidden email]
> > [hidden email]
> > 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)

Peng Xi-2

Re: 350nm laser delivered into a microscope

In reply to this post by George McNamara

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 <[hidden email]> 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
> [hidden email]
> [hidden email]
> 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)
>

Peng Xi-2

Re: 350nm laser delivered into a microscope

In reply to this post by George McNamara

Paul Rigby

Re: 350nm laser delivered into a microscope

In reply to this post by Peng Xi-2

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:[hidden email]] On
Behalf Of Peng Xi
Sent: Wednesday, 6 February 2008 11:50 AM
To: [hidden email]
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 <[hidden email]> wrote:

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
> [hidden email]
> [hidden email]
> 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)
>

George McNamara

Re: 350nm laser delivered into a microscope

In reply to this post by Peng Xi-2

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

Hi Peng,

The Zeiss LSM 510 software says it has 80 mW. That would be split
over two lines, passed through a fiber optic cable, through a
collimator lens assembly, and into the microscope. I do not have a
light meter, so do not know how much reaches the specimen. I usually
use only the 364 nm line, except for days (lenses? specimens?) where
the 364 nm line is very dim.

Best wishes with NADH and FAD imaging.

George

At 09:49 PM 2/5/2008, you wrote:

>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 <[hidden email]> 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
> > [hidden email]
> > [hidden email]
> > 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)
> >

Kevin Braeckmans

Re: 350nm laser delivered into a microscope

In reply to this post by Peng Xi-2

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

Dear Peng,

We have an Ar UV laser on our MRC1024 which is coupled through the side port
of a Nikon TE300 microscope (actually into the scanhead which is at the
sideport). This is (was) standard Bio-Rad design, so going through the
sideport should be no problem. I haven't actually measured the light losses
though ...

Best regards,

Kevin

Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens Peng Xi
> Verzonden: woensdag 6 februari 2008 3:38
> Aan: [hidden email]
> Onderwerp: Re: 350nm laser delivered into a microscope
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Michael,
> Thank you for your reply! I think if it is heavily absorbed when
> using the side port, I'll use the mercury lamp port. I have
> successfullly modified a Nikon TE200 to a two-photon microscope with
> the mercury lamp port, to minimize the dispersion from the reflective
> prism. But still, I think it is much convenient to use the side-port
> for my confocal construction, if the absorption is not an issue... :)
> Thank you!
>
> Best regards,
> 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/
>
>
>
> On 2/5/08, Michael Weber <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Hey Peng,
> >
> > another way of bringing the laser to the object plane would be not
> > through the microscope ports, but directly below the objective via a
> > dichromatic mirror for UV. For example people in Czech Republic
> (Brno)
> > build a little laser coupling device that one can screw between
> > objective and turret. This way you can get around the spectral
> response
> > issues with the microscope prisms.
> >
> > Direct coupling of the laser or scanning system to the microscope
> would
> > allow you to work around fiber coupling issues. Then you probably
> need
> > to use existing ports - maybe one can change the prisms to good
> > dichromatic mirrors?
> >
> > ---
> >
> > Beside that, I would also be interested in news about 350nm diode
> > lasers. We have a setup with a (nearly dead) Enterprise laser and
> where
> > looking for diode lasers to get rid of the heavy and hot beast. But
> the
> > information we got so far from companies and independent people was
> that
> > the current generation of UV diode lasers has too low quality (long-
> term
> > stability, beam shape etc.) for setups like confocals.
> >
> > cheers,
> > Michael
> >
> >
> > George McNamara 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
> > > [hidden email]
> > > [hidden email]
> > > 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)