deep UV excitation (280nm)

[darren.thomson]

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Hello All,

We have a compound which annoyingly excites at 280nm. We need to detect whether it has entered the cell or not.
Is anyone aware of an excitation source this deep? Would we need to go back to mercury lamps and try tease out with a custom filter?

Cheers,

Dr Darren Thomson
Manchester Fungal Infection Group, UK.

[mmodel]

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I am afraid that tryptophan would also fluoresce in this range



Mike Model


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Darren Thomson <[hidden email]>
Sent: Tuesday, January 24, 2017 9:50 AM
To: [hidden email]
Subject: deep UV excitation (280nm)

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Hello All,

We have a compound which annoyingly excites at 280nm. We need to detect whether it has entered the cell or not.
Is anyone aware of an excitation source this deep? Would we need to go back to mercury lamps and try tease out with a custom filter?

Cheers,

Dr Darren Thomson
Manchester Fungal Infection Group, UK.

[Andrea Latini]

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dear Darren,
you can use a Deuterium source. for instance Princeton Instruments sells model DS-421.
"
30-watt light source, 110 V, provides useful UV continuum starting at ~190 nm and continuing out to ~350 nm. Negligible visible light output helps to minimize stray light."

cheers.

Andrea Latini
CrestOptics Spa

[Ryan Schreiner]

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Another issue, in addition to the tryptophan fluorescence noted by Mike, is
the excitation light's transmittance through the objective.  Many
objectives have little to no transmittance of light below 300nm.

Ryan Schreiner

On Tue, Jan 24, 2017 at 10:08 AM Darren Thomson <
[hidden email]> wrote:

> *****
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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello All,
>
> We have a compound which annoyingly excites at 280nm. We need to detect
> whether it has entered the cell or not.
> Is anyone aware of an excitation source this deep? Would we need to go
> back to mercury lamps and try tease out with a custom filter?
>
> Cheers,
>
> Dr Darren Thomson
> Manchester Fungal Infection Group, UK.
>

[George McNamara]

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Hi Darren,

LEDs are now available in the deep UV. You do not need to go through the
objective lens to excite fluorescence (MUSE is typically imaging in the
visible). For example (see web pages for images ... the first image on
the muse site reminds me of this image
http://uvmbored.com/event/rocky-horror-picture-show ):

http://www.musemicro.com

Based on IP developed jointly at Lawrence Livermore and UC Davis, the
MUSE (Microscopy with Ultraviolet Sectioning Excitation) Microscope uses
short-wavelength UV light which penetrates only microns-deep into tissue
eliminating the need for precision-cut, thin specimens and even slides.
In addition, short-wavelength UV light excites many fluorescent dyes
simultaneously, for snap-shot color images. The result is stunning
detailed images conveying a degree of resolution, structure and depth
unachievable until now by any single technology

Sample preparation is simplified from hours to minutes with a process
that does not required trained personnel, organic solvents or related
expense. The technique does not alter samples, is non-destructive and
thus preserves valuable tissue for molecular studies. Moreover, MUSE
Microscope images offer a large field of view for whole-slide-like
digital imaging capability and can be easily and quickly formatted
through application software to provide familiar, H&E appearance, with
diagnostic-quality, subcellular detail to allow easy transition for
pathologists used to viewing standard slides.


http://www.fasebj.org/content/30/1_Supplement/51.3


  MUSE: A New, Fast, Simple Microscopy Method for Slide-Free Histology
  and Surface Topography

 1. Richard M Levenson
    <http://www.fasebj.org/search?author1=Richard+M+Levenson&sortspec=date&submit=Submit>and
 2. Farzad Fereidouni
    <http://www.fasebj.org/search?author1=Farzad+Fereidouni&sortspec=date&submit=Submit>
 3.    Dept. of Pathology and Laboratory Medicine, UC Davis Medical
    Center, Sacramento, CA
 4.


    Abstract

*Introduction*Conventional brightfield or fluorescence microscopy works
best with thin, stained specimens mounted on glass slides, but to
prepare these requires hours of processing and the help of highly
skilled technical personnel. We describe a new, inexpensive form of
light microscopy that can generate high-quality histology and
histopathology images directly from cut surfaces of fresh (or fixed)
tissue samples of any thickness, with less than 1 minute of preparation.

*Background*While alternative microscopy-scale methods, such as confocal
or two-photon, or optical coherence tomography approaches, can generate
reasonable images from thick tissues, the instrumentation is typically
complex, expensive and not widely available. We are developing an
alternative technique, MUSE (Microscopy with UV Surface Excitation),
that relies on simple properties of light, tissue, and fluorescent
stains, and can be implemented at relatively low cost. Thick pieces of
tissue obtained by biopsy, surgery or necropsy can be imaged directly.
The method is non-destructive, and eliminates requirement for
conventional histology processing, formalin fixation,
paraffin-embedding, or thin-sectioning.

*Method*Ultraviolet light at about 280 nm from an LED arranged to
provide oblique illumination, is used to excite just the surface layer
of tissue that has been briefly (~10 seconds) stained with common and
inexpensive fluorescent dyes. Unlike light of longer wavelength, 280-nm
light only penetrates to a depth of 10 microns or less, and thus excites
fluorescent signals, conveniently in the visible range, only from the
cut specimen surface. The images, which can be diffraction-limited, are
captured using conventional microscope optics and a standard color camera

*Results*MUSE samples from fresh (or fixed) tissues stained with general
tissue stains such as rhodamine and Hoechst generate high-resolution
fluorescence images that can be converted in real time from fluorescence
to H&E-like brightfield appearance, for ease of interpretation. However,
fluorescence display mode preserves surface shading and depth cues that
allow for appreciation of surface profiles important in understanding 3D
organization of complex specimens. We have obtained high-quality MUSE
images from normal and diseased tissues from multiple organisms, as well
as from plants, and manufactured materials. Moreover, surface profile
information provides views beyond what can be seen with in standard
thin-sectioned material, generating results that can resemble those
obtainable with scanning electron microscopy (SEM).

*Conclusion*We have developed a new form of optical microscopy that
generates diagnostic-quality histological images, with enhanced content,
from fresh or fixed, but unsectioned tissue, rapidly, with high
resolution, simply and inexpensively. We anticipate that there could be
widespread adoption in preclinical and clinical research facilities, as
well as hospital-based and stand-alone medical settings.


On 1/24/2017 9:15 AM, MODEL, MICHAEL wrote:

> *****
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> *****
>
> I am afraid that tryptophan would also fluoresce in this range
>
>
>
> Mike Model
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Darren Thomson <[hidden email]>
> Sent: Tuesday, January 24, 2017 9:50 AM
> To: [hidden email]
> Subject: deep UV excitation (280nm)
>
> *****
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> *****
>
> Hello All,
>
> We have a compound which annoyingly excites at 280nm. We need to detect whether it has entered the cell or not.
> Is anyone aware of an excitation source this deep? Would we need to go back to mercury lamps and try tease out with a custom filter?
>
> Cheers,
>
> Dr Darren Thomson
> Manchester Fungal Infection Group, UK.

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Johannes Helm]

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Good afternoon,

avant propos:
Is your substance anything fluorescent or will the signal light, you
want to see, be produced in another way?

280nm directly - without using any multi photon approach - could be
challenge, indeed, and for quite a number of reasons so:

a)
There are not so many microscopes around, which are equipped with optics
that would be suitable for this wavelength. The sole one, which I
spontaneously can think of, comes from Zeiss and includes Ultrafluar
objectives and a special tube lens. I have worked on an "Axioskop" once,
which had been equipped with these elements, and also used the old 160mm
Ultrafluar objectives. However, that was in the early 90s ( and I also
had some Bausch & Lomb catadioptric objectives, and I had all those on
loan from Professors Thorbjörn Caspersson and Rudolf Rigler from the
Karolinska Institute, whose help I gratefully mention even here).

b)
Most conventional light sources (Hg-lamps, Xe-lamps or mixed gas lamps)
will not emit the wavelength you want. Even if there might be strong
lines in the Hg spectrum close to your desired wavelengths, the material
from which the arc lamp bulbs are made will absorb deep UV light. I know
that one earlier could purchase 75W Xe arc lamps in quartz bulbs
emitting light down to 320nm from Osram. However, this was many years
ago, and I am not sure whether they are being produced any longer. These
bulbs had to be installed in special lamp housing, which included
collector lenses made from quartz or fused silica, since the standard
lenses would not transmit light of any wavelength that short. Also these
lamps were really good in producing Ozone, which you then had to get rid
of by having the entire lamp being located underneath a suction tube of
the house ventilation.

c)
Illuminating anything biological at 280nm will, as I would guess (and I
might, of course, be wrong) result in all the material shining brightly
in autofluorescence. So, I assume that all your cell would shine in
bluish strong autofluorescence. Unless the substance, which you are
interested in, would have a really large Stokes shift and emit
fluorescence in the yellow or red wavelength region, where I would
assume the 280nm produced autofluorescence to be weak, the signal light,
which you would want to see, would most probably be drowned in
autofluorescence.

e)
Laser light at these deeper UV wavelengths isn't an easy victory,
either. Although there are deep UV lEDs available attaining some
hundreds of Microwatts output power at around 280nm, I am not sure that
they would be suitable for your purpose (but they nevertheless might be,
indeed!). The HeCd laser produces a strong 325nm line, and a version of
the heavy Sabre by Coherent is specified do deliver 350mW@275.4nm, but
at least the latter one really is a big laser compared to anything in
"standard biology laboratories" and I would not assume that you would
have the funding or the infrastructure required for purchasing and
operating such a large machine.


I have never been working with with flow cytometers, but might be - if
you do not need a microscopic "image" and if the Stokes shift of your
substance is large enough to overcome the autofluorescence problem -
there exists a flow cytometer for your wavelength range and might be a
tool suitable for the purpose.


Otherwise, the sole solution, which I could see would be using a multi
photon microscope. The emission wavelengths of "Standard lasers" for
this type of microscope are however too long for generating a 2p process
at 280nm, you would have to go for a three photon approach. The main
problem then will be to, again, suppress un-wanted two photon effects,
which in this case would be "collateral damage". There are, however,
laser sources that would be suitable for generating a 2p process at
280nm, and you might want to hear whether there is possibly any lab,
which would have such a laser rigged up onto an LSM.

I wish you, without any irony or sarcasm, good success!!!!!

Johannes


On 2017-01-24 15:50, Darren Thomson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your
> posting.
> *****
>
> Hello All,
>
> We have a compound which annoyingly excites at 280nm. We need to
> detect whether it has entered the cell or not.
> Is anyone aware of an excitation source this deep? Would we need to go
> back to mercury lamps and try tease out with a custom filter?
>
> Cheers,
>
> Dr Darren Thomson
> Manchester Fungal Infection Group, UK.

--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
University of Oslo
Institute of Basic Medical Sciences
Department of Molecular Medicine
Division of Physiology
P. O. Box 1103 - Blindern
NO-0317 Oslo
Norway

Voice: [phone]
Fax: [phone]

WWW: folk.uio.no/jhelm

[Michael Giacomelli]

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Hi Darren,

We are using 280 nm LEDs for fluorescence imaging from here:

http://www.qphotonics.com/Deep-UV-light-emitting-diode-500-800uW-275nm-UVTOP275.html

Note that the specs are quite conservative.  Ours came rated for ~3x
the power listed on the website.  I do recommend carefully looking for
a current driver however.  The forward voltage on these diodes is >6
V, which is more than some drivers can handle.

Mike

On Tue, Jan 24, 2017 at 10:44 AM, George McNamara
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Darren,
>
> LEDs are now available in the deep UV. You do not need to go through the
> objective lens to excite fluorescence (MUSE is typically imaging in the
> visible). For example (see web pages for images ... the first image on the
> muse site reminds me of this image
> http://uvmbored.com/event/rocky-horror-picture-show ):
>
> http://www.musemicro.com
>
> Based on IP developed jointly at Lawrence Livermore and UC Davis, the MUSE
> (Microscopy with Ultraviolet Sectioning Excitation) Microscope uses
> short-wavelength UV light which penetrates only microns-deep into tissue
> eliminating the need for precision-cut, thin specimens and even slides. In
> addition, short-wavelength UV light excites many fluorescent dyes
> simultaneously, for snap-shot color images. The result is stunning detailed
> images conveying a degree of resolution, structure and depth unachievable
> until now by any single technology
>
> Sample preparation is simplified from hours to minutes with a process that
> does not required trained personnel, organic solvents or related expense.
> The technique does not alter samples, is non-destructive and thus preserves
> valuable tissue for molecular studies. Moreover, MUSE Microscope images
> offer a large field of view for whole-slide-like digital imaging capability
> and can be easily and quickly formatted through application software to
> provide familiar, H&E appearance, with diagnostic-quality, subcellular
> detail to allow easy transition for pathologists used to viewing standard
> slides.
>
>
> http://www.fasebj.org/content/30/1_Supplement/51.3
>
>
>  MUSE: A New, Fast, Simple Microscopy Method for Slide-Free Histology
>  and Surface Topography
>
> 1. Richard M Levenson
>
> <http://www.fasebj.org/search?author1=Richard+M+Levenson&sortspec=date&submit=Submit>and
> 2. Farzad Fereidouni
>
> <http://www.fasebj.org/search?author1=Farzad+Fereidouni&sortspec=date&submit=Submit>
> 3.    Dept. of Pathology and Laboratory Medicine, UC Davis Medical
>    Center, Sacramento, CA
> 4.
>
>
>    Abstract
>
> *Introduction*Conventional brightfield or fluorescence microscopy works best
> with thin, stained specimens mounted on glass slides, but to prepare these
> requires hours of processing and the help of highly skilled technical
> personnel. We describe a new, inexpensive form of light microscopy that can
> generate high-quality histology and histopathology images directly from cut
> surfaces of fresh (or fixed) tissue samples of any thickness, with less than
> 1 minute of preparation.
>
> *Background*While alternative microscopy-scale methods, such as confocal or
> two-photon, or optical coherence tomography approaches, can generate
> reasonable images from thick tissues, the instrumentation is typically
> complex, expensive and not widely available. We are developing an
> alternative technique, MUSE (Microscopy with UV Surface Excitation), that
> relies on simple properties of light, tissue, and fluorescent stains, and
> can be implemented at relatively low cost. Thick pieces of tissue obtained
> by biopsy, surgery or necropsy can be imaged directly. The method is
> non-destructive, and eliminates requirement for conventional histology
> processing, formalin fixation, paraffin-embedding, or thin-sectioning.
>
> *Method*Ultraviolet light at about 280 nm from an LED arranged to provide
> oblique illumination, is used to excite just the surface layer of tissue
> that has been briefly (~10 seconds) stained with common and inexpensive
> fluorescent dyes. Unlike light of longer wavelength, 280-nm light only
> penetrates to a depth of 10 microns or less, and thus excites fluorescent
> signals, conveniently in the visible range, only from the cut specimen
> surface. The images, which can be diffraction-limited, are captured using
> conventional microscope optics and a standard color camera
>
> *Results*MUSE samples from fresh (or fixed) tissues stained with general
> tissue stains such as rhodamine and Hoechst generate high-resolution
> fluorescence images that can be converted in real time from fluorescence to
> H&E-like brightfield appearance, for ease of interpretation. However,
> fluorescence display mode preserves surface shading and depth cues that
> allow for appreciation of surface profiles important in understanding 3D
> organization of complex specimens. We have obtained high-quality MUSE images
> from normal and diseased tissues from multiple organisms, as well as from
> plants, and manufactured materials. Moreover, surface profile information
> provides views beyond what can be seen with in standard thin-sectioned
> material, generating results that can resemble those obtainable with
> scanning electron microscopy (SEM).
>
> *Conclusion*We have developed a new form of optical microscopy that
> generates diagnostic-quality histological images, with enhanced content,
> from fresh or fixed, but unsectioned tissue, rapidly, with high resolution,
> simply and inexpensively. We anticipate that there could be widespread
> adoption in preclinical and clinical research facilities, as well as
> hospital-based and stand-alone medical settings.
>
>
>
> On 1/24/2017 9:15 AM, MODEL, MICHAEL wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> I am afraid that tryptophan would also fluoresce in this range
>>
>>
>>
>> Mike Model
>>
>>
>> ________________________________
>> From: Confocal Microscopy List <[hidden email]> on
>> behalf of Darren Thomson <[hidden email]>
>> Sent: Tuesday, January 24, 2017 9:50 AM
>> To: [hidden email]
>> Subject: deep UV excitation (280nm)
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Cmmodel%40KENT.EDU%7C5447564400ab4be1ed1208d4446aeae2%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=Zmc2DJyp095fti%2BWYgypGVII%2BbTjVqp37ereHJHVBI4%3D&reserved=0
>> Post images on
>> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%7C5447564400ab4be1ed1208d4446aeae2%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=hFwa3D1H6uZ9FPt%2FR0qH7D7LjCPMkY5Pfu7zAn694Bc%3D&reserved=0
>> and include the link in your posting.
>> *****
>>
>> Hello All,
>>
>> We have a compound which annoyingly excites at 280nm. We need to detect
>> whether it has entered the cell or not.
>> Is anyone aware of an excitation source this deep? Would we need to go
>> back to mercury lamps and try tease out with a custom filter?
>>
>> Cheers,
>>
>> Dr Darren Thomson
>> Manchester Fungal Infection Group, UK.
>
>
> --
>
>
> George McNamara, PhD
> Houston, TX 77054
> [hidden email]
> https://www.linkedin.com/in/georgemcnamara
> https://works.bepress.com/gmcnamara/75/
> http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Eric Girard]

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Unless a laser is available, a mercury or deuterium lamp is your best bet.
Point detectors for UVC are available with thin film filters, you may find
a standard.

If you use a laser, we do speckle scrambling.

Best,

Eric Girard
gigaconcept.com

Le 24 janv. 2017 9:50 AM, "Darren Thomson" <[hidden email]>
a écrit :

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello All,
>
> We have a compound which annoyingly excites at 280nm. We need to detect
> whether it has entered the cell or not.
> Is anyone aware of an excitation source this deep? Would we need to go
> back to mercury lamps and try tease out with a custom filter?
>
> Cheers,
>
> Dr Darren Thomson
> Manchester Fungal Infection Group, UK.
>

[Johannes Helm]

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PS: I forgot to mention that there have been a number of companies
designing and producing Schwarzschild type mirror objectives or
catadioptric objectives for the deep UV. Among these were Ealing and OfR
(Optics for Research). Ealing is, as far as I know, defunct but their
lenses are still being sold by a number of companies (can be found on a
search engine).

Best wishes,

Johannes

On 2017-01-24 15:50, Darren Thomson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your
> posting.
> *****
>
> Hello All,
>
> We have a compound which annoyingly excites at 280nm. We need to
> detect whether it has entered the cell or not.
> Is anyone aware of an excitation source this deep? Would we need to go
> back to mercury lamps and try tease out with a custom filter?
>
> Cheers,
>
> Dr Darren Thomson
> Manchester Fungal Infection Group, UK.

--
P. Johannes Helm

Voice: ([phone] (office)
Fax: [phone] (office)

[Kurt Thorn]

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On 1/24/2017 6:50 AM, Darren Thomson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello All,
>
> We have a compound which annoyingly excites at 280nm. We need to detect whether it has entered the cell or not.
> Is anyone aware of an excitation source this deep? Would we need to go back to mercury lamps and try tease out with a custom filter?

Ordinary glass (i.e. coverslips and slides) doesn't transmit below about
310 nm. So this is pretty tricky to do without quartz or other
UV-transparent optics on the excitation side.  We have done this for
lanthanide excitation, however, by building a custom transmitted light
illuminator using quartz optics and a full spectrum Xe lamp for
excitation, using excitation filters from Semrock.  We have also done it
in an epi-illumination geometry using UV LEDs mounted around the
objective, aimed at the sample.  Any parts in the excitation pathway
need to be quartz or fused silica (you can get quartz slides and
coverslips) but if your emission wavelength is > 310 nm then you can use
regular glass (beware, most objectives don't transmit < 340 nm light).  
COP or COC polymers are also an option for UV transmitting materials, as
is PDMS (if you do microfluidics).

You can read more about our approach here:
http://pubs.rsc.org/en/Content/ArticleLanding/2012/LC/C2LC40699C#!divAbstract 
and
http://onlinelibrary.wiley.com/doi/10.1002/adom.201600548/abstract

Feel free to email me with questions - I can provide lots more detail,
including part numbers and other approaches we've tried.

Kurt
>
> Cheers,
>
> Dr Darren Thomson
> Manchester Fungal Infection Group, UK.
>


--
Kurt Thorn
Associate Professor
Director, Nikon Imaging Center
http://thornlab.ucsf.edu/
http://nic.ucsf.edu/blog/

[George McNamara]

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See also Energetiq -- web page has graph vs D2 lamp (Deuterium).

"Very high brightness across spectrumUV-Vis-NIR (170nm - 2100nm)"

http://www.energetiq.com/ldls-laser-driven-light-source-duv-broadband.php



On 1/24/2017 10:06 AM, Eric Girard wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Unless a laser is available, a mercury or deuterium lamp is your best bet.
> Point detectors for UVC are available with thin film filters, you may find
> a standard.
>
> If you use a laser, we do speckle scrambling.
>
> Best,
>
> Eric Girard
> gigaconcept.com
>
> Le 24 janv. 2017 9:50 AM, "Darren Thomson" <[hidden email]>
> a écrit :
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Hello All,
>>
>> We have a compound which annoyingly excites at 280nm. We need to detect
>> whether it has entered the cell or not.
>> Is anyone aware of an excitation source this deep? Would we need to go
>> back to mercury lamps and try tease out with a custom filter?
>>
>> Cheers,
>>
>> Dr Darren Thomson
>> Manchester Fungal Infection Group, UK.
>>

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[maiti]

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In my experience, multiphoton excitation is probably the best way for doing
UV microscopy in live specimens. Three photon works well in a normal
multiphoton microscope, PROVIDED the objective and other optics passes the
fluorescence emission. However, please forget the thumb rule of Lambda/3 for
three photon.
Excitation is better towards 700 nm (REF: Maiti et al., Measuring Serotonin
Distribution in Live Cells with Three-Photon Excitation, Science, 1997, Vol.
275, Issue 5299, pp. 530-532). 740 nm gives a good optimization between cell
survivability and signal. Keep in mind the following too:

1) A good Fluor objective will work OK for Tryptophan (of course , inside a
cell, Trp would not give you any contrast). However, please check the
dichroic, filter etc. for passing the fluorescence wavelength
2) Non-descanned detection is really preferred, as nothing inside a
conventional confocal is really optimized for UV
3) about 30 mW at the back aperture of a high NA objective should give you
reasonable signal, at least it does for intravesicular serotonin in neurons .

(Some references:
Balaji et al., Quantitative measurement of serotonin synthesis and
sequestration in individual live neuronal cells. J Neurochem.95(5) (2005)
1217
Kaushalya et al., Three-photon microscopy shows that somatic release can be a
quantitatively significant component of serotonergic neurotransmission in the
mammalian brain. J Neurosci Res. 2008, 86(15),3469-3480

Sarkar et al., The dynamics of somatic exocytosis in monoaminergic neurons. 
Front. Physiol. 2012, 3( 414), 1-13.)
 

If you want to image deeper in the UV, you will have to use non-
epifluorescent detection, as a normal objective would not pass any
fluorescence  below about 320 nm. It is reasonably easy to modify a scope for
such a thing. We have detected Dopamine (emission ~305 nm) quite effectively
with it.

Ref: Sarkar et al., Label-free dopamine imaging in live rat brain slices.
ACS Chem. Neurosci. 2014, doi: 10.1021/cn5000138 

Thanks, and I will be happy to help with more details off the list, if
needed.
Sudipta Maiti

On Tue, 24 Jan 2017 11:37:08 -0600, George McNamara wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> See also Energetiq -- web page has graph vs D2 lamp (Deuterium).
>
> "Very high brightness across spectrumUV-Vis-NIR (170nm - 2100nm)"
>
> http://www.energetiq.com/ldls-laser-driven-light-source-duv-broadband.php
>
> On 1/24/2017 10:06 AM, Eric Girard wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your posting.
> > *****
> >
> > Unless a laser is available, a mercury or deuterium lamp is your best bet.
> > Point detectors for UVC are available with thin film filters, you may find
> > a standard.
> >
> > If you use a laser, we do speckle scrambling.
> >
> > Best,
> >
> > Eric Girard
> > gigaconcept.com
> >
> > Le 24 janv. 2017 9:50 AM, "Darren Thomson"

<[hidden email]>

> > a écrit :
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your posting.
> >> *****
> >>
> >> Hello All,
> >>
> >> We have a compound which annoyingly excites at 280nm. We need to detect
> >> whether it has entered the cell or not.
> >> Is anyone aware of an excitation source this deep? Would we need to go
> >> back to mercury lamps and try tease out with a custom filter?
> >>
> >> Cheers,
> >>
> >> Dr Darren Thomson
> >> Manchester Fungal Infection Group, UK.
> >>
>
> --
>
> George McNamara, PhD
> Houston, TX 77054
> [hidden email]
> https://www.linkedin.com/in/georgemcnamara
> https://works.bepress.com/gmcnamara/75/
> http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

Prof. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Road, Colaba
Mumbai 400005, India
Ph. [phone]
Alternate e-mail: [hidden email]
webpage: biophotonics.co.in

[jerie]

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Dear Darren,

just in case and because I did not see it in the list of replies, there is
a provider for complete modifications of systems to do deep uv microscopy

http://www.rapp-opto.com/deep-uv-microscopy-109.html

no commercial interest.

Abraços, Jens

Dr. Jens Rietdorf, visiting scientist @ center for technological
development in health CDTS, Oswaldo Cruz Foundation Fiocruz, Rio de Janeiro
Brasil.

On Tue, Jan 24, 2017 at 12:50 PM, Darren Thomson <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello All,
>
> We have a compound which annoyingly excites at 280nm. We need to detect
> whether it has entered the cell or not.
> Is anyone aware of an excitation source this deep? Would we need to go
> back to mercury lamps and try tease out with a custom filter?
>
> Cheers,
>
> Dr Darren Thomson
> Manchester Fungal Infection Group, UK.
>