excitation at 241 and 280

[John Shields]

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Good morning,
We are interested in looking at terbium luminescence and fluorescent
aromatic amino acids.

The excitation spectrum has two peaks at 241 nm and 280 nm (the first
is for Tb itself and the second for aromatic amino acids which belong
to the protein coordinating the metal). Tb emission is fairly broad,
but  ~490nm and ~545nm are the strongest.

Does anyone have experience exciting in those wavelengths and what is required?

THanks
John Shields
Center for Ultrastructural Research
University of Georgia
Athens

[Sudipta Maiti]

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Three photon is an option. We regularly excite serotonin which has a 1-photon
peak around 270 nm. Refs: Stem Cells Dev 2009 18(4), 615-628; J Neurosci Res.
2008, 86(15),3469-3480, and references sited there.
The other option is two-photon with an OPO. We have used this for dopamine.
See: Applied. Optics., 43(12) (2004) 2412-7; Microsc. Res. Tech., 2004. 63(1):
p. 67-71 .
Hope that helps.
Sudipta

On Mon, 10 Oct 2011 08:29:37 -0400, John Shields wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good morning,
> We are interested in looking at terbium luminescence and fluorescent
> aromatic amino acids.
>
> The excitation spectrum has two peaks at 241 nm and 280 nm (the first
> is for Tb itself and the second for aromatic amino acids which belong
> to the protein coordinating the metal). Tb emission is fairly broad,
> but  ~490nm and ~545nm are the strongest.
>
> Does anyone have experience exciting in those wavelengths and what
> is required?
>
> THanks
> John Shields
> Center for Ultrastructural Research
> University of Georgia
> Athens

Dr. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: [phone]
alternate e-mail: [hidden email]
url: www.biophotonics.weebly.com

[yuansheng sun]

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Dear Dr. Maiti,

I am very interested in 3P imaging of tryptophan.  We have a Mira 900 Ti:Sa
laser.  I read one of your earlier publications (Science 275 530-532, 1997)
when you worked in W.W. Webb's lab.  I have a question about the 3P imaging
you described in your J. Neurosci. Res 2008 86(15).

*saturated solution of copper sulfate with 1-cm path length is placed in
front of the PMT to filter out back-scattered infrared light*

Is this solution used as the emission filter?  There is no other emission
filter used?  Is this a key to achieve sufficient SNR and contrast for the
3P imaging of tryptophan?  We tried the 3P imaging (740 nm excitation) of
tryptophan solutions in differentt concentrations and did not get good
signals. When measuring those solutions with a spectrofluorometer (270
nm excitation), we obtained reasonable results.  We used the quartz
(coverslips or cuvettes) in all of our imaging.  Any suggestion would be
greatly appreciated.

Best regards,
Sheng

On Mon, Oct 10, 2011 at 9:43 AM, Sudipta Maiti
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Three photon is an option. We regularly excite serotonin which has a
> 1-photon
> peak around 270 nm. Refs: Stem Cells Dev 2009 18(4), 615-628; J Neurosci
> Res.
> 2008, 86(15),3469-3480, and references sited there.
> The other option is two-photon with an OPO. We have used this for dopamine.
> See: Applied. Optics., 43(12) (2004) 2412-7; Microsc. Res. Tech., 2004.
> 63(1):
> p. 67-71 .
> Hope that helps.
> Sudipta
>
> On Mon, 10 Oct 2011 08:29:37 -0400, John Shields wrote
>  > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Good morning,
> > We are interested in looking at terbium luminescence and fluorescent
> > aromatic amino acids.
> >
> > The excitation spectrum has two peaks at 241 nm and 280 nm (the first
> > is for Tb itself and the second for aromatic amino acids which belong
> > to the protein coordinating the metal). Tb emission is fairly broad,
> > but  ~490nm and ~545nm are the strongest.
> >
> > Does anyone have experience exciting in those wavelengths and what
> > is required?
> >
> > THanks
> > John Shields
> > Center for Ultrastructural Research
> > University of Georgia
> > Athens
>
>
> Dr. Sudipta Maiti
> Dept. of Chemical Sciences
> Tata Institute of Fundamental Research
> Homi Bhabha Raod, Colaba, Mumbai 400005
> Ph. 91-22-2278-2716 / 2539
> Fax: [phone]
> alternate e-mail: [hidden email]
> url: www.biophotonics.weebly.com
>

[Johannes Helm]

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Good evening,

I have once done 334nm excitation and this worked fine using Zeiss
Ultrafluar lenses, old style. However, while, according to Dr. Hoecherl,
then Senior Engineer at Zeiss in Oberkochen, being excellent in
transmission even down to the limits of the VUV, these lenses were not
chromatically "corrected" for confocal resolution. (ref. P. J. Helm, O.
Franksson, K. Carlsson, Pfluegers Arch.,429, 672 (1995))

Also, I had some very old objectives from Bausch & Lomb, which were
excellent (catadioptric and Schwarzschild design). I doubt, however, you
would have them accessible, they were exquisite pieces built in five or
six exp., only. I had them on loan from an old Professor, now retired, and
do not have the slightest idea, where they are.

But perhaps you can test a Zeiss Ultrafluar. In case of inf. corrected
lenses you would also need a suitable tube lens (was available from Zeiss
many years ago).

Best wishes,
Johannes

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good morning,
> We are interested in looking at terbium luminescence and fluorescent
> aromatic amino acids.
>
> The excitation spectrum has two peaks at 241 nm and 280 nm (the first
> is for Tb itself and the second for aromatic amino acids which belong
> to the protein coordinating the metal). Tb emission is fairly broad,
> but  ~490nm and ~545nm are the strongest.
>
> Does anyone have experience exciting in those wavelengths and what is
> required?
>
> THanks
> John Shields
> Center for Ultrastructural Research
> University of Georgia
> Athens
>

--
P. Johannes Helm

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

[Sudipta Maiti]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

1) single photon excitation will almost always  give more signal. It is in situations where you
cannot use single photon that you turn to multiphoton.
2) How good is your MP collection? is your PMT close to your objective? Do you get similar
differences for visible dyes, say rhodamine?
3) contrast is different from signal. In a cell, pretty much everything will have tryptophan (except
the nucleus, which appears dark - histones typically do not have Trp). So you may not see many
features.
4) yes, copper sulphate is used as a very effective emission filter for serotonin. For tryptophan it
is not as great, though it should stIll do.
I guess we can discuss further off the list.
Cheers.
Sudipta

 10 Oct 2011 11:49:48 -0400, yuansheng sun wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Dr. Maiti,
>
> I am very interested in 3P imaging of tryptophan.  We have a Mira
> 900 Ti:Sa laser.  I read one of your earlier publications (Science
> 275 530-532, 1997) when you worked in W.W. Webb's lab.  I have a
> question about the 3P imaging you described in your J. Neurosci. Res
> 2008 86(15).
>
> *saturated solution of copper sulfate with 1-cm path length is
> placed in front of the PMT to filter out back-scattered infrared light*
>
> Is this solution used as the emission filter?  There is no other emission
> filter used?  Is this a key to achieve sufficient SNR and contrast
> for the 3P imaging of tryptophan?  We tried the 3P imaging (740 nm
> excitation) of tryptophan solutions in differentt concentrations and
> did not get good signals. When measuring those solutions with a
> spectrofluorometer (270 nm excitation), we obtained reasonable
> results.  We used the quartz
> (coverslips or cuvettes) in all of our imaging.  Any suggestion
> would be greatly appreciated.
>
> Best regards,
> Sheng
>
> On Mon, Oct 10, 2011 at 9:43 AM, Sudipta Maiti
> <[hidden email]>wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Three photon is an option. We regularly excite serotonin which has a
> > 1-photon
> > peak around 270 nm. Refs: Stem Cells Dev 2009 18(4), 615-628; J Neurosci
> > Res.
> > 2008, 86(15),3469-3480, and references sited there.
> > The other option is two-photon with an OPO. We have used this for dopamine.
> > See: Applied. Optics., 43(12) (2004) 2412-7; Microsc. Res. Tech., 2004.
> > 63(1):
> > p. 67-71 .
> > Hope that helps.
> > Sudipta
> >
> > On Mon, 10 Oct 2011 08:29:37 -0400, John Shields wrote
> >  > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Good morning,
> > > We are interested in looking at terbium luminescence and fluorescent
> > > aromatic amino acids.
> > >
> > > The excitation spectrum has two peaks at 241 nm and 280 nm (the first
> > > is for Tb itself and the second for aromatic amino acids which belong
> > > to the protein coordinating the metal). Tb emission is fairly broad,
> > > but  ~490nm and ~545nm are the strongest.
> > >
> > > Does anyone have experience exciting in those wavelengths and what
> > > is required?
> > >
> > > THanks
> > > John Shields
> > > Center for Ultrastructural Research
> > > University of Georgia
> > > Athens
> >
> >
> > Dr. Sudipta Maiti
> > Dept. of Chemical Sciences
> > Tata Institute of Fundamental Research
> > Homi Bhabha Raod, Colaba, Mumbai 400005
> > Ph. 91-22-2278-2716 / 2539
> > Fax: [phone]
> > alternate e-mail: [hidden email]
> > url: www.biophotonics.weebly.com
> >

Dr. Sudipta Maiti
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: [phone]
alternate e-mail: [hidden email]
url: www.biophotonics.co.in

[Urs Utzinger]

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Since excitation below 380 nm does not easily pass through an
objective lenses you will need to use a "non-standard" approach. You
have several options:

1) You can obtain UV optimized objective lenses but this could become
very costly if you need transmission below 340 nm. You might also need
a specialized light source as cold mirrors (= mirror in a Xenon light
source taking out NIR light) usually do not reflect below 300 nm.
2) You can use quartz glass slides and quartz cover slips and
illuminate through the condenser or replace the condenser with a UV
source. The problem with this approach is that excitation light that
is not absorbed by your sample will be propagating directly to your
sensor and you need very good blocking filters.
3) You can illuminate at oblique angle using a UV laser. The laser
will hit the sample "sideways" and not go through your objective lens.
Your objective lens will need to leave enough working distance and
numerical aperture to project your laser beam onto the sample. You
would only need quartz cover slips.
4) You can use two photon microscopy with a visible laser beam. That
can be accomplished with Ti:Saph-OPO combination. Since the
illumination will be in the visible it will pass the objective lens.
Your fluorophore  has a broad emission and is bright, so even if you
do not capture all emission at the maximum intensity wavelength you
can receive a sufficiently strong signal. A regular cover slip should
work. Two Photon excitation would be preferred over three photon as
efficiency is larger. 3P might work on densely packet organelles. You
will need UV optical filters and suppressing light from the light
source reaching your sensor will require special care.

Urs Utzinger
University of Arizona




On Mon, Oct 10, 2011 at 5:29 AM, John Shields <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good morning,
> We are interested in looking at terbium luminescence and fluorescent
> aromatic amino acids.
>
> The excitation spectrum has two peaks at 241 nm and 280 nm (the first
> is for Tb itself and the second for aromatic amino acids which belong
> to the protein coordinating the metal). Tb emission is fairly broad,
> but  ~490nm and ~545nm are the strongest.
>
> Does anyone have experience exciting in those wavelengths and what is required?
>
> THanks
> John Shields
> Center for Ultrastructural Research
> University of Georgia
> Athens
>

[Iain Johnson]

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The absorption cross-sections for terbium (one or two photon) are
extraordinarily low.  In practice it is necessary to couple the terbium to a
sensitizing dye via a chelator.  The chelator can be synthetic (e.g. DTPA)
or a calcium-binding protein (since Tb3+ is somewhat isomorphous with
Ca++).  The conventional sensitizers such as carbostyril 124 and dipicolinic
acid excite around 300-350 nm in one-photon, but don't appear to work at all
in two-photon.  You can find the state-of-play on the development of
two-photon sensitizers on PubMed e.g.
http://www.ncbi.nlm.nih.gov/pubmed/18491006.

Iain

Iain Johnson Consulting
Eugene, OR


On Mon, Oct 10, 2011 at 5:29 AM, John Shields <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good morning,
> We are interested in looking at terbium luminescence and fluorescent
> aromatic amino acids.
>
> The excitation spectrum has two peaks at 241 nm and 280 nm (the first
> is for Tb itself and the second for aromatic amino acids which belong
> to the protein coordinating the metal). Tb emission is fairly broad,
> but  ~490nm and ~545nm are the strongest.
>
> Does anyone have experience exciting in those wavelengths and what is
> required?
>
> THanks
> John Shields
> Center for Ultrastructural Research
> University of Georgia
> Athens
>

[George McNamara]

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*****

Reinforcing Johannes reply - Zeiss still has ultrafluar's (10x/0.2NA,
40x/0.6NA) on the https://www.micro-shop.zeiss.com website.

Cheung et al 2011 Cytometry - Deep Ultraviolet Mapping of Intracellular
Protein and Nucleic Acid in Femtograms per Pixel, used a Energetiq lamp
as the light source for their work. See also their live cell imaging
paper (reported deep UV as less phototoxic than 300nm+ UV):
Zeskind BJ, Jordan CD, Timp W, Trapani L, Waller G, Horodincu V, Ehrlich
DJ, Matsudaira P. Nucleic acid and protein mass mapping by live-cell
deep-ultraviolet microscopy. Nat Methods 2007;4:567–569.

Standard confocal PMTs are not going to detect tryptophan very well. See
emission spectrum at
http://www.mcb.arizona.edu/ipc/fret/index.html
or
http://www.spectra.arizona.edu/

Extinction coefficient 5,579 M-1cm-1
quantum yield 0.12
so "intrinsic brightness" (Ec*QY/1000) is 0.669 for comparison, Alexa
Fluor 488 is 78,000*0.92/1000 = 71.760.

Iain already commented on Terbium and sensitizers. Terbium is not on the
spectral pages, but is a poor fluorescence reporter (except for the
lovely features of long lifetime and narrow emission spikes - if you
have the right wavelength selector and detector).

Johannes suggestion for using reflection lenses is also excellent. Check
the Internet for current suppliers of these (hopefully that fit your
microscope). There was an article by Piper in January 2011 Microscopy
Today on Catadioptric lenses. Also a December 2004 article by Joseph
Armstrong of KLA Tencor about these lenses.


Best wishes,

George


On 10/10/2011 8:29 AM, John Shields wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good morning,
> We are interested in looking at terbium luminescence and fluorescent
> aromatic amino acids.
>
> The excitation spectrum has two peaks at 241 nm and 280 nm (the first
> is for Tb itself and the second for aromatic amino acids which belong
> to the protein coordinating the metal). Tb emission is fairly broad,
> but  ~490nm and ~545nm are the strongest.
>
> Does anyone have experience exciting in those wavelengths and what is required?
>
> THanks
> John Shields
> Center for Ultrastructural Research
> University of Georgia
> Athens
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami