exciting terbium cryptate with 2-P?

[Rietdorf, Jens]

Dear list,

I want to excite terbium cryptate fluorescence with 2-P; any idea, about
the excitation spectrum of it?

Thanks, jens

[Mario-2]

Jens,

Unless you use short wavelength excitation ¾280 nm it is difficult to excite terbium or any other lanthanide. Multiphoton excitation works pretty well, but you still have to get the equivalent of these relatively short wavelengths, which could mean 3 P not 2 P. It is very helpful to use a sensitizer that helps form part of the cryptate.

As you may know, the most serious impediment to getting good luminescence yield from a lanthanide is to eliminate quenching due to water. In fact, the number of water molecules in the hydration sphere of terbium can be estimated from the luminescence lifetime and is most easily done by replacing increasing fractions of the aqueous phase with D2O (heavy water). The D-O stretching frequency does not couple with the excited state terbium whereas H-O does quite well; hence, the strong quenching by H2O.

Chelated terbium greatly enhances the luminescence yield by excluding H2O, but almost all lanthanide complexes that have any use as luminescers include a sensitizer. For example, 4-aminosalicylic acid when included in the coordination sphere with terbium (but not europium), greatly enhances the luminescence yield when excited at 306 nm (max extinct.). To get the best signal one only needs to worry about exciting the pAS which very efficiently transfers its excitation to terbium. Another sensitizer that works well for both terbium and europium is the coumarin carbostyril 124 (7-Amino-4-methyl-2-quinolone) with excitation max at 328 nm.

Both sensitizers can be excited with multiphoton but depending on your laser this may typically mean 3 photon excitation. For the x-ray excitable probes, I used conjugates of pAS-DTPA and sometimes pAS2-DTPA. The carbostyril version lacking the extra carboxyl and phenoxyl OH tend to be more hydrophobic and are a little harder to make into good aqueous probes with low non-specific binding.

Although the focus of the following concerned x-ray applications, you might check: Moronne, M.M. (1999), Development of x-ray excitable luminescent probes for high resolution scanning x-ray microscopy. J. Ultramicroscopy, 77:23-36.

I spent a lot of time with lanthanide probes; let me know if you need any more help.

Mario

[hidden email]

Dear list,

I want to excite terbium cryptate fluorescence with 2-P; any idea, about
the excitation spectrum of it?

Thanks, jens

--

________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

[George McNamara]

Hi Mario,

Your text makes it sound like 3 photon excitation is evil. As your post and sites with data such as www.tciamerica.com/useful-info/product-lit/L3012E.pdf point out, 1 photon excitation of Europium is around 300 nm (250-350 nm), that is around half that of the 615 nm emission peak - really line - of Europium 3+ ion. When operating a spectral confocal microscope in open pinhole mode, the wavelength selectivity decreases, leading to MP laser excitation light leaking into visible emission bandpass on my Leica MP/SP5 (thanks again Coherent for 3800 mW peak power out of the Chameleon Ultra 2).

Robert leif, Bob Zucker and colleagues have published on multiphoton excitation of lanthanide macrocycles, see www.newportinstruments.com/quantum/pdf/one_and_two04.pdf


***

With respect to 3 photon excitation, a question: does the cube dependence result in smaller excitation volume than 2 photon excitation?

Enjoy,

George
p.s. Today I was operating an SP5 with 405 nm and 633 nm excitation and DAPI (~420-480 nm) and reflection (630-635 nm, "reflection" option on in AOBS) emission, pinhole 1.0 airy units, 10x/0.4 NA objective lens, ~100 um collagen gel with cells specimen. The background of the DAPI emission was amazingly high, until I switched the 633 nm laser to 0%. I ran the study using 2 sequential tracks.

For those who have not read it, I highly recommend:

Second harmonic generating (SHG) nanoprobes for in vivo imaging.
Pantazis P, Maloney J, Wu D, Fraser SE.
Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14535-40. Epub 2010 Jul 28.PMID: 20668245

Text mentions the commercial sources of the BaTiO3 and ZnO (and one other) SHG nanocrystal. Epi-detection works as well as transmitted light detector. I do not recall them mentioning an (epi) NDD detector, so might get even better. The paper cites an earlier Swiss/Caltech paper on conjugating these nanocrystals to antibodies (possibly the authors of the patent application mentioned in the text).

and


Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.
Szent-Gyorgyi C, Schmidt BF, Fitzpatrick JA, Bruchez MP.
J Am Chem Soc. 2010 Aug 18;132(32):11103-9.PMID: 20698676

I hope someone on the list can enlighten me with respect to the author's statement about donor quantum yield not mattering. My expectation is that Cy3B(s) should outperform Cy3(s) as donors. I am also mystified why the dendron is cell permeable.


At 12:18 PM 8/16/2010, you wrote:

Jens,

Unless you use short wavelength excitation ¾280 nm it is difficult to excite terbium or any other lanthanide. Multiphoton excitation works pretty well, but you still have to get the equivalent of these relatively short wavelengths, which could mean 3 P not 2 P. It is very helpful to use a sensitizer that helps form part of the cryptate.

As you may know, the most serious impediment to getting good luminescence yield from a lanthanide is to eliminate quenching due to water. In fact, the number of water molecules in the hydration sphere of terbium can be estimated from the luminescence lifetime and is most easily done by replacing increasing fractions of the aqueous phase with D2O (heavy water). The D-O stretching frequency does not couple with the excited state terbium whereas H-O does quite well; hence, the strong quenching by H2O.

Chelated terbium greatly enhances the luminescence yield by excluding H2O, but almost all lanthanide complexes that have any use as luminescers include a sensitizer. For example, 4-aminosalicylic acid when included in the coordination sphere with terbium (but not europium), greatly enhances the luminescence yield when excited at 306 nm (max extinct.). To get the best signal one only needs to worry about exciting the pAS which very efficiently transfers its excitation to terbium. Another sensitizer that works well for both terbium and europium is the coumarin carbostyril 124 (7-Amino-4-methyl-2-quinolone) with excitation max at 328 nm.

Both sensitizers can be excited with multiphoton but depending on your laser this may typically mean 3 photon excitation. For the x-ray excitable probes, I used conjugates of pAS-DTPA and sometimes pAS2-DTPA. The carbostyril version lacking the extra carboxyl and phenoxyl OH tend to be more hydrophobic and are a little harder to make into good aqueous probes with low non-specific binding.

Although the focus of the following concerned x-ray applications, you might check: Moronne, M.M. (1999), Development of x-ray excitable luminescent probes for high resolution scanning x-ray microscopy. J. Ultramicroscopy, 77:23-36.

I spent a lot of time with lanthanide probes; let me know if you need any more help.

Mario
[hidden email]

Dear list,

I want to excite terbium cryptate fluorescence with 2-P; any idea, about
the excitation spectrum of it?

Thanks, jens

-- 

________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

[Mario-2]

George,

It was certainly not my intent to disparage 3 P excitation, only to emphasize that depending on the sensitizer and cryptates cage structure that the "effective" wavelengths needed to excite lanthanides need to be relatively short compared to typical organic fluorophores.

Another issue is the total throughput lanthanide labels are limited to given their luminescence decay time are on the order of 1-2 msec. not nanoseconds. However, this can be turned to great advantage using time resolved detection which virtually eliminates any background autofluorescence interference.

Mario

Hi Mario,

Your text makes it sound like 3 photon excitation is evil. As your post and sites with data such as  www.tciamerica.com/useful-info/product-lit/L3012E.pdf point out, 1 photon excitation of Europium is around 300 nm (250-350 nm), that is around half that of the 615 nm emission peak - really line - of Europium 3+ ion. When operating a spectral confocal microscope in open pinhole mode, the wavelength selectivity decreases, leading to MP laser excitation light leaking into visible emission bandpass on my Leica MP/SP5 (thanks again Coherent for 3800 mW peak power out of the Chameleon Ultra 2).

Robert leif, Bob Zucker and colleagues have published on multiphoton excitation of lanthanide macrocycles, see  www.newportinstruments.com/quantum/pdf/one_and_two04.pdf


***

With respect to 3 photon excitation, a question: does the cube dependence result in smaller excitation volume than 2 photon excitation?

Enjoy,

George
p.s. Today I was operating an SP5 with 405 nm and 633 nm excitation and DAPI (~420-480 nm) and reflection (630-635 nm, "reflection" option on in AOBS) emission, pinhole 1.0 airy units, 10x/0.4 NA objective lens, ~100 um collagen gel with cells specimen. The background of the DAPI emission was amazingly high, until I switched the 633 nm laser to 0%. I ran the study using 2 sequential tracks.

For those who have not read it, I highly recommend:

Second harmonic generating (SHG) nanoprobes for in vivo imaging.
Pantazis P, Maloney J, Wu D, Fraser SE.
Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14535-40. Epub 2010 Jul 28.PMID: 20668245

Text mentions the commercial sources of the BaTiO3 and ZnO (and one other) SHG nanocrystal. Epi-detection works as well as transmitted light detector. I do not recall them mentioning an (epi) NDD detector, so might get even better. The paper cites an earlier Swiss/Caltech paper on conjugating these nanocrystals to antibodies (possibly the authors of the patent application mentioned in the text).

and


Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.
Szent-Gyorgyi C, Schmidt BF, Fitzpatrick JA, Bruchez MP.
J Am Chem Soc. 2010 Aug 18;132(32):11103-9.PMID: 20698676

I hope someone on the list can enlighten me with respect to the author's statement about donor quantum yield not mattering. My expectation is that Cy3B(s) should outperform Cy3(s) as donors. I am also mystified why the dendron is cell permeable.


At 12:18 PM 8/16/2010, you wrote:

Jens,

Unless you use short wavelength excitation ¾280 nm it is difficult to excite terbium or any other lanthanide. Multiphoton excitation works pretty well, but you still have to get the equivalent of these relatively short wavelengths, which could mean 3 P not 2 P. It is very helpful to use a sensitizer that helps form part of the cryptate.

As you may know, the most serious impediment to getting good luminescence yield from a lanthanide is to eliminate quenching due to water. In fact, the number of water molecules in the hydration sphere of terbium can be estimated from the luminescence lifetime and is most easily done by replacing increasing fractions of the aqueous phase with D2O (heavy water). The D-O stretching frequency does not couple with the excited state terbium whereas H-O does quite well; hence, the strong quenching by H2O.

Chelated terbium greatly enhances the luminescence yield by excluding H2O, but almost all lanthanide complexes that have any use as luminescers include a sensitizer. For example, 4-aminosalicylic acid when included in the coordination sphere with terbium (but not europium), greatly enhances the luminescence yield when excited at 306 nm (max extinct.). To get the best signal one only needs to worry about exciting the pAS which very efficiently transfers its excitation to terbium. Another sensitizer that works well for both terbium and europium is the coumarin carbostyril 124 (7-Amino-4-methyl-2-quinolone) with excitation max at 328 nm.

Both sensitizers can be excited with multiphoton but depending on your laser this may typically mean 3 photon excitation. For the x-ray excitable probes, I used conjugates of pAS-DTPA and sometimes pAS2-DTPA. The carbostyril version lacking the extra carboxyl and phenoxyl OH tend to be more hydrophobic and are a little harder to make into good aqueous probes with low non-specific binding.

Although the focus of the following concerned x-ray applications, you might check: Moronne, M.M. (1999), Development of x-ray excitable luminescent probes for high resolution scanning x-ray microscopy. J. Ultramicroscopy, 77:23-36.

I spent a lot of time with lanthanide probes; let me know if you need any more help.

Mario
[hidden email]

Dear list,

I want to excite terbium cryptate fluorescence with 2-P; any idea, about
the excitation spectrum of it?

Thanks, jens

--

 ________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
 http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
 http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
 http://home.earthlink.net/~geomcnamara

--

________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

[Mike Buchin]

Mario,

 

From another perspective:  we have been working with a few academic sites using both Europium and Terbium with pulse excitation at 365 nm and long wavelength emission. Excitation pulse is 10-20 microseconds via fast high power LEDs.  Emission is collected with a 10-20 microsecond delay for several hundred microseconds using time delayed gating/ image intensified CCD detection.  In one application, we are running a camera at 30 fps with 20-40 excitation/emission pairs per frame.  In some cases, additional photon collection is accomplished by real time summation of each 33 ms exposure.   We have imaged zebrafish and some beads (progressive dilutions) courtesy of Leif et. al. The ability to (temporally) avoid auto fluorescence is certainly a huge plus.  The method is pretty straightforward once the hardware is in place.

 

The downside of direct 365 (vs. a two or three photon method) is the short wavelength/penetration/absorption issue.  On the other hand, the direct method is pretty energy efficient.

 

I hope this adds a little more breadth to the discussion.

 

Mike

 

Michael Buchin

Stanford Photonics, Inc.

Ph: 650-969-5991

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mario
Sent: Saturday, August 21, 2010 1:19 AM
To: [hidden email]
Subject: Re: exciting terbium cryptate with 2-P?

 

George,

 

It was certainly not my intent to disparage 3 P excitation, only to emphasize that depending on the sensitizer and cryptates cage structure that the "effective" wavelengths needed to excite lanthanides need to be relatively short compared to typical organic fluorophores.

 

Another issue is the total throughput lanthanide labels are limited to given their luminescence decay time are on the order of 1-2 msec. not nanoseconds. However, this can be turned to great advantage using time resolved detection which virtually eliminates any background autofluorescence interference.

 

Mario

 

Hi Mario,

Your text makes it sound like 3 photon excitation is evil. As your post and sites with data such as  www.tciamerica.com/useful-info/product-lit/L3012E.pdf point out, 1 photon excitation of Europium is around 300 nm (250-350 nm), that is around half that of the 615 nm emission peak - really line - of Europium 3+ ion. When operating a spectral confocal microscope in open pinhole mode, the wavelength selectivity decreases, leading to MP laser excitation light leaking into visible emission bandpass on my Leica MP/SP5 (thanks again Coherent for 3800 mW peak power out of the Chameleon Ultra 2).

Robert leif, Bob Zucker and colleagues have published on multiphoton excitation of lanthanide macrocycles, see  www.newportinstruments.com/quantum/pdf/one_and_two04.pdf


***

With respect to 3 photon excitation, a question: does the cube dependence result in smaller excitation volume than 2 photon excitation?

Enjoy,

George
p.s. Today I was operating an SP5 with 405 nm and 633 nm excitation and DAPI (~420-480 nm) and reflection (630-635 nm, "reflection" option on in AOBS) emission, pinhole 1.0 airy units, 10x/0.4 NA objective lens, ~100 um collagen gel with cells specimen. The background of the DAPI emission was amazingly high, until I switched the 633 nm laser to 0%. I ran the study using 2 sequential tracks.

For those who have not read it, I highly recommend:

Second harmonic generating (SHG) nanoprobes for in vivo imaging.
Pantazis P, Maloney J, Wu D, Fraser SE.
Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14535-40. Epub 2010 Jul 28.PMID: 20668245

Text mentions the commercial sources of the BaTiO3 and ZnO (and one other) SHG nanocrystal. Epi-detection works as well as transmitted light detector. I do not recall them mentioning an (epi) NDD detector, so might get even better. The paper cites an earlier Swiss/Caltech paper on conjugating these nanocrystals to antibodies (possibly the authors of the patent application mentioned in the text).

and


Fluorogenic dendrons with multiple donor chromophores as bright genetically targeted and activated probes.
Szent-Gyorgyi C, Schmidt BF, Fitzpatrick JA, Bruchez MP.
J Am Chem Soc. 2010 Aug 18;132(32):11103-9.PMID: 20698676

I hope someone on the list can enlighten me with respect to the author's statement about donor quantum yield not mattering. My expectation is that Cy3B(s) should outperform Cy3(s) as donors. I am also mystified why the dendron is cell permeable.


At 12:18 PM 8/16/2010, you wrote:

Jens,

Unless you use short wavelength excitation ¾280 nm it is difficult to excite terbium or any other lanthanide. Multiphoton excitation works pretty well, but you still have to get the equivalent of these relatively short wavelengths, which could mean 3 P not 2 P. It is very helpful to use a sensitizer that helps form part of the cryptate.

As you may know, the most serious impediment to getting good luminescence yield from a lanthanide is to eliminate quenching due to water. In fact, the number of water molecules in the hydration sphere of terbium can be estimated from the luminescence lifetime and is most easily done by replacing increasing fractions of the aqueous phase with D2O (heavy water). The D-O stretching frequency does not couple with the excited state terbium whereas H-O does quite well; hence, the strong quenching by H2O.

Chelated terbium greatly enhances the luminescence yield by excluding H2O, but almost all lanthanide complexes that have any use as luminescers include a sensitizer. For example, 4-aminosalicylic acid when included in the coordination sphere with terbium (but not europium), greatly enhances the luminescence yield when excited at 306 nm (max extinct.). To get the best signal one only needs to worry about exciting the pAS which very efficiently transfers its excitation to terbium. Another sensitizer that works well for both terbium and europium is the coumarin carbostyril 124 (7-Amino-4-methyl-2-quinolone) with excitation max at 328 nm.

Both sensitizers can be excited with multiphoton but depending on your laser this may typically mean 3 photon excitation. For the x-ray excitable probes, I used conjugates of pAS-DTPA and sometimes pAS2-DTPA. The carbostyril version lacking the extra carboxyl and phenoxyl OH tend to be more hydrophobic and are a little harder to make into good aqueous probes with low non-specific binding.

Although the focus of the following concerned x-ray applications, you might check: Moronne, M.M. (1999), Development of x-ray excitable luminescent probes for high resolution scanning x-ray microscopy. J. Ultramicroscopy, 77:23-36.

I spent a lot of time with lanthanide probes; let me know if you need any more help.

Mario
[hidden email]

Dear list,

I want to excite terbium cryptate fluorescence with 2-P; any idea, about
the excitation spectrum of it?

Thanks, jens

--

 ________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
 http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
 http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
 http://home.earthlink.net/~geomcnamara

 

 

-- 

________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]