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Joseph R. Lakowicz - One of the best experts on this subject based on the ideXlab platform.
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Time-resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-Photon Excitation.
Biophysical journal, 1997Co-Authors: Joseph R. Lakowicz, Ignacy Gryczynski, H Malak, M Schrader, P Engelhardt, H Kano, S W HellAbstract:We examined the fluorescence spectral properties of the DNA stains DAPI (4',6-diamidino-2-phenylindole, hydrochloride) and Hoechst 33342 (bis-benzimide, or 2,5'-bi'1H-benzimidazole2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazi nyl)) with two-Photon (2h nu) and three-Photon (3h nu) Excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of Excitation of DAPI bound to DNA changed from two-Photon at 830 nm to three-Photon at 885 nm. In contrast, Hoechst 33342 displayed only two-Photon Excitation from 830 to 885 nm. DAPI-DNA displayed the same emission spectra and decay times for 2h nu and 3h nu Excitation. Hoechst 33342-DNA displayed the same intensity decay for Excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiPhoton Excitation as compared to one-Photon Excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3h nu at 885 nm than for 2h nu at 830 nm. We used 970-nm Excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-Photon Excitation. Three-Photon Excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope.
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Three-Photon Excitation in fluorescence microscopy
Journal of biomedical optics, 1996Co-Authors: Stefan W. Hell, Ignacy Gryczynski, H Malak, Martin Schrader, Karsten Bahlmann, Aleksi E. Soini, Joseph R. LakowiczAbstract:We show experiments proving the feasibility of scanning fluorescence microscopy by three-Photon Excitation. Three-Photon Excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-Photon Excitation is performed at an Excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the Excitation power, also providing intrinsic 3-D imaging. The resolution of a three-Photon Excitation microscope is increased over that of a comparable two-Photon Excitation microscope.
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Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-Photon and two-Photon Excitation
Time-Resolved Laser Spectroscopy in Biochemistry IV, 1994Co-Authors: Ignacy Gryczynski, Joseph R. LakowiczAbstract:We studied the steady state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-Photon (OPE) and two-Photon (TPE) Excitation. Hoechst 33342 was found to display a large cross-section for two-Photon Excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 nm and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for one- and two-Photon Excitation. The anisotropy decay measurements of bis-benzimide, 2,5'-bi-1H-benzimidazole, 2'-(4- ethoxphenyl)-5-(4-methyl-1-piperazinyl) (HOECHST 33342) in ethanol revealed the same correlation times for two-Photon Excitation as observed for one-Photon Excitation. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for two-Photon Excitation than for one-Photon Excitation. The anisotropy spectra of Hoechst 33342 was examined in glycerol at -20 degree(s)C, revealing limiting values close to the theoretical limits for one-Photon (0.4) and two-Photon (0.57) Excitation. The steady-state anisotropy for one-Photon Excitation decreases in the shorter wavelength region (R6G dye laser, 280 to 315 nm), but the two-Photon anisotropy for 560 to 630 nm Excitation remains as high as in the long- wavelength region (690 to 770 nm). This result suggests that one- Photon absorption is due to two electronic transitions, but only one transition contributes to the two-Photon absorption over the wavelength range from 580 to 770 nm.
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calcium dependent fluorescence lifetimes of indo 1 for one and two Photon Excitation of fluorescence
Photochemistry and Photobiology, 1993Co-Authors: Henryk Szmacinski, Ignacy Gryczynski, Joseph R. LakowiczAbstract:We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca2+. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in the multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-Photon and two-Photon Excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-Photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-Photon cross section of Indo-1 is relatively high, about 4 x 10(-49) cm4 s/Photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-Photon Excitation, using either emission wavelength ratios or lifetime imaging methods.
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time resolved fluorescence intensity and anisotropy decays of 2 5 diphenyloxazole by two Photon Excitation and frequency domain fluorometry
The Journal of Physical Chemistry, 1992Co-Authors: Joseph R. Lakowicz, Ignacy Gryczynski, Zygmunt Gryczynski, Eva Danielsen, Mary J WirthAbstract:We report the first time-resolved fluorescence measurements of the intensity and anisotropy decays resulting from two-Photon Excitation. A 10-GHz frequency-domain fluorometer (Rev. Sci. Instrum 1990, 61, 2331), equipped with two focal lenses and an emission monochromator, was used for steady-state and time-resolved measurements of PPO fluorescence. The emission spectra and the intensity decays observed with single- and two-Photon Excitation were essentially identical
Robert J Stanley - One of the best experts on this subject based on the ideXlab platform.
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the two Photon Excitation cross section of 6map a fluorescent adenine analogue
Journal of Physical Chemistry B, 2005Co-Authors: Robert J Stanley, Zhanjia Hou, And Aiping Yang, Mary E HawkinsAbstract:6MAP is a fluorescent analogue of adenine that undergoes Watson−Crick base pairing and base stacking in double-stranded DNA. The one-Photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two Photons. We report the first determination of the two-Photon Excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two Photons. The one-Photon and two-Photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-Photon Excitation shows no measurable quenching up to about 5 μM. The maximum in the two-Photon Excitation spectrum gives a two-Photon cross section, δTPE, of 3.4 ± 0.1 Goeppert−Mayer (G.M.) at ...
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two Photon Excitation of 4 hydroxymethyl 4 5 8 trimethylpsoralen
Photochemistry and Photobiology, 1997Co-Authors: Robert J Stanley, Michelle Lin, Warren K Hoeffler, Steven G Boxer, Michael W Berns, Eugene A BauerAbstract:— Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradiated with a mode-locked titanium: sapphire (Ti: sapphire) laser tuned to 730 nm, an aqueous solution of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-Photon Excitation with UVA Excitation, and its magnitude depends quad-ratically on the intensity of laser Excitation. These results suggest that two-Photon Excitation occurs to a potentially photochemically active state. To estimate the two-Photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm Excitation at room temperature that resulted in a value of 0.045 ± 0.007. The two-Photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 ± 10−50 cm4 s (20 Goppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.
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research note two Photon Excitation of 4 hydroxymethyl 4 5 8 trimethylpsoralen
1997Co-Authors: Robert J Stanley, Michelle Lin, Steven G Boxer, Warren K Hoefflerl, Michael W Bern, Eugene A BauerlAbstract:Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradiated with a mode-locked titanium : sapphire (Ti : sapphire) laser tuned to 730 nm, an aqueous solution of 4‘hydroxymethyl-4,5’,8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-Photon Excitation with UVA Excitation, and its magnitude depends quadratically on the intensity of laser Excitation. These results suggest that two-Photon Excitation occurs to a potentially photochemically active state. To estimate the two-Photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm Excitation at room temperature that resulted in a value of 0.045 * 0.007. The two-Photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 X cm4 s (20 Goppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.
Øyvind Frette - One of the best experts on this subject based on the ideXlab platform.
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testing fluorescence lifetime standards using two Photon Excitation and time domain instrumentation fluorescein quinine sulfate and green fluorescent protein
Journal of Fluorescence, 2018Co-Authors: Arne S. Kristoffersen, Svein Rune Erga, Borge Hamre, Øyvind FretteAbstract:It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-Photon Excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for Excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10− 3 − 10− 5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-Photon Excitation and lifetime data was obtained in the time-domain using time-correlated single-Photon counting.
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Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Rhodamine B, Coumarin 6 and Lucifer Yellow
Journal of Fluorescence, 2014Co-Authors: Arne S. Kristoffersen, Svein Rune Erga, Borge Hamre, Øyvind FretteAbstract:Having good information about fluorescence lifetime standards is essential for anyone performing lifetime experiments. Using lifetime standards in fluorescence spectroscopy is often regarded as a straightforward process, however, many earlier reports are limited in terms of lifetime concentration dependency, solvents and other technical aspects. We have investigated the suitability of the fluorescent dyes rhodamine B, coumarin 6, and lucifer yellow as lifetime standards, especially to be used with two-Photon Excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine which wavelengths we should use for the Excitation and an appropriate detector range. We also measured lifetimes for different concentrations, ranging from 10^−2– 10^−6 M, in both water, ethanol and methanol solutions. We observed that rhodamine B lifetimes depend strongly on concentration. Coumarin 6 provided the most stable lifetimes, with a negligible dependency on concentration and solvent. Lucifer yellow lifetimes were also found to depend little with concentration. Finally, we found that a mix of two fluorophores (rhodamine B/coumarin 6, rhodamine B/lucifer yellow, and coumarin 6/lucifer yellow) all yielded very similar lifetimes from a double-exponential decay as the separate lifetimes measured from a single-exponential decay. All lifetime measurements were made using two-Photon Excitation and obtaining lifetime data in the time-domain using time-correlated single-Photon counting.
Ignacy Gryczynski - One of the best experts on this subject based on the ideXlab platform.
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Time-resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-Photon Excitation.
Biophysical journal, 1997Co-Authors: Joseph R. Lakowicz, Ignacy Gryczynski, H Malak, M Schrader, P Engelhardt, H Kano, S W HellAbstract:We examined the fluorescence spectral properties of the DNA stains DAPI (4',6-diamidino-2-phenylindole, hydrochloride) and Hoechst 33342 (bis-benzimide, or 2,5'-bi'1H-benzimidazole2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazi nyl)) with two-Photon (2h nu) and three-Photon (3h nu) Excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of Excitation of DAPI bound to DNA changed from two-Photon at 830 nm to three-Photon at 885 nm. In contrast, Hoechst 33342 displayed only two-Photon Excitation from 830 to 885 nm. DAPI-DNA displayed the same emission spectra and decay times for 2h nu and 3h nu Excitation. Hoechst 33342-DNA displayed the same intensity decay for Excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiPhoton Excitation as compared to one-Photon Excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3h nu at 885 nm than for 2h nu at 830 nm. We used 970-nm Excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-Photon Excitation. Three-Photon Excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope.
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Three-Photon Excitation in fluorescence microscopy
Journal of biomedical optics, 1996Co-Authors: Stefan W. Hell, Ignacy Gryczynski, H Malak, Martin Schrader, Karsten Bahlmann, Aleksi E. Soini, Joseph R. LakowiczAbstract:We show experiments proving the feasibility of scanning fluorescence microscopy by three-Photon Excitation. Three-Photon Excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-Photon Excitation is performed at an Excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the Excitation power, also providing intrinsic 3-D imaging. The resolution of a three-Photon Excitation microscope is increased over that of a comparable two-Photon Excitation microscope.
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Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-Photon and two-Photon Excitation
Time-Resolved Laser Spectroscopy in Biochemistry IV, 1994Co-Authors: Ignacy Gryczynski, Joseph R. LakowiczAbstract:We studied the steady state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-Photon (OPE) and two-Photon (TPE) Excitation. Hoechst 33342 was found to display a large cross-section for two-Photon Excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 nm and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for one- and two-Photon Excitation. The anisotropy decay measurements of bis-benzimide, 2,5'-bi-1H-benzimidazole, 2'-(4- ethoxphenyl)-5-(4-methyl-1-piperazinyl) (HOECHST 33342) in ethanol revealed the same correlation times for two-Photon Excitation as observed for one-Photon Excitation. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for two-Photon Excitation than for one-Photon Excitation. The anisotropy spectra of Hoechst 33342 was examined in glycerol at -20 degree(s)C, revealing limiting values close to the theoretical limits for one-Photon (0.4) and two-Photon (0.57) Excitation. The steady-state anisotropy for one-Photon Excitation decreases in the shorter wavelength region (R6G dye laser, 280 to 315 nm), but the two-Photon anisotropy for 560 to 630 nm Excitation remains as high as in the long- wavelength region (690 to 770 nm). This result suggests that one- Photon absorption is due to two electronic transitions, but only one transition contributes to the two-Photon absorption over the wavelength range from 580 to 770 nm.
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calcium dependent fluorescence lifetimes of indo 1 for one and two Photon Excitation of fluorescence
Photochemistry and Photobiology, 1993Co-Authors: Henryk Szmacinski, Ignacy Gryczynski, Joseph R. LakowiczAbstract:We characterized the fluorescence intensity decays of Indo-1, which is commonly used as an emission wavelength-ratiometric calcium probe. The apparent lifetime of the long-wavelength side of the emission of Indo-1 is dependent on Ca2+. This long-wavelength emission displays the characteristics of an excited-state reaction, that is, a negative preexponential component in the multiexponential analysis. The emission spectra and lifetime of Indo-1 appear to be identical for one-Photon and two-Photon Excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-Photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-Photon cross section of Indo-1 is relatively high, about 4 x 10(-49) cm4 s/Photon molecule at 690 nm for both the calcium-free and calcium-bound forms. Hence, Indo-1 can be used for calcium imaging based on one- or two-Photon Excitation, using either emission wavelength ratios or lifetime imaging methods.
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time resolved fluorescence intensity and anisotropy decays of 2 5 diphenyloxazole by two Photon Excitation and frequency domain fluorometry
The Journal of Physical Chemistry, 1992Co-Authors: Joseph R. Lakowicz, Ignacy Gryczynski, Zygmunt Gryczynski, Eva Danielsen, Mary J WirthAbstract:We report the first time-resolved fluorescence measurements of the intensity and anisotropy decays resulting from two-Photon Excitation. A 10-GHz frequency-domain fluorometer (Rev. Sci. Instrum 1990, 61, 2331), equipped with two focal lenses and an emission monochromator, was used for steady-state and time-resolved measurements of PPO fluorescence. The emission spectra and the intensity decays observed with single- and two-Photon Excitation were essentially identical
Eugene A Bauer - One of the best experts on this subject based on the ideXlab platform.
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two Photon Excitation of 4 hydroxymethyl 4 5 8 trimethylpsoralen
Photochemistry and Photobiology, 1997Co-Authors: Robert J Stanley, Michelle Lin, Warren K Hoeffler, Steven G Boxer, Michael W Berns, Eugene A BauerAbstract:— Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradiated with a mode-locked titanium: sapphire (Ti: sapphire) laser tuned to 730 nm, an aqueous solution of 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-Photon Excitation with UVA Excitation, and its magnitude depends quad-ratically on the intensity of laser Excitation. These results suggest that two-Photon Excitation occurs to a potentially photochemically active state. To estimate the two-Photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm Excitation at room temperature that resulted in a value of 0.045 ± 0.007. The two-Photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 ± 10−50 cm4 s (20 Goppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.