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- Theory of two-photon induced fluorescence anisotropy decay i...Theory of two-photon induced fluorescence anisotropy decay in membranes
Biophysical Journal, Volume 64, Issue 5, 1 May 1993, Pages 1567-1575
Sun-Yung Chen and B.Wieb Van Der Meer
AbstractWe report the first theoretical description for the time-dependent fluorescence anisotropy decay resulted from two-photon excitation (()) for fluorophores in macroscopically isotropic and oriented membranes. In case of two-photon excitation, the initial value of the fluorescence anisotropy (0) immediately after excitation by a flash of polarized light is a function of the components of the two-photon absorption transition tensor [unk] and the projections of the emission transition moment to the principal axes of [unk]. The components of [unk] depend on the symmetries of all molecular states relevant to the two-photon absorption process. The maximal value of (0) is proven to be as large as 0.61 in contrast to 0.4 for the conventional one-photon induced fluorescence anisotropy (0). It is shown that only for some special cases the ratio of the two-photon () over the conventional one-photon () will be a constant at all times for fluorophores in macroscopically isotropic membrane systems. In oriented membrane systems, an additional order parameter <> can be determined by the use of angle-resolved fluorescence depolarization measurements resulted from two-photon excitation. The advantages of measuring time-resolved fluorescence anisotropy decays or angle-resolved fluorescence depolarization ratios by two-photon excitation for the study of orientational dynamics in isotropic or oriented membranes are discussed from the theoretical point of view.
Abstract | PDF (696 kb) - Time-Resolved Fluorescence Spectroscopy and Imaging of DNA L...Time-Resolved Fluorescence Spectroscopy and Imaging of DNA Labeled with DAPI and Hoechst 33342 Using Three-Photon Excitation
Biophysical Journal, Volume 72, Issue 2, 1 February 1997, Pages 567-578
Joseph R. Lakowicz, Ignacy Gryczynski, Henryk Malak, Martin Schrader, Peter Engelhardt, Hiroski Kano and Stefan W. Hell
AbstractWe 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-H-benzimidazole2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)) with two-photon (2hν) and three-photon (3hν) 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 2hν and 3hν 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 3hν at 885 nm than for 2hν 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.
Abstract | PDF (1735 kb) - Three-photon induced fluorescence of the calcium probe Indo-...Three-photon induced fluorescence of the calcium probe Indo-1
Biophysical Journal, Volume 70, Issue 1, 1 January 1996, Pages 547-555
H. Szmacinski, I. Gryczynski and J.R. Lakowicz
AbstractWe report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.
Abstract | PDF (858 kb) - …more
Copyright © 1996 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 71, Issue 6, 3448-3453, 1 December 1996
doi:10.1016/S0006-3495(96)79540-1
Research Article
Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation
I. Gryczynski, H. Malak, J.R. Lakowicz, H.C. Cheung, J. Robinson and P.K. Umeda
Abstract
We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.
