Two-photon Fluorescence Excitation Spectra Research Articles

Hidden Electronic Excited State of Enhanced Green Fluorescent Protein

Precise two-photon absorption spectra of the green fluorescent protein (GFP) and the mutants sapphire-GFP (T203I) and enhanced GFP (S65T/F64L), as well as a model compound for the chromophore, 4'-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) were measured by multiplex two-photon absorption spectroscopy. The observed TPA bands of the anionic forms of enhanced GFP and HBDI were significantly shifted to the higher energy compared with the lowest-energy bands in one-photon absorption spectra. This result indicated the existence of a hidden electronic excited state in the vicinity of the lowest excited singlet (S1) state of the anionic form of the GFP chromophore, which is the origin of the blue shift of the two-photon absorption spectra as well as two-photon fluorescence excitation spectra.

Supercontinuum Dynamically Visualizes a Dividing Single Cell

During cell division, various organelles behave dynamically. Visualization of these dynamic behaviors of organelles is a promising one step forward for understanding life at the molecular level. One- or two-photon excited fluorescence microscopy has so far been used for visualizing these cell dynamics. The fluorescent probe introduced into a living cell can visualize the spatial distribution of a target molecule in real time, enabling the tracing of cell dynamics at the molecular level. Introducing a fluorescent probe into a cell, however, may alter the physical and chemical conditions of the cell. Here we show a new method for direct (no need for staining cells) visualization of living cell processes with coherent anti-Stokes Raman scattering (CARS) spectroscopy. A new light source, supercontinuum generated from a photonic crystal fiber, has facilitated ultrabroadband (>3500 cm(-1)) multiplex CARS spectroscopy and imaging with high molecular specificity. Using this multiplex CARS technique, we have been successful in tracing the whole cell division process, the splitting of a mother cell into two daughter cells, appearance and disappearance of septum, and dynamic distribution changes of organelles consisting of lipid membrane. The supercontinuum has also facilitated simultaneous measurement of the CARS and two-photon excited fluorescence (TPEF) spectra, enabling what we call multiple nonlinear spectral imaging. Three-dimensional image reconstruction of a living cell with high speed is now possible to elucidate more detailed molecular-level dynamics inside a dividing living cell.

Stepwise Two-photon Excited Fluorescence from Higher Excited States of Chlorophylls in Photosynthetic Antenna Complexes

Stepwise two-photon excited fluorescence (TPEF) spectra of the photosynthetic antenna complexes PCP, CP47, CP29, and light-harvesting complex II (LHC II) were measured. TPEF emitted from higher excited states of chlorophyll (Chl) a and b was elicited via consecutive absorption of two photons in the Chl a/b Qy range induced by tunable 100-fs laser pulses. Global analyses of the TPEF line shapes with a model function for monomeric Chl a in a proteinaceous environment allow distinction between contributions from monomeric Chls a and b, strongly excitonically coupled Chls a, and Chl a/b heterodimers/-oligomers. The analyses indicate that the longest wavelength-absorbing Chl species in the Qy region of LHC II is a Chl a homodimer with additional contributions from adjacent Chl b. Likewise, in CP47 a spectral form at approximately 680 nm (that is, however, not the red-most species) is also due to strongly coupled Chls a. In contrast to LHC II, the red-most Chl subband of CP29 is due to a monomeric Chl a. The two Chls b in CP29 exhibit marked differences: a Chl b absorbing at approximately 650 nm is not excitonically coupled to other Chls. Based on this finding, the refractive index of its microenvironment can be determined to be 1.48. The second Chl b in CP29 (absorbing at approximately 640 nm) is strongly coupled to Chl a. Implications of the findings with respect to excitation energy transfer pathways and rates are discussed. Moreover, the results will be related to most recent structural analyses.

Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy

We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin.

Photochemical Production of Nitric Oxide via Two-Photon Excitation with NIR Light

Herein we demonstrate the successful photochemical generation of nitric oxide via two-photon excitation (TPE) from the supramolecular complex PPIX-RSE ({mu-S,mu-S'-protoporphyrin-IX-bis(2-thioethyl)diester]tetranitrosyl-diiron). The TPE fluorescence spectra indicate efficient energy transfer from the PPIX antennae to the iron sulfur nitrosyl cluster. Further evidence of NO release is demonstrated using a nitric oxide specific electrode and ESI+ MS.

Zeeman spectra of the à 1Au←X̃ 1Ag transition of trans-glyoxal studied by Doppler-free two-photon fluorescence excitation spectroscopy

Doppler-free two-photon fluorescence excitation spectra of the A 1Au(v7′=1)←X 1Ag(v″=0) transition of trans-glyoxal were measured by propagating a laser beam either polarized parallel (π pump) or perpendicular (σ pump) to the magnetic field of 6 T. π pump and σ pump dependence of the Zeeman spectra of the Q(K)Q(J=6) lines for K=0–6 was measured. Intensities of the Zeeman components of the Q(6)Q(6) lines were observed to have the maxima at the high and low wave number ends and the minimum at the middle for π pump, and the minima at the high and low wave number ends and the maximum at the middle for σ pump. By comparing calculated and observed patterns of the Zeeman spectra, it became clear that the transition tensor M00 is dominant and the effective intermediate state is 1 1Bu(ππ*). K dependence of the Zeeman spectra of the Q(K)Q(J=15) lines was measured, and the Zeeman splittings were observed to increase proportionally to K2. J dependence of the Zeeman spectra of the Q(K=J)Q(J) lines was measured for J=...

One- and two-photon excited dual fluorescence properties of zinc(ii) and cadmium(ii) complexes containing 4-dipropylaminobenzaldehyde thiosemicarbazone

A new ligand, 4-dipropylaminobenzaldehyde thiosemicarbazone (HL) and its complexes (ZnL2, CdL2), which exhibit intense two-photon excited (TPE) dual fluorescence using 800 nm laser pulses in the femtosecond regime, were synthesized and fully characterized. The measured power dependence of the fluorescence signals provides direct evidence for TPE. The two-photon excited dual fluorescence spectra were compared and contrasted with the corresponding results obtained from one-photon excitation. Emission peaks of the ligand and its complexes were assigned with the aid of PM3 calculations.

Two-photon-excited fluorescence of zinc(II), cadmium(II) complexes containing phenothiazine ligand

Two new ligands, S-benzyl-β-N-[10-ethylphenothiazine-3-methylene] dithiocarbazate (HL 1 ), S-methyl-β-N-[10-ethylphenothiazine-3-methylene] dithiocarbazate (HL2), and their complexes (ZnL 2 1 , ZnL 2 2 , CdL 2 2 ), which exhibit intensive two-photon excited (TPE) fluorescence at 800 nm laser pulses in the femtosecond regime, are synthesized and fully characterized. The measured power dependence of the fluorescence signals provides a direct evidence for TPE. All of them exhibit a large two-photon absorptive crosssection. The two-photon-excited fluorescence spectra of them are compared and contrasted with the corresponding results obtained from the one-photon excitation. Emission peaks of the ligands and their complexes are observed mostly at the region 550—595 nm in the TPE fluorescence spectra.

Two-photon excitation studies of hypocrellins for photodynamic therapy

The photophysical and photochemical properties of hypocrellins (HA and HB) are examined with two-photon excitations at 800 nm using femtosecond pulses from a Ti:sapphire laser. The two-photon excited fluorescence spectra of HA and HB are very similar to those obtained by one-photon excitation, which may indicate that the two-photon induced photodynamic processes of hypocrellins are similar to one-photon induced photodynamic processes. The two-photon excitation cross sections of HA and HB are measured at 800 nm as about 34.8×10 −50 cm 4 s/photon and 21.3×10 −50 cm 4 s/photon, respectively. The large two-photon cross sections of both HA and HB, suggest that the hypocrellins can be potential two-photon phototherapeutic agents. As an example for two-photon photodynamic therapy of hypocrellins, we also further examine the cell-damaging effects of HA upon two-photon illumination. Our preliminary results of cell viability test indicate hypocrellins can effectively damage the Hela cells under two-photon illumination.

One- and two-photon spectra of jet-cooled 2,3-dimethylindole: 1L b and 1L a assignments

One-photon and polarized two-photon fluorescence excitation spectra and the dispersed fluorescence spectrum of jet-cooled 2,3-dimethylindole are reported. The two-photon intensity ratios for linear versus circular polarized excitation show that the origin is primarily of 1L b character and that lines with predominantly 1L a character appear at 342, 356, 371, 383, 396, and 406 cm −1 above the 1L b origin. These lines exhibit what appears to be methyl rotor tunneling spitting. The ab initio computed dispersed fluorescence spectrum for the 1L b state agrees well with the observed dispersed fluorescence reported here; that computed for 1L a does not. Large splitting of certain vibrational modes due to mixing with methyl motions is responsible for marked differences in the dispersed fluorescence compared to that of indole and 3-methylindole.

Multi-photon fluorescence microscopy — the response of plant cells to high intensity illumination

Multi-photon fluorescence microscopy has been cited for its advantage in increased depth penetration due to low linear absorption and scattering coefficient of biological specimen in the near infrared (NIR) range. Because of the need of high peak power for efficiently exciting two-photon fluorescence, the relationship between cell damage and peak power has become an interesting and much debated topic in the applications of multi-photon fluorescence microscopy. It is conceivable that at high illumination intensity, non-linear photochemical processes have impacts on cell physiology and viability in ways much different from low illumination in the linear domain. In this article, we discuss some of the issues in two-photon fluorescence microscopy, including the degree of transparency of the specimen, a comparison of single- and two-photon excited fluorescence spectra, and the cell damage under high intensity illumination, using plant cells as a model.

Two-Photon Spectroscopy of Jet-Cooled Benzonitrile: New Results for a Charge Transfer Molecule

We compare the first jet-cooled two-photon fluorescence excitation spectrum of benzonitrile with its one-photon spectrum measured by resonance-enhanced multi-photon ionization (REMPI). Both spectra include the pure electronic transition and one vibrationally induced transition. The inducing mode is the ν

Two-Photon Fluorescence Excitation Spectrum of 1,6-Methano-[10]annulene

The two-photon fluorescence excitation spectrum of 1,6-methano-[10]annulene has been measured in fluid solution at room temperature between 25 000 and 40 000 cm-1 and in rigid solution at 77 and 15 K between 25 000 and 28 000 cm-1. The two-photon polarization ratios of both spectra have been determined. Spectral assignments have been made on the basis of two-photon polarization data, ab initio calculations, and comparison with one-photon data. The four excited states below 5 eV have been identified. Calculated one- and two-photon allowed intensities are in good agreement with results derived from the alternant hydrocarbon theory. The S0 → S1 two-photon intensity is mainly vibronically induced by b1 modes. The most active b1 vibration, the “Kekule”-type mode responsible for double bond localization in 1,6-methano-[10]annulene, shifts to ≈1540 cm-1 in S1 from the ground state 1355 cm-1 value. Applying two-photon polarization results to 1,6-methano-[10]annulene (C2v symmetry), the tensor elements of the strongest totally symmetric S0 → S1 (B1 × b1 × a1) transition have been estimated.

Near-infrared two-photon excitation of protoporphyrin IX: photodynamics and photoproduct generation.

The spectroscopy and photochemistry of protoporphyrin IX in ethanol and in Triton X-100 micelle solution have been examined using near-infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. We have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.

Observation of the S1 state of spheroidene in LH2 by two-photon fluorescence excitation

Abstract The two-photon fluorescence excitation spectrum of LH2, measured by directly exciting the carotenoid S 1 transition and monitoring fluorescence from the bacteriochlorophyll, provides the first direct experimental verification of carotenoid S 1 to bacteriochlorophyll energy transfer. It also provides an estimate of the in situ spheroidene S 1 transition energy of 13 900±150 cm −1 , slightly red-shifted from solution estimates. The relative abilities of the carotenoids spheroidene (10 conjugated double bonds) and rhodopin glucoside (11 conjugated double bonds) to transfer energy via their S 1 states are discussed in terms of spectral overlap factors and competing processes such as S 1 →S 0 internal conversion.

Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm

We report on two-photon fluorescence excitation (TPE) action cross sections for five widely used molecular fluorophores. Measurements were performed by use of ultrashort (~100-fs) Ti:sapphire pulsed excitation over the range 690-960 nm. TPE spectra were obtained by comparison with a fluorescein calibration standard. Large cross sections were found for the cyanine reagent Cy 3 (~140 GM) and for Rhodamine 6G (~150 GM), both at 700 nm [1 GM = 10(-50) (cm(4) s)/photon]. Several fluorophores show interesting and desirable blue shifts with respect to twice the one-photon absorption wavelength. Fluorophore fluorescence intensities showed no significant departure (?4%) from quadratic illumination power dependence, indicating genuine two-photon processes. Implications of these measurements for two-photon laser-scanning microscopy are discussed.

Excited-state proton transfer in 1-naphthol·(NH 3) n complexes: the threshold size is n=4

Resonant two-photon ionization, fluorescence and fluorescence-detected excitation spectra of 1-naphthol·(NH 3) n clusters are reported and interpreted with emphasis on determining the smallest clusters for which excited-state proton transfer (ESPT) can occur. Consistent with the earliest studies but in contrast to more recent work, the ESPT threshold is found at n=4, not n=3. The confusion is shown to have arisen from weak, broad fluorescence of n=1–3 clusters extending to ∼400 nm. Excitation spectra of genuine ESPT emission at longer wavelengths gives no indication of contributions from n<4.

Two-photon-induced fluorescence.

Nonresonant two-photon electronic spectroscopy of polyatomic molecules is reviewed for the period since 1979. Emphasis is placed on studies that expose patterns in the two-photon fluorescence (also ionization, optoacoustic) excitation spectra of aromatic hydrocarbons and the effect of vibrations and substitution, particularly within the framework of pseudoparity rules. A section is devoted to biological molecules and the emerging use of two-photon-induced fluorescence anisotropy. Relevant theoretical results are discussed, with emphasis on quantum chemical predictions of vibronic coupling and substituent effects on two-photon absorptivity and tensor properties of individual molecules. This chapter includes higher-order spectroscopy, and a limited number of three- and four-photon studies are discussed.

Nonlinear Spectroscopy on a Single Quantum System: Two-Photon Absorption of a Single Molecule

Two-photon fluorescence excitation spectra of single diphenyloctatetraene molecules trapped in an n -tetradecane matrix were measured at cryogenic temperatures. The purely electronic zero-phonon line (transition at 444 nanometers) of these single molecules with a width of about 60 megahertz was excited by a continuous-wave, single-mode laser at 888 nanometers. Even though the two-photon absorption cross section is extremely small, a high photon count rate and low background allowed nonlinear spectroscopy to be extended to the single-molecule level. This experiment also suggests the possibility of two-photon single-molecule scanning microscopy.

Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm

Measurements of two-photon fluorescence excitation (TPE) spectra are presented for 11 common molecular fluorophores in the excitation wavelength range 690 nm < λ < 1050 nm. Results of excitation by ∼100-fs pulses of a mode-locked Ti:sapphire laser are corroborated by single-mode cw Ti:sapphire excitation data in the range 710 nm < λ < 840 nm. Absolute values of the TPE cross section for Rhodamine B and Fluorescein are obtained by comparison with one-photon-excited fluorescence, assuming equal emission quantum efficiencies. TPE action cross sections for the other nine fluorophores are also determined. No differences between one-photon- and two-photon-excited fluorescence emission spectra are found. TPE emission spectra are independent of excitation wavelength. With both pulsed and cw excitation the fluorescence emission intensities are strictly proportional to the square of the excitation intensity to within ±4% for excitation intensities sufficiently below excited-state saturation.