Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed

Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.     

Fluorescence resonance energy transfer in a novel two-photon absorbing system

A novel fluorescence resonance energy transfer (FRET) system containing a two-photon absorbing dye and a nile red chromophore has been synthesized. Upon two-photon excitation by laser at 815 nm this molecule displays efficient energy transfer from the two-photon absorbing dye to the nile red moiety, with an 8-fold increase in emission compared to the model compound. Similarly, single-photon excitation of the two-photon absorbing moiety at 405 nm results in >99% energy-transfer efficiency, along with a 3.4-fold increase in nile red emission compared to direct excitation of the nile red chromophore at 540 nm. This system provides an effective way to use IR radiation to excite molecules that, by themselves, have little or no two-photon absorption.     

FLUORESCENCE OF TYROSINE AND TRYPTOPHAN IN PROTEINS USING ONE- AND TWO-PHOTON EXCITATION

Abstract— We examined the emission spectra of tyrosine- and tryptophan-containing proteins using one-photon (270–310 nm) and two-photon (565–610 nm) excitation. Emission spectra for two-photon excitation of native and denatured human serum albumin and of three purine nucleoside phosphorylases indicated an absence of the tyrosine emission normally seen for one-photon excitation below 290 nm. We examined the one-photon and two-photon excitation spectra of tyrosine-tryptophan mixtures to determine the origin of selective excitation of the tryptophan residues. These results confirmed a short-wavelength shift of the tyrosine two-photon excitation spectrum relative to that of tryptophan, as recently reported by Rehms and Callis (1993) Chem. Phys. Lett . 208 , 276–282.     

Metal ion sensing novel calix[4]crown fluoroionophore with a two-photon absorption property

1,3-Alternate calix[4]arene-based fluorescent chemosensors bearing two-photon absorbing chromophores have been synthesized, and their sensing behaviors toward metal ions were investigated via absorption band shifts as well as one- and two-photon fluorescence changes. Free ligands absorb the light at 461 nm and weakly emit their fluorescence at 600 nm when excited by UV-vis radiation at 461 nm, but no two-photon excited fluorescence is emitted by excitation at 780 nm. Addition of an Al(3+) or Pb(2+) ion to a solution of the ligand causes a blue-shifted absorption and enhanced fluorescence due to a declined resonance energy transfer (RET) upon excitation by one- and two-photon processes. Addition of a Pb(2+) ion to a solution of 1.K(+) results in a higher fluorescence intensity than the original 1.Pb(2+) complex regardless of one- or two-photon excitation, due to the allosteric effect induced by the complexation of K(+) with a crown loop.     

Two-photon excitation of 2,5-diphenyloxazole using a low power green solid state laser

This Letter concerns two-photon excitation of 2,5-Diphenyloxazole (PPO) upon illumination from a pulsed 532 nm solid state laser, with an average power of 30 mW, and a repetition rate of 20 MHz. A very agreeable emission spectrum position and shape has been achieved for PPO receiving one- and two-photon excitation, which suggests that the same excited state is involved for both excitation modes. Also, a perfect quadratic dependence of laser power in the emission intensity function has been recorded. We tested the application of a small solid state green laser to two-photon induced time-resolved fluorescence, revealing the emission anisotropy of PPO to be considerably higher for two-photon than for one-photon excitation.     

Resonance CARS study of the structure of "green" and "red" chromophores within the red fluorescent protein DsRed

Vibrational spectra of red fluorescent protein DsRed have been studied for the first time by polarization-sensitive multiplex coherent anti-Stokes Raman scattering at two excitation wavelengths, 545 and 583 nm, in resonance with the absorption bands of the immature "green" and mature "red" protein chromophores. Overall vibrational patterns of both DsRed chromophores were found to be similar to each other and to differ from that of S65T-GFP at pH8. The combined analysis of our CARS data and published structural information suggest that both "green" and "red" DsRed species possess an extended chromophore structure. Consequently, our data suggest that pi-bonding system extension during isomerization around the cis peptide bond between Phe 65 and Gln 66 is a necessary but not sufficient step in DsRed chromophore maturation.     

Two-Color Two-Photon Excitation of Fluorescence

Abstract— We report the observation of two-photon excitation of an organic fluorophore with two different wavelengths, a phenomenon we refer to as two-color two-photon (2C2P) excitation. Ultraviolet emission of p -Merphenyl at 340 nm was observed when the sample was illuminated with both 375 and 750 nm pulses from a picosecond dye laser. The emission of p -terphenyl was about 100-fold and more than 1000-fold less for illumination at only 375 or 750 nm, respectively. Observation of the 2C2P signal required temporal and spatial overlap of the 375 and 750 nm pulses. The amplitude of the signal depended on the polarization of each beam. 2C2P excitation can have applications in fluorescence microscopy and elsewhere when spatially localized excitation is desirable.     

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 x 10(-50) cm(4) s/photon and 21.3 x 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.     

The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue

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 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes.     

Two-Photon Excitation of 4'-Hydroxymethyl-4,5',8-Trimethylpsoralen

Abstract— 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 Göppert-Mayer). The excited-state photophysics and photochemistry of psoralens suggest potential applications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa.     

Selective fluorescence recovery after bleaching of single E2GFP proteins induced by two-photon excitation.

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.     

RAPID COMMUNICATION

Abstract— We show that the calcium fiuorophore Indo-1 can be excited by simultaneous absorption of three-photons at 885 nra, a wavelength readily available from Ti:sapphire lasers. Three-photon excitation was demonstrated by the emission intensity of Indo-1 which depended on the cube of the laser power, and by a higher anisotropy than was observed for two-photon excitation. Excitation of Indo-1 becomes a two-photon process when the wavelength is decreased to 820 nm. Three-photon excitation was accomplished at a low 17μ concentration of Indo-1. Examination of the spatial profile of the excited Indo-1 showed a smaller volume for three- versus two-photon excitation. These results suggest that three-photon excitation may be useful in fluorescence microscopy using the long wavelength output of Tksapphire lasers, and may provide higher spatial resolution than available using two-photon excitation.     

Femtosecond pump-probe experiments on trapped flavin: optical control of dissociation

Femtosecond pump-probe experiments are performed on flavin biomolecules isolated in an ion trap. Mass spectra of the photoinduced fragments show that the fragmentation pathways can be modified using two-color two-photon excitation. In particular, when an infrared probe pulse (810 nm) is added subsequent to the first excitation step (excitation of the S(1) state of flavin mononucleotide at 405 nm), branching ratios between lumichrome and lumiflavin production are inverted relative to the single excitation case.     

Singlet oxygen generation via two-photon excited FRET

A new approach to two-photon excited photodynamic therapy has been developed. A dendritic array of eight donor chromophores capable of two-photon absorption (TPA) was covalently attached to a central porphyrin acceptor. Steady-state fluorescence measurements demonstrated that the donor chromophores transfer excited-state energy to the porphyrin with 97% efficiency. Two-photon excitation of the donor chromophores at 780 nm resulted in a dramatic increase in porphyrin fluorescence relative to a porphyrin model compound. Enhanced singlet oxygen luminescence was observed from oxygen-saturated solutions of the target compound under two-photon excitation conditions.     

Tracking of mercury ions in living cells with a fluorescent chemodosimeter under single- or two-photon excitation

Tracking of Hg²⁺ in solutions as well as in living cells was conducted with a fluorescent chemodosimeter by measuring the spectral shift of its fluorescence under single- or two-photon excitation. The spectral hypsochromic shifts of this chemodosimeter when reacting with Hg²⁺ were found to be about 50 nm in acetonitrile/water solutions and 32 nm in Euglena gracilis 277 living cells. This chemodosimeter shows high sensitivity and selectivity, and is not influenced by the pH values. It can signal Hg²⁺ in solutions down to the ppb range under either single-photon excitation (SPE) at 405 nm or two-photon excitation (TPE) at 800 nm. However, with low cellular chemodosimeter concentrations, the SPE spectra were disturbed by the auto-fluorescence from the native fluorophore in the cell, while the TPE spectra were still of high quality since the two-photon absorption cross section of this chemodosimeter is much larger than that of the native fluorophores in the cell.     

Two-photon and multi-photon excitation of naphthalene in the vapor phase,studied by excitation and fluorescence spectra

Naphthalene vapor is irradiated by μsec dye laser pulses of 150 kW peak power and a spectral bandwidth of 0.3 nm. A two-photon excitation spectrum is detected by monitoring the near UV fluorescence as a function of laser wavelength which is tuned between 570 and 610 nm. The fluorescence obtained by irradiation into the strongest band of the two-photon spectrum could be spectroscopically resolved using a bandwidth of 80 cm^?1. The spectrum exhibits vibrational structure which lies on a strong non-resolved background. From information in both spectra it can be definitely concluded that vibronic levels of B_3u × b_3u species in the lowest singlet state are predominantly excited in a two-photon process. The non-resolved background in the fluorescence spectrum is attributed to subsequent excitation of the two-photon state by a third photon. Further stepwise excitation in the strong radiation field of the laser is also taken into account.     

NIR-Ⅱ Excitation and NIR-I Emission Based Two-photon Fluorescence Lifetime Microscopic Imaging Using Aggregation-induced Emission Dots

Near-infrared(NIR)lights are powerful tools to conduct deep-tissue imaging since NIR-Ⅰ wavelengths hold less photon absorption and NIR-Ⅱ wavelengths serve low photon scattering in the biological tissues compared with visible lights.Two-photon fluorescence lifetime microscopy(2PFLM)can utilize NIR-Ⅱ excitation and NIR-Ⅰ emission at the same time with the assistance of a well-designed fluorescent agent.Aggregation induced emission(AIE)dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA)cross-section as aggregated dots.Herein,we report two-photon fluorescence lifetime microscopic imaging with NIR-Ⅱ excitation and NIR-Ⅰ emission using a novel deep-red AIE dye.The AIE-gens held a 2PA cross-section as large as 1.61×10⁴GM at 1040 nm.Prepared AIE dots had a two-photon fluorescence peak at 790 nm and a stable lifetime of 2.2 ns under the excitation of 1040 nm femtosecond laser.The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system.Abundant vessels as small as 3.17μm were still observed with a nice signal-background ratio at the depth of 750μm.Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.     

Quantum-sized gold clusters as efficient two-photon absorbers

The two-photon absorption properties of Au25 cluster has been investigated with the aid of two-photon excited fluorescence in the communication wavelength region with a cross-section of 2700 GM at 1290 nm. Additional visible fluorescence has been discovered for small gold clusters which is two-photon allowed (after excitation at 800 nm), and the absolute cross-section has been determined for gold clusters with number of gold atoms varying from 25 to all the way up to 2406 using one and two-photon excited time-resolved fluorescence upconversion measurements. Record high TPA cross-sections have been measured for quantum sized clusters making them suitable for two-photon imaging as well as other applications such as optical power limiting and lithography.     

Ultrafast excited state dynamics of modified phthalocyanines: p-HPcZn and p-HPcCo

Two modified metallophthalocyanines (MPcs) containing sulfonic naphthoxy substituents were synthesized. The measurements of transient absorption and time-resolved photoluminescence were used to study the ultrafast response and excited state dynamics of two MPcs in dimethyl sulfoxide (DMSO) solution, which were predominantly in the monomeric form. Under excitation at 400 nm, these molecules experience vibrational relaxation to the bottom of the first excited state and then the excitation rapidly converts to the low-lying charge-transfer (CT) state and finally reaches the triplet states. Under excitation at 800 nm, they show a two-photon absorption character, and their excited state dynamics exhibit strong dependence on the probe wavelength. The main results with 400 nm pumping are similar to the results with 800 nm pumping. For p-HPcZn, weak two-photon photoluminescence was also observed with a lifetime of 52 +/- 2 ps. A four-level model was used to illustrate the excited state dynamics of p-HPcZn, while a five-level model was suggested for p-HPcCo molecule.     

Resonance hyper-Raman excitation profiles and two-photon states of a donor-acceptor substituted polyene

Resonance Raman and resonance hyper-Raman spectra and excitation profiles have been measured for a "push-pull" donor-acceptor substituted conjugated polyene bearing a julolidine donor group and a nitrophenyl acceptor group, in acetone at excitation wavelengths from 485 to 356 nm (two-photon wavelengths for the nonlinear spectra). These wavelengths span the strong visible to near-UV linear absorption spectrum, which appears to involve at least three different electronic transitions. The relative intensities of different vibrational bands vary considerably across the excitation spectrum, with the hyper-Raman spectra showing greater variation than the linear Raman. A previously derived theory of resonance hyper-Raman intensities is modified to include contributions from purely vibrational levels of the ground electronic state as intermediate states in the two-photon absorption process. These contributions are found to have only a slight effect on the hyper-Rayleigh intensities and profiles, but they significantly influence some of the hyper-Raman profiles. The absorption spectrum and the Raman, hyper-Rayleigh, and hyper-Raman excitation profiles are quantitatively simulated under the assumption that three excited electronic states contribute to the one- and two-photon absorption in this region. The transition centered near 400 nm is largely localized on the nitrophenyl group, while the transitions near 475 and 355 nm are more delocalized.