Two-photon fluorescence excitation spectroscopy by pulse shaping ultrabroad-bandwidth femtosecond laser pulses (original) (raw)
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Physica Scripta, 2009
A short overview of the principles and applications of the two-colour two-photon (2C2P) excitation of fluorescence by using femtosecond pulses is given. Fluorescence is generated by the simultaneous absorption of an 800 nm photon and a 400 nm photon of overlapping laser beams of a titanium:sapphire laser. Two examples of its application are presented: firstly, it is used to monitor the enzymatic cleavage of bovine serum albumin (BSA) by elastase. The fluorescent amino acid tryptophan present in BSA is excited corresponding to an effective one-photon wavelength of 266 nm. Secondly, it is shown how one can utilize the different polarizations of the excited beams for determining the symmetry of the excited states of molecules, exemplarily shown for p-terphenyl in cyclohexane. Further applications and experiments for 2C2P are suggested for using it in UV-fluorescence microscopy and for determining the properties of the electronic states of biomolecules by using differently polarized photons.
Optical Response of Fluorescent Molecules Studied by Synthetic Femtosecond Laser Pulses
The Journal of Physical Chemistry Letters, 2012
The optical response of the fluorescent molecule IR144 in solution is probed by pairs of collinear pulses with intensity just above the linear dependence using two different pulse shaping methods. The first approach mimics a Michelson interferometer, while the second approach, known as multiple independent comb shaping (MICS), eliminates spectral interference. The comparison of interfering and noninterfering pulses reveals that linear interference between the pulses leads to the loss of experimental information at early delay times. In both cases, the delay between the pulses is controlled with attosecond resolution and the sample fluorescence and stimulated emission are monitored simultaneously. An out-of-phase behavior is observed for fluorescence and stimulated emission, with the fluorescence signal having a minimum at zero time delay. Experimental findings are modeled using a two-level system with relaxation that closely matches the phase difference between fluorescence and stimulated emission and the relative intensities of the measured effects.
Two-Color Two-Photon Excitation Using Femtosecond Laser Pulses
The Journal of Physical Chemistry B, 2008
The use of two-color two-photon (2c2p) excitation easily extends the wavelength range of Ti:sapphire lasers to the UV, widening the scope of its applications especially in biological sciences. We report observation of 2c2p excitation fluorescence of p-terphenyl (PTP), 2-methyl-5-t-butyl-p-quaterphenyl (DMQ) and tryptophan upon excitation with 400 and 800 nm wavelengths using the second harmonic and fundamental wavelength of a mode-locked Ti:sapphire femtosecond laser. This excitation is energetically equivalent to a one-photon excitation wavelength at 266 nm. The fluorescence signal is observed only when both wavelengths are spatially and temporally overlapping. Adjustment of the relative delay of the two laser pulses renders a cross correlation curve which is in good agreement with the pulse width of our laser. The fluorescence signal is linearly dependent on the intensity of each of the two colors but quadratically on the total incident illumination power of both colors. In fluorescence microscopy, the use of a combination of intense IR and low-intensity blue light as a substitute for UV light for excitation can have numerous advantages. Additionally, the effect of differently polarized excitation photons relative to each other is demonstrated. This offers information about different transition symmetries and yields deeper insight into the two-photon excitation process.
Multiphoton excitation cross‐sections of molecular fluorophores
Bioimaging, 1996
Nonlinear excitation of fluorophores through molecular absorption of two or three near-infra-red photons from the tightly focused femtosecond pulses of a mode-locked laser offers the cellular biologist an unprecedented panoply of biomolecular indicators for microscopic imaging and cellular analysis. Measurements of the two-photon excitation spectra of 25 ultraviolet and visible absorbing fluorophores from 690 to 1050 nm reveal useful cross sections for near infra-red excitation, providing an artist's palette of emission markers, chemical indicators, and native cellular absorbers for living biological preparations. Measurements of three-photon fluorophore excitation spectra now suggest relatively benign wavelengths to excite deeper UV fluorophores. The inherent optical sectioning capabilities of focused nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background. Measured nonlinear excitation spectra are described and implications to nonlinear microscopy for biological imaging are defined.
Polarized Fluorescence in Tryptophan Excited by Two-Photon Femtosecond Laser Pulses
Optics and Spectroscopy, 2018
The decay of polarized fluorescence of tryptophan in water-propylene glycol solution two-photon excited by femtosecond laser pulses was explored. Linearly and circularly polarized light in the wavelength band of 385-510 nm was used for excitation. Processing of the experimental fluorescence signals as functions of time and light polarization provided an opportunity to identify two characteristic lifetimes characterizing the fluorescence intensity decay and the rotational diffusion time τ rot. The spectral dependences of the anisotropy parameter Ω and anisotropy parameters r for linearly and circularly polarized excitation have been determined. It is shown that the fluorescence temporal dependence is well described by the sum of two exponents with amplitudes a 1 and a 2 and characteristic times τ f1 and τ f2 at all wavelengths in the studied spectral range. The experimental dependences of parameters Ω, r, τ f1 , and a 2 /a 1 are nontrivial, which indicates the complexity of the excitation relaxation processes. The obtained results can be used for interpretation of the experiments and prove of the theoretical models describing the intensity and polarization of fluorescence of tryptophan molecules in solutions and organic compounds under optical excitation.
Review of Scientific Instruments, 2015
Superfluorescence appears as an intense background in femtosecond time-resolved fluorescence noncollinear optical parametric amplification spectroscopy, which severely interferes the reliable acquisition of the time-resolved fluorescence spectra especially for an optically dilute sample. Superfluorescence originates from the optical amplification of the vacuum quantum noise, which would be inevitably concomitant with the amplified fluorescence photons during the optical parametric amplification process. Here, we report the development of a femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectrometer assisted with a 32-channel lock-in amplifier for efficient rejection of the superfluorescence background. With this spectrometer, the superfluorescence background signal can be significantly reduced to 1/300-1/100 when the seeding fluorescence is modulated. An integrated 32-bundle optical fiber is used as a linear array light receiver connected to 32 photodiodes in one-to-one mode, and the photodiodes are further coupled to a home-built 32-channel synchronous digital lock-in amplifier. As an implementation, time-resolved fluorescence spectra for rhodamine 6G dye in ethanol solution at an optically dilute concentration of 10 −5 M excited at 510 nm with an excitation intensity of 70 nJ/pulse have been successfully recorded, and the detection limit at a pump intensity of 60 µJ/pulse was determined as about 13 photons/pulse. Concentration dependent redshift starting at 30 ps after the excitation in time-resolved fluorescence spectra of this dye has also been observed, which can be attributed to the formation of the excimer at a higher concentration, while the blueshift in the earlier time within 10 ps is attributed to the solvation process.
The Journal of Chemical Physics, 2004
We report the use of spectrally resolved femtosecond two-color three-pulse photon echoes as a potentially powerful multidimensional technique for studying vibrational and electronic dynamics in complex molecules. The wavelengths of the pump and probe laser pulses are found to have a dramatic effect on the spectrum of the photon echo signal and can be chosen to select different sets of energy levels in the vibrational manifold, allowing a study of the dynamics and vibrational splitting in either the ground or the excited state. The technique is applied to studies of the dynamics of vibrational electronic states in the dye molecule Rhodamine 101 in methanol.
Spectrally Resolved Femtosecond 2-COLOUR 3-PULSE Photon Echoes for Studies of Molecular Dynamics
2004
We present a new multidimensional femtosecond spectroscopy technique based on spectrally resolved 2-colour 3-pulse photon echoes for investigating molecular dynamics in a variety of systems including proteins. In this technique the sample is illuminated by two femtosecond 'pump' pulses with wave vectors k 1 , k 2 and wavelength O pump and a femtosecond 'probe' pulse with wave vector k 3 and wavelength O probe. Nonlinear signals are generated in the phasematching directions k 4 =-k 1 + k 2 + k 3 and k 6 =-k 3 + k 1 + k 2. These signals are analysed in spectrometers equipped with CCD detectors and the spectra of the signals are recorded for various values of (i) the delay t 12 between pulses 1 and 2, (ii) the delay t 23 between pulses 2 and 3, and (iii) the wavelengths O pump , O probe. The technique has been used for studying vibrational and electronic dynamics of dye molecules, such as cresyl violet in methanol, and ultra-fast transient processes that occur during the photo-dissociation of carbonmonoxy myoglobin (MbCO) into myoglobin (Mb) and CO.
Journal of Biomedical Optics, 2011
Controlling two-photon molecular fluorescence leading to selective fluorophore excitation has been a long sought after goal in fluorescence microscopy. In this letter, we thoroughly explore selective fluorescence suppression through simultaneous two-photon absorption by two different fluorophores followed by selective one-photon stimulated emission for one particular fluorophore. We achieve this by precisely controlling the time delay between two identical ultrafast near infrared laser pulses. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).
Australasian Physics & Engineering Sciences in Medicine, 2004
We present a new multidimensional femtosecond spectroscopy technique based on spectrally resolved 2-colour 3-pulse photon echoes for investigating molecular dynamics in a variety of systems including proteins. In this technique the sample is illuminated by two femtosecond 'pump' pulses with wave vectors k 1 , k 2 and wavelength λ pump and a femtosecond 'probe' pulse with wave vector k 3 and wavelength λ probe. Nonlinear signals are generated in the phasematching directions k 4 =-k 1 + k 2 + k 3 and k 6 =-k 3 + k 1 + k 2. These signals are analysed in spectrometers equipped with CCD detectors and the spectra of the signals are recorded for various values of (i) the delay t 12 between pulses 1 and 2, (ii) the delay t 23 between pulses 2 and 3, and (iii) the wavelengths λ pump , λ probe. The technique has been used for studying vibrational and electronic dynamics of dye molecules, such as cresyl violet in methanol, and ultra-fast transient processes that occur during the photo-dissociation of carbonmonoxy myoglobin (MbCO) into myoglobin (Mb) and CO.