Two-photon Excited Fluorescence Signal Research Articles

In-vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber

A supercontinuum light source generated with a femtosecond Ti:Sapphire oscillator has been used to obtain both vibrational and two-photon excitation fluorescence (TPEF) images of a living cell simultaneously at different wavelengths. Owing to an ultrabroadband spectral profile of the supercontinuum, multiple vibrational resonances have been detected through coherent anti-Stokes Raman scattering (CARS) process. In addition to the multiplex CARS process, multiple electronic states can be excited due to the broadband electronic two-photon excitation using the supercontinuum, giving rise to a two-photon excitation fluorescence (TPEF) signal. Using a living yeast cell whose nucleus is labeled by green fluorescent protein (GFP), we have succeeded in visualizing organelles such as mitochondria, septum, and nucleus through the CARS and the TPEF processes. The supercontinuum enables us to perform unique multi-nonlinear optical imaging through two different nonlinear optical processes.

Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging

We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. An added benefit of our technique is that it leads to somewhat improved image resolution.

Two-photon autofluorescence spectroscopy and second-harmonic generation of epithelial tissue

A spectroscopy system is developed for studying the two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) of epithelial tissue in backscattering geometry. Our findings show that TPEF signals from epithelial and underlying stromal layers exhibit different spectral characteristics, providing information on the biomorphology and biochemistry of tissue. The SHG signal serves as a sensitive indicator of collagen to separate the epithelial layer from underlying stroma. The polarization dependence of the SHG signal reveals a well-ordered orientation of collagen fibers in the stromal layer. The results demonstrate the potential of depth-resolved TPEF and SHG in determining the pathology of epithelial tissue.

Two- and three-photon excited fluorescence in Y-shaped molecules

In this work, we have studied the two- and three-photon excited fluorescence on a new series of Y-shaped chromophores, using pulses at 750 and 1400 nm from an optical parametric amplifier pumped by 150 fs pulses from a Ti:sapphire chirped pulse amplified system. The measured two- and three-photon absorption cross-sections are in the order of 1000 × 10 −50 cm 4 s and 5 × 10 −78 cm 6 s 2, respectively. Besides, the two-photon excited fluorescence signal, achieved using low energy pulses (Ti:sapphire modelocked oscillator), were used in an evolutionary strategy to optimize either the laser pulse or the two-photon excited fluorescence.

Metal-ion sensing fluorophores with large two-photon absorption cross sections: aza-crown ether substituted donor-acceptor-donor distyrylbenzenes.

Chromophores based on a donor-acceptor-donor structure possessing a large two-photon absorption cross section and one or two mono-aza-15-crown-5 ether moieties, which can bind metal cations, have been synthesized. The influence of Mg(2+) binding on their one- and two-photon spectroscopic properties has been investigated. Upon binding, the two-photon action cross sections at 810 nm decrease by a factor of up to 50 at high Mg(2+) concentrations and this results in a large contrast in the two-photon excited fluorescence signal between the bound and unbound forms, for excitation in the range of 730 to 860 nm. Experimental and computational results indicate that there is a significant reduction of the electron donating strength of the aza-crown nitrogen atom(s) upon metal ion binding and that this leads to a blue shift in the position as well as a reduction in the strength of the lowest-energy two-photon absorption band. The molecules reported here can serve as models for the design of improved two-photon excitable metal-ion sensing fluorophores.

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.

Thermal-lens-induced anomalous solvent’s effect on fluorescence produced by two-photon continuous-wave laser excitation

Measurements of two-photon-excited fluorescence (TPF) of fluorescein and Rhodamine 6G in various solvents were performed with a continuous-wave (cw) laser for excitation and an acousto-optic tunable filter for spectral dispersion. Interestingly, the cw laser excitation produced an unwanted thermal-lens effect when the measurements were performed in solvents that absorb the excitation laser light (e.g., alcohols and water, because these solvents absorb the 780-nm excitation light through the overtone and combination transitions of the O-H group). The defocusing effect of the thermal lens leads to a decrease in the TPF signal. Because the strength of the thermal lens depends on the thermo-optical properties (dn/dT and thermal conductivity) of the solvent, its interference makes the effect of solvents on the TPF much different from those on one-photon-excited fluorescence. However, the thermal-lens interference will not limit the application of this cw laser excited TPF technique because, even when measurements were performed in solvents that absorb cw excitation laser light, the thermal-lens interference was observed only in solvents such as nonpolar organic solvents that have relatively better thermo-optical properties. Interference was not observed in water, which is the most widely used solvent for the TPF technique (because water has poor thermo-optical properties).

Effect of scattering on nonlinear optical scanning microscopy imaging of highly scattering media

The intensity of two-photon excited fluorescence (TPF) generated by ultrashort laser pulses was measured as a function of the depth of a focal point inside highly scattering media. The purpose was to investigate the spatial location of TPF in a scattering medium. Owing to the scattering, the intensity of the incident beam as well as the generated TPF signal was attenuated exponentially as the focal point was scanned into the medium. As the scattering strength of the medium was increased, the TPF was not confined to the focal region and had a wider distribution. These observations show that the scattering will result in the degradation of the ability of optical depth sectioning of nonlinear optical scanning microscopy.