Three-photon Excitation Fluorescence Research Articles

Excellent Multiphoton Excitation Fluorescence with Large Multiphoton Absorption Cross Sections of Arginine-Modified Gold Nanoclusters for Bioimaging.

Fluorescent gold nanoclusters (Au NCs) with excellent one-photon and multiphoton properties have been demonstrated as promising candidates in many application fields. However, small multiphoton absorption (MPA) cross sections and weak multiphoton excitation (MPE) fluorescence impede their practical applications under near-infrared (NIR) excitation for biological imaging. Here, we report the regulated one-photon and multiphoton properties and mechanisms of arginine-stabilized 6-aza-2-thiothymine Au NCs (Arg/ATT-Au NCs) and the applications for MPE fluorescence imaging. The introduction of arginine into the capping layer of ATT-Au NCs significantly modifies the electronic structure, the absorption cross sections, and the relaxation dynamics of the lowest excited state, drastically reducing the nonradiative relaxation, suppressing the blinking, and greatly enhancing the fluorescence. Besides the improved one-photon properties, Arg/ATT-Au NCs demonstrate remarkable MPE fluorescence with a large MPA cross section. The two-photon (λex = 850 nm), three-photon (λex = 1400 nm), and four-photon (λex = 1700 nm) absorption cross sections have been determined to be 6.1 × 10-47 cm4 s1 photon-1, 1.5 × 10-78 cm6 s2 photon-2, and 5.5 × 10-108 cm8 s3 photon-3, respectively, much higher than those of conventional organic compounds and previously reported Au NCs. Moreover, Arg/ATT-Au NCs have been successfully applied in two-photon and three-photon excitation fluorescence imaging of living cells with NIR excitation. The manifold advantages of small size, high quantum yield, suppressed blinking, good photostability and cytocompatibility, large MPA cross sections, and excellent MPE fluorescence imaging performances make fluorescent Arg/ATT-Au NCs a great candidate of imaging probes with vis-NIR excitation.

Simultaneous label-free autofluorescence-multiharmonic microscopy driven by femtosecond sources based on self-phase modulation enabled spectral selection

Nonlinear optical microscopy technique has unique advantages in tissue imaging, such as enhanced contrast, high resolution, and label-free deep optical sectioning capabilities. Nonlinear optical microscopy also has multiple imaging modalities, corresponding to various components in biological tissues. Unfortunately, its wide applications are hindered due to the lack of broadly tunable femtosecond sources designed for driving multimodalities simultaneously. To solve this challenge, we propose a new wavelength conversion approach—self-phase modulation (SPM) enabled spectral selection, dubbed as SESS. The SESS employs SPM to broaden the input spectrum in a short fiber, and the broadened spectrum features well-isolated spectral lobes. Using the suitable optical filters to select the outermost spectral lobes produces nearly transform-limited femtosecond pulses. In this work, we demonstrate a fiber-optic SESS source for multimodal nonlinear optical microscopy. Based on a 43-MHz Yb-fiber laser, this SESS source can emit 990-nm, 84-fs pulses with >5-nJ energy and ~84-fs pulse duration; it can also produce 1110-nm, 48-fs pulses with 15-nJ energy. The 990-nm pulses are used to drive two-photon excitation fluorescence of many important fluorophores and second-harmonic generation microscopy, which, combined with image splicing technology, enables us to obtain a large field of view image of the gastric tissue. We also employ the 1110-nm pulses to drive simultaneous label-free autofluorescence-multiharmonic microscopy for multimodal imaging of gastric tissue. Two-photon excitation fluorescence, three-photon excitation fluorescence, second-harmonic generation and third-harmonic generation signals of gastric tissue are simultaneously excited efficiently. Such a multimodal nonlinear optical microscopy driven by SESS sources becomes a powerful tool in biomedical imaging.

Compact fiber-based multi-photon endoscope working at 1700 nm.

We present the design, implementation and performance analysis of a compact multi-photon endoscope based on a piezo electric scanning tube. A miniature objective lens with a long working distance and a high numerical aperture (≈ 0.5) is designed to provide a diffraction limited spot size. Furthermore, a 1700 nm wavelength femtosecond fiber laser is used as an excitation source to overcome the scattering of biological tissues and reduce water absorption. Therefore, the novel optical system along with the unique wavelength allows us to increase the imaging depth. We demonstrate that the endoscope is capable of performing third and second harmonic generation (THG/SHG) and three-photon excitation fluorescence (3PEF) imaging over a large field of view (> 400 μm) with high lateral resolution (2.2 μm). The compact and lightweight probe design makes it suitable for minimally-invasive in-vivo imaging as a potential alternative to surgical biopsies.

Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

Temporal focusing microscopy using three-photon excitation fluorescence with a 92-fs Yb-fiber chirped pulse amplifier.

Temporal focusing (TF) microscopy is a wide-field two-photon excitation fluorescence (2PEF) microscopy technique, the optical sectioning capability of which is lower than that of point-scanning 2PEF microscopy. Here we demonstrate TF microscopy using three-photon excitation fluorescence (3PEF), which enhances the optical sectioning capability. As an excitation light source for the 3PEF, we developed an Yb-fiber chirped pulse amplifier, which produces 92-fs 9.0-μJ 1060-nm pulses at a repetition rate of 200 kHz. The optical sectioning capability was improved by a factor of 1.3 compared with that of 2PEF-TF microscopy. We also demonstrate dual-color imaging with both 2PEF and 3PEF.

Hierarchy of Periodic Patterns in the Twist-bend Nematic Phase of Mesogenic Dimers

Several attractive properties of hydrocarbon-linked mesogenic dimers have been discovered, a major feature of these has been the ability of dimers under confinement to form periodic patterns. At least six different types of stripe-like periodic patterns can be observed in these materials under various experimental conditions. Our most recent finding is the discovery of micrometre-scale periodic patterns at the N-Ntb phase transition interface, and sub-micrometre scale patterns in the Ntb phase. Layer-like sub-micrometre patterns were observed using three-photon excitation fluorescence polarizing microscopy, and these are being reported here.

Application of three-photon excitation FCS to the study of protein oligomerization.

Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three-photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in the presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three-photon excitation process was verified from the slope of the log–log plot of intensity against laser power.

Three-photon excitation source at 1250 nm generated in a dual zero dispersion wavelength nonlinear fiber.

We demonstrate 1250 nm pulses generated in dual-zero dispersion photonic crystal fiber capable of three-photon excitation fluorescence microscopy. The total power conversion efficiency from the 28 fs seed pulse centered at 1075 nm to pulses at 1250 nm, including coupling losses from the nonlinear fiber, is 35%, with up to 67% power conversion efficiency of the fiber coupled light. Frequency-resolved optical gating measurements characterize 1250 nm pulses at 0.6 nJ and 2 nJ, illustrating the change in nonlinear spectral phase accumulation with pulse energy even for nonlinear fiber lengths < 50 mm. The 0.6 nJ pulse has a 26 fs duration and is the shortest nonlinear fiber derived 1250 nm pulse yet reported (to the best of our knowledge). The short pulse durations and energies make these pulses a viable route to producing light at 1250 nm for multiphoton microscopy, which we we demonstrate here, via a three-photon excitation fluorescence microscope.

Label-free multi-photon imaging using a compact femtosecond fiber laser mode-locked by carbon nanotube saturable absorber

We demonstrate label-free multi-photon imaging of biological samples using a compact Er(3+)-doped femtosecond fiber laser mode-locked by a single-walled carbon nanotube (CNT). These compact and low cost lasers have been developed by various groups but they have not been exploited for multiphoton microscopy. Here, it is shown that various multiphoton imaging modalities (e.g. second harmonic generation (SHG), third harmonic generation (THG), two-photon excitation fluorescence (TPEF), and three-photon excitation fluorescence (3PEF)) can be effectively performed on various biological samples using a compact handheld CNT mode-locked femtosecond fiber laser operating in the telecommunication window near 1560nm. We also show for the first time that chlorophyll fluorescence in plant leaves and diatoms can be observed using 1560nm laser excitation via three-photon absorption.

Optical generation, templating, and polymerization of three-dimensional arrays of liquid-crystal defects decorated by plasmonic nanoparticles

Defects in liquid crystals are used to model topological entities ranging from Skyrmions in high-energy physics to early-universe cosmic strings, as well as find practical applications in self-assembly of diffraction gratings and in scaffolding of plasmonic nanoparticles, but they are hard to control and organize into three-dimensional lattices. We laterally scan focused laser beams to produce periodic arrays of twist-stabilized defects forming either linear (fingers) or axially symmetric (torons) configurations in partially polymerizable liquid crystal films. Polymerization allows for stabilization of these structures and the formation of three-dimensional arrays of defects by stacking of the thin cholesteric films on top of each other. In the process of fabrication of such arrays, we polymerize the liquid crystal film with an array of torons or fingers and then sequentially produce and photopolymerize new liquid crystal layers on top of it, thus obtaining a three-dimensional structure of twist-stabilized defects in a layer-by-layer fashion. Templating by the polymerized layer spontaneously yields ordered organization of fingers and torons in the new cholesteric layer, thus enabling a three-dimensional ordered structure of defects. Nondestructive three-dimensional imaging of director fields by use of three-photon excitation fluorescence polarizing microscopy reveals the nature of topological singularities and physical underpinnings behind the observed templating effect. Three-dimensional patterning of defects templates the self-assembly of plasmonic nanoparticles into individual singularities and their arrays, laying the groundwork for potential applications in nanophotonics, plasmonics, metamaterial fabrication, and nanoscale energy conversion.

Three-Photon Excitation Fluorescence Lifetime Imaging Using Time-Correlated Single Photon Counting Method

Paper has been removed due to double publication.

Nonlinear imaging microscopy techniques as diagnostic tools for art conservation studies

We present results of the implementation of three-photon excitation fluorescence (3PEF) and third harmonic generation imaging measurements for the precise and nondestructive detection of natural and synthetic varnish layers, which are used for the surface protection of painted artifacts. For this purpose, we employ as an excitation source a compact femtosecond laser operating at 1028 nm. Two-dimensional images of the multilayer structures from different samples are depicted. The third harmonic signals show the interface between the different materials, when its refractive index mismatch is high enough. The depths of different layers of varnishes, presenting similar refractive index, are distinguishable with an axial resolution of approximately 1 microm by employing 3PEF measurements.

Rapid modeling of two-dimensional multi-photon excitation microscopic imaging through turbid medium

A rapid model to study two-dimensional (2-D) multi-photon excitation (MPE) fluorescence microscopic imaging through turbid medium has been presented. This model combines traditional Monte Carlo method with 2-D point spread function (PSF). Planar fluorescent object was used to calculate the 2-D PSF, which avoided the simulation of point scanning in the direct Monte Carlo method. In the meantime, this rapid model has generalized program logic and need only one-step Monte Carlo calculation for the simulations of single, two- and three-photon excitation fluorescence microscopic imaging. Experimental results showed that the rapid model using convolution operation was much faster than the direct Monte Carlo method. Similar computation efficiency was achieved for different order of MPE. This model provides high potential in the simulation of MPE fluorescence imaging.

Diamond deposition in low-pressure acetylene flames: in situ temperature and species concentration measurements by laser diagnostics and molecular beam mass spectrometry

Diamond deposition in a flat, premixed acetylene–oxygen–argon flame at 50 mbar was investigated to characterize the reactive gas phase in the vicinity of the substrate. For this, flames with and without a substrate present were analyzed as a function of stoichiometry; also, the distance between the substrate and the burner was varied. Optimal conditions for the deposition of diamond films were found for oxygen–acetylene ratios of 1.3 and 1.4 and at distances between substrate and burner of 8, 9, and 10 mm. The flame structure in this region was investigated. In particular, gas temperature and OH radical concentrations were measured by laser-induced fluorescence (LIF). Furthermore, hydrogen atoms were monitored using three-photon excitation and subsequent fluorescence detection. Molecular beam mass spectrometry was employed to obtain an overview of stable species and hydrocarbon intermediates. The results provide a substantial experimental basis for comparison with theoretical models and are consistent with earlier observations, which stress the importance of H and CH 3 for the diamond deposition process. In addition, the observations indicate the participation of hydrocarbon species with more than 2 carbon atoms, e.g., C 3H 3, C 4H 3, and C x H 2 with x = 4, 6, or 8, in the gas-phase reactions controlling the deposition of diamond; an active role for these species in diamond chemical vapor deposition (CVD) has not been discussed before. As a first interpretation, diamond formation seems to be controlled by a counterbalance between OH and hydrocarbon intermediates at a position in the flame where sufficient H-atoms and CH 3 radicals are present to support diamond film growth.

Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser

We demonstrate that three-photon excitation images of both fixed and living biological specimens can be readily obtained using an all-solid-state Nd:YLF laser excitation source. Optically sectioned images of fixed Caenorhabditis elegans embryos stained with DAPI and embryos triple-labeled with DAPI, fluorescein and Texas Red are presented. Time series images of a living LLC-PK cell stained with Hoechst 33342 during the progression from metaphase to telophase are also presented. The mode of excitation was inferred from the power-law of anthracene and Hoechst 33342 fluorescence versus incident laser power and an axial resolution comparison of anthracene fluorescence with two-photon excited Calcium Crimson fluorescence. Multiphoton excitation imaging is an attractive method for optically sectioning live specimens because of the lower levels of phototoxicity produced compared to other optical sectioning techniques. The combination of two- and three-photon excitation extends the capabilities of a multiple- photon imaging system since a single wavelength can provide localized excitation of a wide variety of fluorophores whose collective emission spectra can span the entire visible spectrum.

Three-photon excitation in fluorescence microscopy

We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.