Nonlinear Fluorescence Research Articles

Wavelength-selective and high-contrast multicolour fluorescence photoswitching in a mixture of photochromic nanoparticles.

Herein, we report on wavelength-selective and high-contrast multicolour fluorescence photoswitching between white-, orange-, cyan-, and dark-colours in a mixture of two distinguishable emission-coloured photochromic nanoparticles composed of different pairs of a photoswitching unit and a fluorescence unit.

Intense Signal Modulation of Nonlinear Optical Scattering and Multiphoton Fluorescence by Ultrasound Irradiation

Tagging linear fluorescence signals by ultrasound has been proposed to achieve improved spatial resolution at known imaging depths. An alternative way to enhance imaging resolution is the use of nonlinear optical methods such as second-harmonic imaging microscopy and two-photon excitation fluorescence. Extending further along these lines, we have studied the fundamental aspects of the effect of ultrasound irradiation on two nonlinear optical effects. In contrast to the focused transducers that are traditionally used in the study of ultrasound -mediated fluorescence that cause a small fluorescence modulation, we have made use of a simple, flat disc-shaped transducer to generate standing waves in the sample cell. Upon ultrasound irradiation, a prompt increase of the hyper-Rayleigh scattering signal with 35% was observed. The signal enhancement shows a clear correlation with the standing wave pattern, as characterized by laser beam deflection scanning measurements. The viability of different possible mechani...

Nonlinear Absorption and Fluorescence in ZnO and ZnO–Au Nanostructures

This paper investigates whether coupling gold nanoparticles to nanostructured zinc oxide enhances fluorescence emission, to produce efficient nanoscale UV emitters that radiate through nonlinear optical absorption processes. The z‐scan technique is used to measure nonlinear absorption simultaneously with measurements of nonlinear‐absorption‐induced fluorescence in zinc oxide nanostructures and hybrid zinc oxide–gold nanostructures. This allows for better determination of the nonlinear excitation and energy dissipation pathways. The measurements show that the nonlinear absorption of the hybrid structures is enhanced by up to two orders of magnitude compared to the pure zinc oxide nanostructures. However, it is found that the increase in nonlinear absorption does not correspond to an equivalent increase in fluorescence due to the competition between two‐photon absorption and excited state absorption.

Plasmonic enhancement and polarization dependence of nonlinear upconversion emissions from single gold nanorod@SiO2@CaF2:Yb3+,Er3+ hybrid core\u2013shell\u2013satellite nanostructures

Lanthanide-doped upconversion nanocrystals (UCNCs) have recently become an attractive nonlinear fluorescence material for use in bioimaging because of their tunable spectral characteristics and exceptional photostability. Plasmonic materials are often introduced into the vicinity of UCNCs to increase their emission intensity by means of enlarging the absorption cross-section and accelerating the radiative decay rate. Moreover, plasmonic nanostructures (e.g., gold nanorods, GNRs) can also influence the polarization state of the UC fluorescence—an effect that is of fundamental importance for fluorescence polarization-based imaging methods yet has not been discussed previously. To study this effect, we synthesized GNR@SiO2@CaF2:Yb3+,Er3+ hybrid core–shell–satellite nanostructures with precise control over the thickness of the SiO2 shell. We evaluated the shell thickness-dependent plasmonic enhancement of the emission intensity in ensemble and studied the plasmonic modulation of the emission polarization at the single-particle level. The hybrid plasmonic UC nanostructures with an optimal shell thickness exhibit an improved bioimaging performance compared with bare UCNCs, and we observed a polarized nature of the light at both UC emission bands, which stems from the relationship between the excitation polarization and GNR orientation. We used electrodynamic simulations combined with Förster resonance energy transfer theory to fully explain the observed effect. Our results provide extensive insights into how the coherent interaction between the emission dipoles of UCNCs and the plasmonic dipoles of the GNR determines the emission polarization state in various situations and thus open the way to the accurate control of the UC emission anisotropy for a wide range of bioimaging and biosensing applications.

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging.

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

A new method of assessing the surgical margin in rectal carcinoma—using nonlinear optical microscopy

Nowadays, surgical resection is still the most effective treatment strategy for rectal carcinoma and one of the most important factors affecting whether the operation is successful or not is the surgical margin determination, especially in the distal rectal carcinoma which should take the sphincter-preserving issue into consideration. However, until recently no reliable evaluation method has been developed for this purpose. There are some shortcomings in intraoperative negative surgical margin assessment such as either lack of enough detailed information of biological tissues or the fact that it is time-consuming. Multiphoton microscopy (MPM)—nonlinear optical microscopy, which is based on the nonlinear optical process two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), has the ability to label freely and noninvasively visualize tissue micro-architecture at the sub-cellular level. The advantage of providing high contrast and high resolution biomedical image in real time makes MPM have a wide range of applications in life sciences. In this study, we introduced MPM to identify the boundary between normal and abnormal rectal tissues. MPM images clearly exhibit biological tissue microstructure and its morphological changes in the regions of our interest, which enable it to determine the surgical margin in rectal carcinoma. It can be foreseen that once MPM imaging system is used in clinical examination, it will greatly improve the accuracy of surgical resection.

Differentiation of Positional Isomers of Propyl Alcohols Using Filament-Induced Fluorescence**Supported by the National Natural Science Foundation of China under Grant Nos 61427816 and 61235003, the Research Fund for the Doctoral Program of Higher Education of China under Grant No 20130061110047, and the Open Fund of the State Key Laboratory of High Field Laser Physics.

We experimentally demonstrate the recognition of positional isomers of propyl alcohol vapor through nonlinear fluorescence induced by high-intensity femtosecond laser filaments in air. By measuring characteristic fluorescence of n-propyl and isopropyl alcohol vapors produced by femtosecond filament excitation, it is found that they show identical spectra, that is, those from molecular bands of CH, C2, NH, OH and CN, while the relative intensities are different. By comparing the ratios of the CH and C2 signals, the two propyl alcohol isomers are differentiated. The different signal intensities are ascribed to different ionization potentials of the two isomer molecules, leading to different production efficiencies of fluorescing fragments.

Label-free identification of the hippocampus and surrounding structures by multiphoton microscopy

The hippocampus is one of the essential neuroanatomical substrates and plays an important role in different neurological illnesses. In this work, multiphoton microscopy (MPM) based on intrinsic nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was applied to label-freely detect the entire hippocampus and surrounding structures in high-magnification imaging, as well as acquire large-scale MPM images at subcellular resolution. It was found that MPM has the capability to identify cornu ammonis, dentate gyrus (DG), alveus, and fimbria of the entire hippocampus, choroid plexus in lateral ventricles, and white matter tracts. MPM also can be used to quantitatively describe the differences of the cellular nucleus in the cornu ammonis and the DG, further identify the morphological features of hippocampal subfields. In addition, the surrounding structures of the hippocampus including the lateral ventricles and white matter serve as useful information to determine the position of the hippocampus. Our results suggest that with the development of the clinical feasibility of two-photon fiberscopes and microendoscope probes, MPM has the potential for in vivo intraoperative identification and monitoring of hippocampus-related lesions without the need for tissue labelling or fluorescent markers.

High-resolution, three-dimensional imaging of pigments and support in paper and textiles

Historic works on paper, illuminated (painted) or unpainted manuscripts, and textiles are fragile and nearly impossible to sample. Non-invasive techniques such as visible microscopy, X-ray fluorescence, Raman, and reflectance spectroscopy are commonly used to determine the artist material present. Recently, nonlinear optical ultrafast pump-probe microscopy was shown to provide non-invasive, high-resolution mapping of pigments in historic paintings to investigate paint stratigraphy. In this paper, we combine our pump-probe contrast with nonlinear fluorescence and second-harmonic generation contrasts exhibited by fibrous supports composed of natural bio-materials (cellulose, collagen, or lignin). Hence, our multi-modal nonlinear microscope is able to simultaneously investigate pigments in conjunction with their support in three dimensions with micrometer-scale spatial resolution. Here we examine the utility of nonlinear pump-probe microscopy by studying a series of mock-up samples, including indigo-dyed cotton cloth, ultramarine blue painted on various types of paper, and papers painted with mixtures and layers of the two pigments. In each case we find that we can combine pump-probe pigment contrast with nonlinear optical fiber contrast to obtain spatial information that is otherwise unavailable to the conservator.

DesR349P Mutation Results in Ultrastructural Disruptions and Compromise of Skeletal Muscle Biomechanics Already at Preclinical Stages in Young Mice before the Onset of Protein Aggregation

Mutations in the extra-sarcomeric protein desmin give rise to protein aggregate myopathies that interfere with cellular function. The R350P mutation is the most common human desminopathy. A murine DesR349P model of human DesR350P desminopathy was previously engineered. In this study, we followed the hypothesis of whether muscle cytoarchitecture and biomechanical properties are already altered in preclinical stages of R349P desminopathy. We applied non-linear second harmonic generation (SHG) and 2-photon fluorescence morphometry analysis to single fibres of fast- and slow-twitch muscle to analyze sarcomeric architecture and nuclear morphology. We found a vast disruption of the lateral sarcomere lattice (indicated by large ‘vernier’ densities) and myofibrillar angular deviations (indicated by cosine angle sums) in single DesR349P fibres. Homozygous fibres were more severely affected than heterozygous. The former showed a marked nuclear pathology with spheric nuclei, increased nuclear number and density. For biomechanics, we assessed active and passive properties. Axial stiffness of DesR349P muscle was much increased, both in fibre and myofibrillar homozygous bundles. Lateral stiffness of the membrane complex in myoblasts was increased in cells heterozygous for DesR349P while homozygous were similar to wt. Caffeine-induced force was compromised in heterozygous fibre bundles but increased in homozygous bundles. This was explained by a marked shift of myofibrillar Ca2+ sensitivity: heterozygous bundles being less sensitive but homozygous more sensitive to Ca2+. This points towards a specific compensatory mechanism in homozygous mutation to cope with otherwise compromised force production already at preclinical stages of desminopathy. Our findings provide a very first complete picture to explain compromised force in heterozygous DesR350P patients: increased muscle stiffness, higher susceptibility towards stretch-induced injury, disrupted myofibrillar alignment and compromised active force production.

Improvement of precision and accuracy of temperature imaging in sooting flames using two-line atomic fluorescence (TLAF)

Severe interference to the fluorescence signal from seeded indium has previously limited the application of current non-linear excitation regime two-line atomic fluorescence (NTLAF) to low sooting flames. The present paper reports new details of these interferences, from spatially- and temporally-resolved spectral measurements in a laminar, non-premixed sooting C2H4 flame burning in air. Three types of interference to the indium LIF signal have been identified: laser-induced fluorescence (LIF) from polycyclic aromatic hydrocarbons (PAH), elastic laser scattering (ELS) and laser-induced incandescence (LII) from soot particles. This knowledge was used to propose new methods to suppress interferences and select customized narrow-band filters for the signal collection, with a narrow bandwidth of ∼1.2 nm (full-width at half-maximum, FWHM) and a high peak transmission of ∼95%. The efficacy of the narrow-band filters for interference suppression in planar NTLAF measurements was assessed by comparing these measurements with those obtained with a set of conventional band-pass filters (FWHM = 10 nm), in both a soot-free flame and a sooting flame. The application of the narrow-band filters was found to increase both the accuracy and the precision of the temperature measurement, so that measurement accuracy that is due to the presence of interferences has been improved from 198 K to 10 K at a location where the soot volume fraction (fv) is 1.2 ppm. An average reduction of 40% in the standard deviation of measured temperature in single-shot measurements has also been achieved with the use of high transmission filters. With the use of narrow-band filters, NTLAF can be reliably applied in laboratory target sooting flames with peak soot volume fraction of several ppm.

Metallo complexes of meso-phenothiazinylporphyrins: Synthesis, linear and nonlinear optical properties

Herein we described the synthesis and the structural characterization of series of metallo-meso-(phenothiazin-3yl)porphyrins containing Ni(II), Cu(II), Zn(II) or Pd(II) coordinated in the central cavity of the macrocycle and an increasing number of peripheral phenothiazine units (from one up to four). Their photophysical properties including UV–vis linear (one photon) and nonlinear (two photons) absorption and fluorescence emissions were investigated in solution or solid state. Fluorescence lifetimes of the new metallo-phenothiazinylporphyrins in the solid state were measured using a time correlated single-photon counting method. Nonlinear optical properties were observed using a Titanium: Sapphire femtosecond laser. Two-photon excited fluorescence emissions of red light for selected Zn(II)-, Cu(II)- and Pd(II)-meso-phenothiazinylporphyrins, as well as second harmonic generation in the case of some Cu(II)- and Pd(II)-meso-phenothiazinylporphyrins are reported.

Broadband transient absorption spectroscopy with 1- and 2-photon excitations: Relaxation paths and cross sections of a triphenylamine dye in solution.

1-photon (382 nm) and 2-photon (752 nm) excitations to the S1 state are applied to record and compare transient absorption spectra of a push-pull triphenylamine (TrP) dye in solution. After 1-photon excitation, ultrafast vibrational and structural molecular relaxations are detected on a 0.1 ps time scale in nonpolar hexane, while in polar acetonitrile, the spectral evolution is dominated by dipolar solvation. Upon 2-photon excitation, transient spectra in hexane reveal an unexpected growth of stimulated emission (SE) and excited-state absorption (ESA) bands. The behavior is explained by strong population transfer S1 → Sn due to resonant absorption of a third pump photon. Subsequent Sn → S1 internal conversion (with τ1 = 1 ps) prepares a very hot S1 state which cools down with τ2 = 13 ps. The pump pulse energy dependence proves the 2-photon origin of the bleach signal. At the same time, SE and ESA are strongly affected by higher-order pump absorptions that should be taken into account in nonlinear fluorescence applications. The 2-photon excitation cross sections σ(2) = 32 ⋅ 10(-50) cm(4) s at 752 nm are evaluated from the bleach signal.

Nonlinear optical property and fluorescence quenching behavior of PVP capped ZnS nanoparticles co-doped with Mn2+ and Sm3+

ZnS nanoparticles co-doped with different percentages of Mn2+ and Sm3+ were synthesized by the chemical co-precipitation method using polyvinylpyrrolidone (PVP) as capping agent. Cubic zinc blende phase of the samples was confirmed from X-ray diffraction. The strong interaction between PVP and ZnS nanoparticles was studied from Fourier Transform Infrared (FTIR) spectrum. The band gap values of ZnS and co-doped ZnS nanoparticles were calculated from UV‐Visible spectra. The photoluminescence spectra of pure ZnS nanoparticles showed an emission at 436nm and when doped with Mn2+ and Sm3+ an extra peak with high intensity was observed at 596nm. On increasing the mole percentage of dopants the intensity of the extra peak showed an enhancement until a certain concentration and then a reduction with further increase in concentration. The binding parameters were determined by Stern‐Volmer relation. The nonlinear absorption coefficients of the doped and undoped samples were calculated using Z-scan technique.

Layer-resolved colorectal tissues using nonlinear microscopy.

In this work, multiphoton microscopy (MPM), based on the nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to evaluate the feasibility of using MPM to distinguish layers of the bowel wall. It was found that MPM has the ability to identify the four-layer microstructures of colorectal tissues including mucosa, submucosa, muscularis propria, and serosa as there are many intrinsic signal sources in each layer. Our results also showed the capability of using the quantitative analyses of MPM images for quantifying some feature parameters including the nuclear area, nuclear-to-cytoplasmic ratio, and optical redox ratio. This work demonstrates that MPM has the potential in noninvasively monitoring the development and progression of colorectal diseases and then guiding effective treatment.

Improved resolution in fluorescence microscopy with the FRET pairs by time gating.

We investigate the temporal information of nonlinear donor fluorescence in the FRET frustration microscopy. The arrival time of the donor fluorescence varies with the distribution of the excitation laser. The differences of the arrival times between different positions in the excitation spot can be further enhanced in the case of adding a depletion beam upon the FRET probes. The spatial information is encoded in the temporal dynamics of the fluorescent photons from donor molecules and time-gating in detection can be used to increase the spatial resolution.

Nonlinear fluorescence imaging by photoinduced charge separation

Manipulation of the optical property of fluorescent probes has been a powerful strategy to establish super-resolution microscopy. We describe a new strategy to realize a probe with a nonlinear fluorescence response by using photoinduced charge separation. In this scheme, the first photon is used for the generation of the charge-separated state and the second photon is for fluorescence excitation. This stepwise two-photon absorption was confirmed by detection of a second-order nonlinear fluorescence response. Transient absorption spectra studies and simulation indicate that fluorescence is emitted through the photophysical pathways we proposed. Fluorescence imaging of biological cells showed marked improvements in image contrast and resolution, demonstrating the usefulness of the fluorescent probe in laser scanning confocal microscopy.

Colloidal nanoplatelets: Energy transfer is speeded up in 2D.

When efficient energy transfer under high carrier densities is required, two-dimensional nanoplatelets are the material of choice, combining an exceptional suppression of nonlinear fluorescence quenching with ultrafast transfer capabilities.

Growth, linear–non-linear optical, fluorescence, thermal and electrical studies of glycine-doped bis-thiourea cadmium formate crystal for electro-optic device applications

In the present investigation, glycine has been doped in bis-thiourea cadmium formate (BTCF) and single crystal was grown by slow solution evaporation technique. The incorporation of glycine in BTCF crystal was confirmed by Fourier transform infrared (FTIR) analysis. The UV-visible transmittance spectrum of pure and doped BTCF crystals was recorded to comparatively analyse the optical transparency, optical bandgap and other optical constants. The third-order non-linear behaviour of grown crystals has been investigated at 632.8 nm using Z-scan technique, and vital third-order non-linear optical (NLO) constants were calculated. The second harmonic generation (SHG) efficiency of doped BTCF crystal is found to be 2.15 times that of KDP material. The fluorescence studies of doped BTCF crystal confirmed the red colour emission in the visible region. The thermal stability of grown crystals was determined by means of thermogravimetric analysis (TGA). The dielectric measurement studies were performed at room temperature. The surface morphology of doped BTCF crystal was analysed by scanning electron microscopy (SEM) technique.