Two-photon Excited Fluorescence Research Articles

Ligand-core NLO-phores: a combined experimental and theoretical approach to the two-photon absorption and two-photon excited emission properties of small-ligated silver nanoclusters.

We report a combined experimental and theoretical study of the two-photon absorption and excited emission properties of monodisperse ligand stabilized Ag11, Ag15 and Ag31 nanoclusters in aqueous solutions. The nanoclusters were synthesized using a cyclic reduction under oxidative conditions and separated by vertical gel electrophoresis. The two-photon absorption cross-sections of these protected noble metal nanoclusters measured within the biologically attractive 750-900 nm window are several orders of magnitude larger than that reported for commercially available standard organic dyes. The two-photon excited fluorescence spectra are also presented for excitation wavelengths within the same excitation spectral window. They exhibit size-tunability. Because the fundamental photophysical mechanisms underlying these multiphoton processes in ligand protected clusters with only a few metal atoms are not fully understood yet, a theoretical model is proposed to identify the key driving elements. Elements that regulate the dipole moments and the nonlinear optical properties are the nanocluster size, its structure and the charge distribution on both the metal core and the bound ligands. We coined this new class of NLO materials as "Ligand-Core" NLO-phores.

Imaging the structure and organization of mouse cerebellum and brain stem with second harmonic generation microscopy

To visualize the structure and organization of the brain is a fundamental requirement in the research of neuroscience. Here, combining with two-photon excitation fluorescence microscopy and transgenetic mouse GAD67, we demonstrate a custom-built second harmonic generation (SHG) microscope to discriminate brain layers and sub regions in the cerebellum and brain stem slices with cellular resolution. In particular, the cell densities of neurons in different brain layers are extracted due to the cell soma appearing as dark shadow on an SHG image. Further, the axon initial segments of the Purkinje cell are easily recognized without labeling, which would be useful for guiding micropipettes for electrophysiology.

Small water-soluble pyrimidine hexafluorophosphate derivatives with high two-photon absorption activities in the near-IR region and their biological applications

Two imidazole–pyrimidine hexafluorophosphate derivatives (6P and 10P) are synthesized. The 2PA cross-section values (σ) were obtained by Z-scan and two-photon excited fluorescence methods. 6P and 10P can be used to quantify and image organelles in living cells and tissues.

CW Optical Limiting Study in Disperse Yellow Dye-doped PMMA-MA Polymer Films

<div><p><em>Molecules with high two-photon absorption (TPA) cross section and the molecules which have reverse saturation absorption (RSA) cross section are of great interest today because of their application in three-dimensional optical data storage and photonic switches. During last forty years, several techniques have been developed to determine the two-photon absorption cross-sections and reverse saturation absorption cross-sections of the materials. This includes direct methods, such as nonlinear transmission and Z-scan methods and indirect methods, such as two-photon excited fluorescence and two-photon pump-probe transient absorption spectroscopy. During recent years, the organic dye-doped polymer films getting more attention due to their advantages to fabricate photonics devices. In this paper, we have studied the nonlinear optical properties like nonlinear absorption and nonlinear refraction of an azo dye Disperse yellow-7 (DY-7) doped in Polymethyl methacrylate-methacrylic acid (PMMA-MA) polymer matrix using open aperture and closed aperture Z-scan experimental methods using continuous wave (CW) laser. The optical limiting properties of these films are also studied in Type 1 and Type 2 configurations at different input power using continuous wave (CW) laser beams of 532 nm wavelength.</em></p></div>

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...

Generalized vector wave theory for ultrahigh resolution confocal optical microscopy.

Polarization modulation of a tightly focused beam in a confocal imaging scheme is considered for incident and collected light fields. Rigorous vector wave theory of a confocal optical microscopy is developed, which provides clear physical pictures without the requirement for fragmentary calculations. Multiple spatial modulations on polarization, phase, or amplitude of the illuminating and the detected beams can be mathematically described by a uniform expression. Linear and nonlinear excitation schemes are derived with tailored excitation and detection fields within this generalized theory, whose results show that the ultimate resolution achieved with the linear excitation can reach one-fifth of the excitation wavelength (or λ/5), while the nonlinear excitation scheme gives rise to a resolution better than λ/12 for two-photon fluorescence excitation and λ/20 for three-photon fluorescence excitation. Hence the resolution of optical microscopy with a near-infrared excitation can routinely reach sub-60 nm. In addition, simulations for confocal laser scanning microscopy are carried out with the linear excitation scheme and the fluorescent one, respectively.

Enhanced Emission from Single Isolated Gold Quantum Dots Investigated Using Two-Photon-Excited Fluorescence Near-Field Scanning Optical Microscopy.

New approaches in molecular nanoscopy are greatly desired for interrogation of biological, organic, and inorganic objects with sizes below the diffraction limit. Our current work investigates emergent monolayer-protected gold quantum dots (nanoclusters, NCs) composed of 25 Au atoms by utilizing two-photon-excited fluorescence (TPEF) near-field scanning optical microscopy (NSOM) at single NC concentrations. Here, we demonstrate an approach to synthesize and isolate single NCs on solid glass substrates. Subsequent investigation of the NCs using TPEF NSOM reveals that, even when they are separated by distances of several tens of nanometers, we can excite and interrogate single NCs individually. Interestingly, we observe an enhanced two-photon absorption (TPA) cross section for single Au25 NCs that can be attributed to few-atom local field effects and to local field-induced microscopic cascading, indicating their potential for use in ultrasensitive sensing, disease diagnostics, cancer cell therapy, and molecular computers. Finally, we report room-temperature aperture-based TPEF NSOM imaging of these NCs for the first time at 30 nm point resolution, which is a ∼5-fold improvement compared to the previous best result for the same technique. This report unveils the unique combination of an unusually large TPA cross section and the high photostability of Au NCs to (non-destructively) investigate stable isolated single NCs using TPEF NSOM. This is the first reported optical study of monolayer-protected single quantum clusters, opening some very promising opportunities in spectroscopy of nanosized objects, bioimaging, ultrasensitive sensing, molecular computers, and high-density data storage.

Study protocol for a prospective, non-controlled, multicentre clinical study to evaluate the diagnostic accuracy of a stepwise two-photon excited melanin fluorescence in pigmented lesions suspicious for melanoma (FLIMMA study)

IntroductionNon-invasive, nanosecond, stepwise two-photon laser excitation of skin tissue was shown to induce melanin fluorescence spectra that allow for the differentiation of melanocytic nevi from cutaneous melanoma.Methods and analysisThis prospective,...

The use of Masson's trichrome staining, second harmonic imaging and two‐photon excited fluorescence of collagen in distinguishing intestinal tuberculosis from Crohn's disease

Differentiation between Crohn's disease (CD) and intestinal tuberculosis (ITB) continues to be difficult. The present study investigated the collagen fibre characteristics of CD and ITB using Masson's trichrome staining, second harmonic generation (SHG) imaging and two-photon excited fluorescence (TPEF) imaging with the aim of distinguishing between them. The characteristics of collagen fibres in intestinal specimens from patients with CD, ITB and healthy controls were compared using Masson's trichrome staining and SHG and TPEF imaging. Masson's trichrome staining showed that the content of collagen fibre (540.92 [139.61-1681.93] vs 236.17 [72.94-1108.32], P<0.05) and fibre deposits (888.92 [315.89-3172.9] vs 498.98 [38.82-5802.31], P<0.05) were both higher in ITB than in CD. The content of collagen fibre (594.677 [139.61-1681.93] vs 107.425 [4.66-988.7], P<0.05) and fibre deposits (1118.4661 [315.89-5802.31] vs 340.575 [29.62-1188.87], P<0.05) were significantly higher in lesions with granulomata than in those without. The SHG/TPEF images demonstrated that the percentage of fibrosis in ITB was also significantly higher than in CD (P<0.05), in both surgical (13.363%±5.303% vs 8.322%±5.078%, P=0.044) and endoscopic specimens (mean rank 13.5 vs 7.5, P=0.023). The SHG/TPEF imaging described different distribution patterns of collagen between CD and ITB; in the former this was irregular in clumps while in ITB the collagen was arranged around caseating granulomata. The evaluation of fibrosis in CD and ITB by Masson's trichrome staining and SHG and TPEF imaging appears to distinguish between these two diseases.

Toward High-Efficient Red Emissive Carbon Dots: Facile Preparation, Unique Properties, and Applications as Multifunctional Theranostic Agents

The achievement of high-efficient pure red emissive carbon dots (CDs) is still a great challenge as well as one of the most critical issues that hinders widespread applications of CDs. Herein, a facile approach for the preparation of high-efficient red emissive CDs (R-CDs) is reported, and they exhibit numerous unique features including pure red emission (λmax ≈ 640 nm), respectable quantum yield (22.9%), low cytotoxicity, two-photon excited fluorescence (TPEF), and high photothermal conversion efficiency (43.9% under irradiation of 671 nm laser). Moreover, the chemical composition and photophysical properties of the R-CDs are detailed characterized and analyzed, and from which their photoluminescence mechanism is proposed. Interestingly, the R-CDs are found to particularly light up RNA-rich nucleolus both in one-photon and two-photon modes as well as show excellent counterstain compatibilities with other classical subcellular dyes. The localization of the R-CDs in nucleolus is supported by ribonuclease d...

One-step separation-free detection of carcinoembryonic antigen in whole serum: Combination of two-photon excitation fluorescence and optical trapping

Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8pg/mL (45 fM) with a wide linear range from 0.01 to 60ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method.

Four asymmetric bis-branched triphenylamine derivatives with charge transfer from one branch to the other: Two-photon emissions and bio-imaging applications

Four asymmetric bis-branched triphenylamine-cored compounds with different electron-withdrawing groups have been synthesized and their optical properties have been systematically investigated. The two-photon absorption cross-section (σ2PA) values were measured by two-photon excited fluorescence which revealed that the four compounds possessed large σ2PA values from 2011 to 4173 GM in the near-infrared region. Theoretical calculations revealed that the compounds were all provided with interesting intramolecular charge transfer generated from one branch to the other which contributed to the large σ2PA values. Cytotoxicity tests indicated that all these compounds had good biocompatibility for living cells, both one- and two-photon fluorescence microscopy studies showed the potential application of the four new compounds in cell imaging due to the large σ2PA and appropriate biocompatibility.

Safety assessment in macaques of light exposures for functional two-photon ophthalmoscopy in humans.

Two-photon ophthalmoscopy has potential for in vivo assessment of function of normal and diseased retina. However, light safety of the sub-100 fs laser typically used is a major concern and safety standards are not well established. To test the feasibility of safe in vivo two-photon excitation fluorescence (TPEF) imaging of photoreceptors in humans, we examined the effects of ultrashort pulsed light and the required light levels with a variety of clinical and high resolution imaging methods in macaques. The only measure that revealed a significant effect due to exposure to pulsed light within existing safety standards was infrared autofluorescence (IRAF) intensity. No other structural or functional alterations were detected by other imaging techniques for any of the exposures. Photoreceptors and retinal pigment epithelium appeared normal in adaptive optics images. No effect of repeated exposures on TPEF time course was detected, suggesting that visual cycle function was maintained. If IRAF reduction is hazardous, it is the only hurdle to applying two-photon retinal imaging in humans. To date, no harmful effects of IRAF reduction have been detected.

Effect of SLM pixelation on two-photon fluorescence by applying an off-centered quadratic spectral phase mask

Spatial light modulators (SLMs) have become a valuable tool to shape ultrashort laser pulses, which have found innumerous applications in different areas of ultrafast science. Despite the fact that this family of devices can be used to produce almost any arbitrary pulse shape, as long as enough bandwidth is available, there are limitations related to their discrete nature that may lead to distortions in the expected pulse shape. Here we investigate such collateral temporal distortions by quantifying the effect imposed by the discrete nature (pixelation) of liquid-crystal-based SLMs on the two-photon excited fluorescence signal of a conjugated polymer, when the central position of a quadratic spectral phase mask is scanned across the laser bandwidth. We conclude that the observed temporal distortions are related to replica pulses generated by an interplay between the additional linear phase resultant of an off-centered quadratic phase mask and pixelation of the applied phase mask.

Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity

Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O6-benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time.

A two-photon fluorescent probe for Cu2+ based on dansyl moiety and its application in bioimaging

A new and simple fluorescent probe for detection of Cu2+ has been synthesized and characterized. The probe displays high selectivity and sensitivity for Cu2+ with remarkably weakened fluorescence intensity and red shift. Moreover, this probe has been successfully applied to detect Cu2+ by one- and two-photon excited fluorescence with low detection limits (<0.2μM). Furthermore, two-photon confocal fluorescence imaging using A549 cells showed that the probe could be applied for monitoring Cu2+ ions in living cells.

Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.

The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo.

Combining Two-Photon-Activated Fluorescence Resonance Energy Transfer and Near-Infrared Photothermal Effect of Unimolecular Micelles for Enhanced Photodynamic Therapy.

Recent years have witnessed significant progress in the field of two-photon-activated photodynamic therapy (2P-PDT). However, the traditional photosensitizer (PS)-based 2P-PDT remains a critical challenge in clinics due to its low two-photon absorption (2PA) cross sections. Here, we propose that the therapeutic activity of current PSs can be enhanced through a combination of two-photon excited fluorescence resonance energy transfer (FRET) strategy and photothermal effect of near-infrared (NIR) light. A core-shell unimolecular micelle with a large two-photon-absorbing conjugated polymer core and thermoresponsive shell was constructed as a high two-photon light-harvesting material. After PSs were grafted onto the surface of a unimolecular micelle, the FRET process from the conjugated core to PSs could be readily switched "on" to kill cancer by the collapsed thermoresponsive shell due to the photothermal effect of NIR light. Such NIR-triggered FRET leads to an enhanced 2PA activity of the traditional PSs and, in turn, amplifies their cytotoxic singlet oxygen generation. Eventually, both in vitro and in vivo PDT efficiencies treated with the thermoresponsive micelles were dramatically enhanced under NIR light irradiation, as compared to pure PSs excited by traditional visible light. Such a facile and simple methodology for the enhancement of the photodynamic antitumor effect holds great promises for cancer therapy with further development.

Details of the Collagen and Elastin Architecture in the Human Limbal Conjunctiva, Tenon's Capsule and Sclera Revealed by Two-Photon Excited Fluorescence Microscopy.

To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera. The limbal conjunctiva, Tenon's capsule, and sclera of human donor corneal buttons were imaged with an inverted two-photon excited fluorescence microscope. No fixation process was necessary. The laser (Ti:sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through a 425/30-nm and a 525/45-nm emission filter, respectively. Multiple, consecutive, and overlapping (z-stack) images were acquired. Collagen signals were collected with SHG, whereas elastin signals were collected with AF. The size and density of collagen bundles varied widely depending on depth: increasing from conjunctiva to sclera. In superficial image planes, collagen bundles were <10 μm in width, in a loose, disorganized arrangement. In deeper image planes (episclera and superficial sclera), collagen bundles were thicker (near 100 μm in width) and densely packed. Comparatively, elastin fibers were thinner and sparse. The orientation of elastin fibers was independent of collagen fibers in superficial layers; but in deep sclera, elastin fibers wove through collagen interbundle gaps. At the limbus, both collagen and elastin fibers were relatively compact and were distributed perpendicular to the limbal annulus. Two-photon excited fluorescence microscopy has enabled us to understand in greater detail the collagen and elastin architecture of the human limbal conjunctiva, Tenon's capsule, and sclera.

Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy.

Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation.