Two-photon excited fluorescence of a conjugated polyelectrolyte and its application in cell imaging

The two-photon excited fluorescence of a conjugated polyelectrolyte (CPE), PPESO3, was studied in methanol and in water. The photophysical and amplified quenching properties of the CPE observed under two-photon excitation were comparable to the results obtained under one-photon excited conditions. Two-photon fluorescence microscopy performed with PPESO3-coated silica nanoparticles in HeLa cells provided images with significantly improved resolution compared to one-photon microscopy, demonstrating the utility of the CPE as a fluorescent probe in two-photon fluorescence cell imaging.     

Imaging theory of nonlinear Raman confocal microscopy

A novel nonlinear Raman confocal microscopy utilizing Raman induced Kerr effect spectroscopy (RIKES) is presented in this paper. The imaging theory of RIKES confocal microscopy with Gaussian beam is derived. The imaging properties of RIKES confocal microscopy and the impact of different beam waist widths of Gaussian beam on the lateral and axial resolution have been analyzed in detail. It is proved that RIKES confocal microscopy has high sensitivity and high resolution, besides capability to characterize inherent structural features, such as vibration mode, vibration orientation, and optically induced molecular reorientation etc. Therefore, nonlinear Raman confocal microscopy that is based on RIKES has potential to provide a novel characteristic imaging method comparable to the existing imaging techniques based on other nonlinear optical processes, such as two-photon fluorescence, second harmonic generation (SHG) and coherent anti-Stoke Raman scattering (CARS).     

Adaptively controlled supercontinuum pulse from a microstructure fiber for two-photon excited fluorescence microscopy

Selective fluorescence excitation of specific molecular species is demonstrated by using coherent control of two-photon excitation with supercontinuum pulses generated with a microstructure fiber. Pulse shaping prior to pulse propagation through the fiber is controlled by a self-learning optimization loop so that the highest fluorescence signal contrast between two fluorescent proteins is obtainable. The self-learning optimization loop successfully controls both the optical nonlinarity of the microstructure fiber and the two-photon excitation of the fluorescent proteins.     

Surface enhanced coherent anti-stokes Raman scattering on nanostructured gold surfaces

Coherent anti-Stokes Raman spectroscopy (CARS) is a well-known tool in multiphoton imaging and nonlinear spectroscopy. In this work we combine CARS with plasmonic surface enhancement on reproducible nanostructured surfaces. We demonstrate strong correlation between plasmon resonances and surface-enhanced CARS (SECARS) intensities on our nanostructured surfaces and show that an enhancement of ～10(5) can be obtained over standard CARS. Furthermore, we find SECARS to be >10(3) times more sensitive than surface-enhanced Raman Spectroscopy (SERS). We also demonstrate SECARS imaging of molecular monolayers. Our work paves the way for reliable single molecule Raman spectroscopy and fast molecular imaging on plasmonic surfaces.     

Novel hydrophilic bis(1,2,3-triazolyl)fluorenyl probe for in vitro zinc ion sensing

A hydrophilic bis(1,2,3-triazolyl)fluorene derivative was synthesized as a multi-photon-absorbing, zinc-ion-sensing fluorescent probe. The fluorescence response was approximately five-fold greater in presence of Zn(2+), resulting in a large binding constant (1 × 10(9)) for a 1:2 ligand to zinc complex. A four-fold increase in the two-photon absorption cross section was achieved upon binding Zn(2+). In vitro two-photon fluorescence microscopy imaging revealed a significant fluorescence increase upon introduction of Zn(2+) into HeLa cells and reversible Zn(2+) binding, demonstrating the potential of this probe for zinc ion sensing.     

Excitation saturation in two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for the study of molecular dynamics at the single-molecule level. Because photophysical dynamics often dramatically influence FCS measurements, the role of various photophysical processes in FCS measurements must be understood to accurately interpret FCS data. We describe the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the effects of excitation saturation on the size and shape of the two-photon fluorescence observation volume and derive a new analytical expression for fluorescence correlation functions that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.     

基于分子工程能量转移构建的比率型双光子荧光探针在生物成像中的应用

设计和合成可被应用于生物系统中各种分析物的比例检测与成像的基于能量转移二元体系的比率型双光子荧光探针是一项至关重要的任务。因此,对近10 a基于荧光共振能量转移(FRET)或跨键能量转移(TBET)构建的比率型双光子荧光探针在生物成像中的应用进行综述。未来的研究方向是基于FRET/TBET构建新型双光子比率型荧光探针,并将其应用于生物分析和疾病诊断领域。     

Real-time monitoring of intracellular mRNA hybridization inside single living cells

A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA-DNA hybridization inside living cells using molecular beacons. A MB specific for beta-actin mRNA has been designed and synthesized. After microinjection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with beta-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the beta-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligonucleotide/mRNA interactions inside single living cells.     

Dynamic SERS imaging of cellular transport pathways with endocytosed gold nanoparticles

Dynamic SERS imaging inside a living cell is demonstrated with the use of a gold nanoparticle, which travels through the intracellular space to probe local molecular information over time. Simultaneous tracking of particle motion and SERS spectroscopy allows us to detect intracellular molecules at 65 nm spatial resolution and 50 ms temporal resolution, providing molecular maps of organelle transport and lisosomal accumulation. Multiplex spectral and trajectory imaging will enable imaging of specific dynamic biological functions such as membrane protein diffusion, nuclear entry, and rearrangement of cellular cytoskeleton.     

CdTe量子点的微波合成及其在双光子显微成像中的应用

采用微波辐射加热的方法,以亚碲酸钠(Na2TeO3)作碲源,以谷胱甘肽(GSH)作稳定剂,在水相中合成出高质量的CdTe量子点。所合成量子点的发射波长从515～630nm可调,荧光量子产率(PLQYs)最高达95%。利用X射线粉末衍射(XRD)、高分辨透射电镜(HRTEM)、紫外-可见吸收光谱(UV-Vis)和荧光发射光谱(PL)等技术表征产物的物相结构和光学性质。用双光子激发荧光法研究CdTe量子点的双光子吸收性质。用双光子激发荧光成像技术,以发红光的CdTe量子点作为双光子荧光探针成功标记了人肺腺癌细胞(A549)。     

In Vivo NIR-II Fluorescence Lifetime Imaging of Whole-Body Vascular Using High Quantum Yield Lanthanide-Doped Nanoparticles

Second near infrared (NIR-II, 1000–1700 nm) fluorescence lifetime imaging is a powerful tool for biosensing, anti-counterfeiting, and multiplex imaging. However, the low photoluminescence quantum yield (PLQY) of fluorescence probes in NIR-II region limits its data collecting efficiency and accuracy, especially in multiplex molecular imaging in vivo. To solve this problem, lanthanide-doped nanoparticles (NPs) β-NaErF₄: 2%Ce@NaYbF₄@NaYF₄ with high PLQY and tunable PL lifetime through multi-ion doping and core–shell structural design, are presented. The obtained internal PLQY can reach up to 50.1% in cyclohexane and 9.2% in water under excitation at 980 nm. Inspired by the above results, a fast NIR-II fluorescence lifetime imaging of whole-body vascular in mice is successfully performed by using the homebuilt fluorescence lifetime imaging system, which reveals a murine abdominal capillary network with low background. A further demonstration of fluorescence lifetime multiplex imaging is carried out in molecular imaging of atherosclerosis cells and different organs in vivo through NPs conjugating with specific peptides and different injection modalities, respectively. These results demonstrate that the high PLQY NPs combined with the homebuilt fluorescence lifetime imaging system can realize a fast and high signal-to-noise fluorescence lifetime imaging; thus, opening a road for multiplex molecular imaging of atherosclerosis.     

Multimodal coherent anti-Stokes Raman spectroscopic imaging with a fiber optical parametric oscillator

We report on multimodal coherent anti-Stokes Raman scattering (CARS) imaging with a source composed of a femtosecond fiber laser and a photonic crystal fiber (PCF)-based optical parametric oscillator (FOPO). By switching between two PCFs with different zero dispersion wavelengths, a tunable signal beam from the FOPO covering the range from 840 to 930 nm was produced. By combining the femtosecond fiber laser and the FOPO output, simultaneous CARS imaging of a myelin sheath and two-photon excitation fluorescence imaging of a labeled axons in rat spinal cord have been demonstrated at the speed of 20 μs per pixel.     

Analysis of individual acidic organelles by capillary electrophoresis with laser-induced fluorescence detection facilitated by the endocytosis of fluorescently labeled microspheres

Submicrometer-sized fluorescent microspheres were loaded into the acidic organelles of NS-1 mouse myeloma cells via endocytosis. Confocal microscopy imaging showed that microspheres colocalized nearly perfectly with LysoTracker Red, a probe that stains acidic organelles. Unlike LysoTracker dyes that seem to leak from acidic organelles upon cell disruption, microspheres are retained within these organelles, facilitating their analysis following isolation. Using capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF), the electrophoretic mobilities of acidic organelles were individually calculated and fluorescence intensities individually measured. When cells were incubated for sufficient time to allow for endocytosis (48 h) with 3.9 x 10(3) microspheres/cell, replicate CE-LIF analyses of the corresponding isolated fraction indicated a dramatic increase in the number of detected events (n = 1990 +/- 234) and in the overall fluorescence intensity of the individual events (0.38 +/- 0.01 RFU; average +/- SD; n = 3) over the corresponding <10-min incubations (n = 60; 0.21 RFU, respectively). In addition, a treatment with 4-fold increase in microsphere density (1.6 x 10(4) microspheres/cell), increased the number of detected individual events (n = 3427 +/- 101) and altered only slightly the fluorescence intensity and electrophoretic mobility distributions. The individual electrophoretic mobility values ranged from -1.45 x 10(-)(4) to -3.0 x 10(-)(4) cm(2) V(-)(1) s(-)(1) while the individual fluorescence values ranged from 0.1 V to over 8 V, demonstrating the benefit of detecting organelles individually rather than averaging their properties over single cells or bulk homogenates.     

Simultaneous two-photon spectral and lifetime fluorescence microscopy

When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope. Fluorescence lifetime images from 16 individual wavelength components of the emission spectrum can be acquired with 10-nm resolution on a pixel-by-pixel basis. The instrument provides a unique visualization of cellular structures and processes through spectrally and temporally resolved information and may ultimately find applications in live cell and tissue imaging.     

Quantitative one-dimensional imaging using picosecond dual-broadband pure-rotational coherent anti-Stokes Raman spectroscopy

We employ picosecond dual-broadband pure-rotational coherent anti-Stokes Raman spectroscopy (CARS) in a one-dimensional (1D) imaging configuration. Temperature and O(2):N(2) concentration ratios are measured along a 1D line of up to 12 mm in length. The images consist of up to 330 individual rotational CARS (RCARS) spectra, corresponding to 330 spatially resolved volume elements in the probe volume. Signal levels are sufficient for the collection of single-laser-pulse images at temperatures of up to approximately 1200 K and shot-averaged images at flame temperatures, demonstrated at 2100 K. The precision of picosecond pure-rotational 1D imaging CARS is assessed by acquiring a series of 100 single-laser-pulse images in a heated flow of N(2) from 410 K-1200 K and evaluating a single volume element for temperature in each image. Accuracy is demonstrated by comparing temperatures from the evaluated averaged spectra to thermocouple readings in the heated flow. Deviations from the thermocouple of <30 K in the evaluated temperature were found at up to 1205 K. Accuracy and single-shot precision are compared to those reported for single-point nanosecond dual-broadband pure-RCARS and nanosecond 1D vibrational CARS.     

Towards a FAST-CARS anthrax detector: Analysis of cars generation from DPA

Abstract

Femtosecond coherent anti-Stokes Raman spectroscopy (CARS) of dipicolinic acid (DPA), a marker molecule for bacterial spores of anthrax, has been investigated. The temporal evolution of the CARS response of DPA was examined using two ultrafast pulses to ‘pump’ the DPA and a third pulse to probe the response. In one set of experiments, a single detector recorded the temporal behaviour of the signal as a function of different pump conditions. In another set of experiments, the full anti-Stokes spectrum was recorded as a function of probe delay. The data from both experiments displayed a strong, instantaneous response and a structured transient response. The implications of these data for molecular detection are discussed.     

Selective imaging of active pharmaceutical ingredients in powdered blends with common excipients utilizing two-photon excited ultraviolet-fluorescence and ultraviolet-second order nonlinear optical imaging of chiral crystals

Second order nonlinear optical imaging of chiral crystals (SONICC) and two-photon excited fluorescence measurements [both autofluorescence and two-photon excited UV-fluorescence (TPE-UVF)] were assessed for the selective detection of APIs relative to common pharmaceutical excipients. Active pharmaceutical ingredients (APIs) compose only a small percentage of most tabulated formulations, yet the API distribution within the tablet can affect drug release and tablet stability. Complementary measurements using either UV-SONICC (266 nm detection) or TPE-UVF were shown to generate signals >50-fold more intense for a model API (griseofulvin) than those produced by common pharmaceutical excipients. The combined product of the measurements produced signals >10(4)-fold greater than the excipients studied. UV-SONICC or TPE-UVF produced greater selectivity than analogous measurements with visible-light detection, attributed to the presence of aromatic moieties within the API exhibiting strong one and two photon absorption at ~266 nm. Complementary SONICC and fluorescence measurements allowed for the sensitive detection of the three-dimensional distribution of tadalafil within a Cialis tablet to a depth of >140 μm.     

Facile and Scalable Preparation of Fluorescent Carbon Dots for Multifunctional Applications

The synthesis of fluorescent nanomaterials has received considerable attention due to the great potential of these materials for a wide range of applications, from chemical sensing through bioimaging to optoelectronics. Herein, we report a facile and scalable approach to prepare fluorescent carbon dots (FCDs) via a one-pot reaction of citric acid with ethylenediamine at 150 °C under ambient air pressure. The resultant FCDs possess an optical bandgap of 3.4 eV and exhibit strong excitation-wavelength-independent blue emission (λ_Em = 450 nm) under either one- or two-photon excitation. Owing to their low cytotoxicity and long fluorescence lifetime, these FCDs were successfully used as internalized fluorescent probes in human cancer cell lines (HeLa cells) for two-photon excited imaging of cells by fluorescence lifetime imaging microscopy with a high-contrast resolution. They were also homogenously mixed with commercial inks and used to draw fluorescent patterns on normal papers and on many other substrates (e.g., certain flexible plastic films, textiles, and clothes). Thus, these nanomaterials are promising for use in solid-state fluorescent sensing, security labeling, and wearable optoelectronics.     

Spatial effects in two-photon excitation transient absorption spectroscopy

We investigate the influence of the transverse mode of the pump beam and the geometry of the pump–probe configuration on the femtosecond two-photon excitation transient absorption (TA) spectroscopy. Strong modulation of the transient TA dynamics may result from the multi-peak distribution of the transverse mode of the pump beam, the large pump beam size, and the large separation angle between the pump and the probe beams, which is sensitive to the pulse length. Quantitative characterization of this kind of spatial effect is of general importance for the correct interpretation of the photophysical mechanisms that can be revealed by the two-photon TA measurements.