Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation microscopy

Simultaneous two-photon excited fluorescence (TPF) and second-harmonic generation (SHG) imaging is demonstrated using a single femtosecond laser and a scanning microscope. This composite nonlinear microscopic technique was applied to imaging DNA and chromosomes, and it was shown that the two different interaction mechanisms provide complementary information on the structure and nonlinear properties of these biological materials, beyond that achievable using either TPF or SHG imaging alone. The use of separate modes of detection, in reflection and transmission respectively, and the simultaneous nature of the acquisition of the two images allows pure TPF and SHG images in precise registration to be obtained.     

Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.     

单量子双能级系统和双态系统关联函数的研究

以具有简单三能级结构染料分子的单量子系统为研究对象,用带有边界条件的速率方程来反映低功率单色连续激光激励下单分子发射荧光的动力学过程.该方法可以通过时间分辨光子探测获得处理单分子动力学随机过程.研究给出了一种通过求解反映单量子系统动力学过程的物理量——延迟函数计算关联函数的方法,并给出了双能级系统和双态系统延迟函数和关联函数的具体表达式.研究表明,在实际测量中只要获得关联函数曲线,就可以通过数据拟合计算出分子跃迁的动力学参量.     

Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Raman imaging. The distribution of the UV-absorbing fluorophore Hoechst 33342 in the apoptotic HeLa cells is measured in the combined cw two-photon-excited fluorescence and Raman microscopy modes. The 647-nm line of a Kr-ion laser is used to excite both the Raman scattering and the two-photon-excited fluorescence emission. The lateral and axial resolutions in the two imaging modes are compared by use of the Gaussian beam approximation and backprojection of the focal volume through the confocal pinhole.     

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging

We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.     

Two-photon fluorescence measurements in a hexagonal CdS crystal

Measurements of two-photon fluorescence (TPF) in a CdS crystal using a dye laser pumped by a Q-switched ruby laser are reported. The TPF is measured as a function of source intensity and wavelength. The total fluorescence shows linear and non-linear dependence on the source intensity; from these measurements the TPF part is determined. The measured transition of the crystal's fundamental gap is 2.5 ± 0.02 eV and another forbidden one-photon but allowed two-photon transition is found at 3.24 ± 0.02 eV.     

Enhanced two-photon fluorescence excitation by resonant grating waveguide structures

Enhanced two-photon fluorescence (TPF) spectroscopy with novel high-finesse resonant polymeric grating waveguide structures (GWSs) is presented. Under resonant conditions the field enhancement at the surface of a GWS can be exploited for TPF spectroscopy without the need for highly focused laser excitation light. We compare the TPF obtained by placing a drop of tetramethylrhodamine (TMR) on top of a GWS with that obtained with TMR on top of a glass substrate. Our procedure and results indicate that the detection of TPF can be improved by a factor of 10 with resonant GWSs.     

Two-photon fluorescence technique of ultra-short laser pulse measurement and the efficiency of nonlinear optical processes

A simple relation is established between the efficiency of an optical doubler excited by a multimode laser with partial mode-locking and the parameters of a two-photon fluorescence (TPF) trace of laser radiation. In general, the efficiency of two quantum processes can be obtained from the measurements of the contrast and the bright spot size of the TPF trace. In several cases these parameters of the TPF trace may be used for approximate estimation of the third and fourth harmonic generation and the stimulated scattering efficiency. It is also shown that for quasi-periodic random radiation the efficiency of nonlinear processes of arbitrary order can be calculated by using a set of correlation functions which depend on one argument only.     

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.     

Polarization-induced control of two-photon excited fluorescence in a chiral polybinaphthyl

The fluorescence behavior of a chiral polybinaphthyl excited with 100 fs 800 nm laser pulses was investigated in tetrahydrofuran solution. The peak fluorescence intensity versus the input irradiance was measured to meet a square dependence, giving evidence for two-photon excited fluorescence (TPF). The variations of the TPF intensity were found to be strongly modulated by the different polarized incident lights and tightly depend on the linearly polarized component of the incident light. Furthermore, combining with the characteristics of chiral molecules, the two-photon polarization ratio was studied to reveal the symmetry of the involved excited states.     

Giant enhancement of two-photon fluorescence induced by resonant double grating waveguide structures

We report a 350-fold enhancement of ultra-short-pulse-excited two-photon fluorescence (TPF) using a resonant double grating waveguide structure (DGWS). These structures show vanishing transmission and maximum reflection under resonance conditions, i.e. specific wavelength, polarisation and angular orientation of the incident light. This guided mode phenomenon is characterised by a large field enhancement inducing an enormous TPF signal of fluorescent molecules at the waveguide surface, as compared to direct non-resonant excitation. We demonstrate that high spectral acceptance for ultra-short pulses with broad spectral bandwidths can be achieved by a specifically designed DGWS, and that neither beam focussing nor high laser power is necessary for TPF excitation. Due to the high enhancement of more than two orders of magnitude, DGWS can be considered as a powerful platform for TPF applications such as biosensing and microarray technology. PACS 42.62.Be; 42.79.Dj; 42.79.Gn     

Super-Resolution Two-Photon Fluorescence Tomography Through the Phase-Shifted Optical Beatings of Bessel Beams for High-Resolution Deeper Tissue 3D Imaging

A novel z-scanning-free epi-detected super-resolution two-photon fluorescence tomography (TPFT) technique enabling super-resolution deeper tissue 3D imaging is reported. To accomplish this, a unique method is conceived by generating the phase-shifted optical beatings of Bessel beams (PS-OB³) with a spatial light modulator (SLM) to break the diffraction limit for enhancing both the lateral and axial resolutions as well as improving the penetration depth in TPFT for super-resolution deeper tissue imaging. By electronically varying the optical beating frequency and the phase shifts of the beating patterns through SLM, the depth-resolved TPF signals about the volumetric tissue are encoded in the spatial frequency domain and hence, a series of depth-resolved TPF images can be retrieved by implementing inverse fast Fourier transform without a need of mechanical depth-scanning. PS-OB³ TPFT provides ≈1.3- and 2-fold improvements in lateral and axial resolutions in comparison with conventional point-scan TPF imaging. It is also illustrated that the epi-detected PS-OB³ TPFT imaging with inherent scattering-resilient properties of the Bessel beams employed gives over 2-fold improvement in imaging depth in porcine brain tissue compared to conventional point-scan Gaussian beam TPF imaging. The z-scanning-free optical sectioning ability of PS-OB³ method developed in TPFT is universal, which can be readily extended to practically any other nonlinear optical imaging modalities for super-resolution deeper 3D imaging in biological and biomedical tissues.     

Broadband amplitude-modulated laser source with high output power for FM/cw lidar transmitter

We are building a long-range FM/cw nonscanning imaging lidar breadboard. This lidar system achieves ranging based on a frequency modulation/continuous wave (FM/cw) technique, implemented by an amplitude modulated mid-IR diode laser transmitter with a linear frequency modulation (LFM) of the subcarrier. Firstly, various schemes of light beam modulation are analyzed. Secondly, we put forward a laser modulation scheme whose core was formed by a 1.55 μm electro-absorption modulated laser diode (EML) and an erbium-doped optical fiber amplifier (EDFA), then a corresponding experimental system architecture and components for light beam modulation and detection are established. Finally, a corresponding experiment of laser beam modulation is completed for the first time. In our experiment, the EML amplitude is modulated by a 200 MHz to 800 MHz LFM signal, whose amplitude value is 2.05 V. The average output power of the modulated laser obtained in the experiment is 10 W, peak power is 16.4 W, and the average modulation depth is 78%. The results of tests predict that this laser modulation scheme is likely to improve the imaging range of the FM/cw lidar.     

Picosecond-pulse-induced two-photon fluorescence enhancement in biological material by application of grating waveguide structures

We report enhancement of two-photon fluorescence (TPF) excitation in fluorescent dyes and fluorescently labeled biomolecules by exploiting the optical properties of double grating waveguide structures (DGWSs). Picosecond laser pulses generate a large evanescent field based on the guided mode phenomenon in the resonant DGWSs, which induces strong TPF signals from fluorescent dyes at the waveguide surface. By recording enhanced TPF signals of Rhodamine B and Lucifer Yellow under resonance conditions, a detection sensitivity of concentrations of approximately one dye molecule per 0.1 microm2 was achieved. For the first time to our knowledge, enhanced TPF signals of a Lucifer Yellow-labeled biomolecule (human self-peptide) in an aqueous environment are demonstrated. These results strongly encourage the use of DGWSs as enhancement platforms in modern biophysics and biotechnology for investigations of biological membranes and cells.     

A high-power electro-absorption modulated 2d-array laser source for FM/cw scannerless lidar

A scannerless amplitude-modulated (AM) semiconductor lidar (or laser radar) using frequency-modulation continuous wave ranging (FM/cw) techniques is one of the most promising research directions at present. In this lidar system, the high-power AM laser source is a key component of the lidar transmitter. First of all, we present here the architecture of an FM/cw scannerless AM ladar and summarize and analyze various schemes of high-power laser modulation. Then we put forward a new method for obtaining a high-power AM laser output, that is, to compose a two-dimensional (2D) array light source with laser diodes (LDs) and electro-absorption modulation modules (EAMs). Finally, the optical properties of electro-absorption modulated laser arrays are briefly analyzed. The analysis shows that the modulated laser arrays have the features of integration and miniaturization and can realize incoherent light illumination in the far field. This laser array is potentially a more ideal high-power AM laser source applied to an FM/cw scannerless imaging lidar.     

Saturation-dips in the weak calcium intercombination line

Lamb-dips have been observed in the weak calcium intercombination line by monitoring resonance fluorescence in an atomic beam excited by light of a stabilized cw dye laser inside or outside the laser cavity. A highly sensitive detection scheme is described.     

以萘为π-中心的双芪类衍生物双光子上转换荧光性能研究

研究了2个新的双光子上转换荧光分子—1,4-双-(9-乙基咔唑基)萘(简称为BECVN)和1,4-双-(4’-N,N-二甲氨基苯乙烯基)萘(简称为BMABN)的单光子、双光子光谱性质。在～375 nm Xe灯光源激发下,两样品的DMF溶液发出很强的蓝、绿色荧光(峰位492～541 nm),视感效果非常明显。BMABN分子的线性吸收/发射光谱的峰位与BECVN分子相比,均发生红移;相对荧光量子产率(Φ_f)比BECVN降低了7.4倍。在飞秒钛宝石激光器泵浦下(760 nm),两样品的DMF溶液发出强的双光子上转换荧光发射,峰位与单光子荧光峰位相比发生红移(500～556 nm)。BMABN的双光子荧光强度和双光子吸收截面分别是BECVN的3倍和30.4倍。     

High efficient system for studying multiphoton absorption

An optical arrangement for studying two-photon absorption in liquids is presented. The main feature of this new arrangement is fuller utilization of fluorescence light. This makes possible application of very low power cw laser in such experiments.     

Affinement d'une raie de fluorescence de Pr3+: LaAlO3 par excitation laser

A cw dye laser has been used for fluorescence line narrowing experiments by resonant excitation on Pr³⁺ ions in LaAlO₃. Scanning the excitation radiation through the absorption line width corresponding to the transition ³H₄ (0 cm^-1) → ¹D₂ (16 694 cm^-1) while fluorescence was observed, has put into evidence a deformation of the symmetry around Pr³⁺ ions, different for every site.This experiment shows the power of cw dye laser spectroscopy for investigating optical spectra in crystals.     

Rb（7^2D）态与基态K原子的碰撞能量转移

Ｋ－Ｒｂ混合蒸汽中，使用Ｒｂ光谱灯和染料激光器，将基态Ｒｂ原子二步激发到７＾２Ｄ态用荧光法测量了过程Ｒｂ（７＾２Ｄ）＋Ｋ（４Ｓ）→Ｒｂ（５Ｓ）＋Ｋ（７Ｓ，５Ｄ），的碰撞转移截面，Ｋ７Ｓ，５Ｄ对Ｒｂ７＾２Ｄ的荧光比中，含Ｋ７Ｓ＾←→５Ｄ碰撞转移的影响，第二个实验可以消除这个影响，利用Ｋ光谱灯和染料激光器产生Ｋ７Ｓ或５Ｄ态，探测Ｋ７Ｓ（５Ｄ）对５Ｄ（７Ｓ）的荧光比，Ｒｂ７Ｄ→Ｋ７Ｓ，５Ｄ碰撞转移截面（