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.     

Membrane potential detection with second-harmonic generation and two-photon excited fluorescence: A theoretical comparison

We theoretically compare the performance of TPEF and SHG microscopy for membrane potential imaging. We argue that electrochromic TPEF and SHG membrane potential responses are reflections of the same phenomenon, and can be described in a unified manner as resulting from the linear Stark effect. We also show that TPEF and SHG exhibit similar sensitivities in the case of both electrochromic and orientational response mechanisms. Despite their similar sensitivities, SHG nevertheless presents advantages over TPEF for membrane potential imaging because of its remarkable spatial and spectral contrast, and because of its insensitivity to non-radiative excited-state damping mechanisms.     

Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy

We demonstrate that simultaneous second-harmonic generation (SHG) and two-photon-excited fluorescence (TPEF) can be used to rapidly image biological membranes labeled with a styryl dye. The SHG power is made compatible with the TPEF power by use of near-resonance excitation, in accord with a model based on the theory of phased-array antennas, which shows that the SHG radiation is highly structured. Because of its sensitivity to local asymmetry, SHG microscopy promises to be a powerful tool for the study of membrane dynamics.     

Nonlinear spectral imaging of human normal skin,basal cell carcinoma and squamous cell carcinoma based on two-photon excited fluorescence and second-harmonic generation

In this work, we use nonlinear spectral imaging based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) for analyzing the morphology of collagen and elastin and their biochemical variations in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and normal skin tissue. It was found in this work that there existed apparent differences among BCC, SCC and normal skin in terms of their thickness of the keratin and epithelial layers, their size of elastic fibers, as well as their distribution and spectral characteristics of collagen. These differences can potentially be used to distinguish BCC and SCC from normal skin, and to discriminate between BCC and SCC, as well as to evaluate treatment responses.     

Zebrafish imaging and two-photon fluorescence imaging using ZnSe quantum dots

This study is to report a ZnSe quantum dot with a large two-photon absorption cross section and good biocompatibility,which can be used in bioimaging.Fluorescence emission at 410 nm is observed in the quantum dot under 760-nm laser excitation.These biocompatible quantum dots exhibit a two-photon cross-section of 9.1×10~5 GM(1 GM=10~(-50) cm~4·s/photon).Two-photon excited laser scanning microscopic images show that cells co-cultured with ZnSe quantum dots are found in the blue channel at a fluorescence intensity that is 14.5 times that of control cells not cocultured with quantum dots.After incubating zebrafish larvae with ZnSe quantum dots for 24 h,the fluorescence intensity of the yolk sac stimulated by ultraviolet light is 2.9 times that of the control group.The proposed material shows a great potential application in biological imaging.     

Simultaneous measurements of second-harmonic generation and two-photon photoelectric emission from Au

Received: 1 October 1998 / Accepted: 30 October 1998     

多色双光子激发荧光显微技术实验研究

双光子激发荧光(two-photon excited fluorescence, TPEF)显微是一种非线性光学显微技术, 具有高的时间分辨率和空间分辨率、高的信噪比和固有的三维层析分辨能力等优点. 传统的TPEF显微一般采用波长可调谐的超短脉冲激光器作为光源. 在实际应用中, 利用TPEF显微技术研究含有多种荧光团或未知成分的待测样品, 往往需要多次改变激发光的波长以获得对各种荧光团的最佳激发. 为了同时获取不同荧光团的荧光信号, 利用超连续谱激光光源实现了多色TPEF显微成像, 实验中无需调节波长, 能够同时获得具有两种不同发射波长的荧光标记的铃兰根茎切片样品的TPEF图像. 实验结果表明, 与传统的TPEF显微相比, 该方法能够同时获取含有多种荧光团的待测样品的高对比度TPEF图像, 具有系统结构简单、操作简便、信息量大等优点, 在生物医学和材料科学等领域具有广阔的应用前景.     

Local second-harmonic generation enhancement on gold nanostructures probed by two-photon microscopy

The localization of surface second-harmonic generation (S-SHG) enhancements from granular gold structures that exhibit local plasmon resonance was investigated. A two-photon microscopy technique was used to perform high spatial resolution S-SHG imaging. The magnitude and the spatial density of S-SHG enhancement confined in submicroscopic regions are strongly dependent on the morphology of the gold's surface. Polarization measurements of local S-SHG responses reveal the local field anisotropy in enhancement regions and furthermore prove the incoherent and strongly depolarized nature of the emission, which is attributed to ultrafast fluctuations of the enhancement location in the focal volume.     

Investigation of transient two-photon excited fluorescence in biological tissue

The fluorescence power from biological tissue excited by a femtosecond laser pulse compared with excitation power does not appear to obey a simple quadratic relationship given by the steady non-linear theory.A more reliable analysis is developed based on transient two-photon absorption because the response time of two-photon absorption is longer than the width of a femtosecond pulse.Good agreement is obtained between the theoretical analysis and the experimental results of fluorescence power versus excitation power.This letter offers potential value to non-linear optics in biological tissues.     

基于双光子激发荧光的单分子探测技术研究

介绍采用双光子激发荧光方法进行单分子探测的原理和自行研制的实验装置,激发光聚焦和荧光收集采用共焦方式。选择香豆素C445水溶液作为研究对象,从样品流速、浓度、激光功率、信噪比和检测限等方面探讨了双光子激发荧光的特性。该谱仪目前己达到探测灵敏区内C445的平均分子数为1.5个的检测限     

Quantitative imaging of mixing dynamics in microfluidic droplets using two-photon fluorescence lifetime imaging

Droplet-based microfluidic systems enable miniaturization of chemical reactions in femtoliter to picoliter volume compartments. Quantifying mixing dynamics of the reagents in droplets is critical to determine the system performance. In this Letter, we developed a two-photon excitation fluorescence lifetime imaging technique to quantitatively image the mixing dynamics in micro?uidic droplets. A cross/autocorrelation method was used to reconstruct a high-quality fluorescence lifetime image of the droplet. The fluorescence decay was analyzed for accurate determination of the mixing ratio at each pixel of the image.     

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.     

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.     

Three-dimensional second-harmonic generation imaging with femtosecond laser pulses

A three-dimensional reflectance scanning optical microscope based on the nonlinear optical phenomenon of second-harmonic generation is presented. A mode-locked Ti:sapphire laser producing <90-fs pulses at ~790nm was used, and the images were constructed by scanning of an object, which possessed local second-order nonlinearity, relative to a focused spot from the laser. The second-harmonic light at ~395nm generated by the specimen was separated from the fundamental beam by use of dichroic and interference filters and was detected by a photodiode. The technique was then used to characterize the distribution of second-order nonlinearity and microstructure of the nonlinear material lithium triborate.     

In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope

We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9 g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.     

Sensitive detection of H2 molecules by two-photon excited laser-induced fluorescence

The sensitive detection of H₂ molecules was demonstrated by means of twophoton excited laser-induces fluorescence spectroscopy with a narrow-band ArF excimer laser. A detection limit of 2×10¹⁴ cm^–3 was obtained with an excitation power of 150 kW. This is already comparable with that obtained by the coherent anti-Stokes Raman scattering (CARS). This technique was successfully applied to measure a spatial distribution of H₂ in a town-gas burner.     

Strong coupling in macrocyclic thiophene investigated by time-resolved two-photon excited fluorescence

We report the femtosecond dynamics of fluorescence anisotropy excited through the two-photon absorption (TPA), which provides direct signatures of delocalized electronic excitations for symmetrical macromolecular architectures. Two-photon excited fluorescence anisotropy is strongly correlated with the orientation and value of the transition moment from the excited state to the second and higher lying states. For macromolecular systems it leads to a relatively low initial fluorescence anisotropy and specific femtosecond anisotropy dynamics. We have experimentally demonstrated qualitatively different anisotropy dynamics for two- and one-photon absorption excitations for strongly coupled ring architecture prospective for artificial-light-harvesting applications and possessing an enhanced TPA-absorption cross section.     

Flow imaging by use of femtosecond-laser-induced two-photon fluorescence

A novel technique is demonstrated for the imaging of turbulent flows in which a single window to the flow is the only optical access required. A femtosecond laser is used to excite two-photon fluorescence in a disodium-fluorescein-seeded water jet. The fluorescence signal is generated at only the focal point of the laser because of the highly nonlinear nature of the two-photon absorption, and it is collected in a direction counterpropagating to the excitation beam. Tight focusing of the laser is used to limit the probe volume, and the two-dimensional mean and rms concentration images are collected by raster scanning the laser.     

Three-dimensional chemical concentration maps in a microfluidic device using two-photon absorption fluorescence imaging

Two-photon absorption fluorescence is employed within a microfluidic device to create a three-dimensional chemical concentration map for mixing uniformity characterization. This multiphoton technique images fluorescence intensity directly and provides a simple, rapid, and readily employed route to composition characterization within microfluidic systems.