Antecedents of two-photon excitation laser scanning microscopy

In 1931, Maria G?ppert-Mayer published her doctoral dissertation on the theory of two-photon quantum transitions (two-photon absorption and emission) in atoms. This report describes and analyzes the theoretical and experimental work on nonlinear optics, in particular two-photon excitation processes, that occurred between 1931 and the experimental implementation of two-photon excitation microscopy by the group of Webb in 1990. In addition to Maria G?ppert-Mayer's theoretical work, the invention of the laser has a key role in the development of two-photon microscopy. Nonlinear effects were previously observed in different frequency domains (low-frequency electric and magnetic fields and magnetization), but the high electric field strength afforded by lasers was necessary to demonstrate many nonlinear effects in the optical frequency range. In 1978, the first high-resolution nonlinear microscope with depth resolution was described by the Oxford group. Sheppard and Kompfner published a study in Applied Optics describing microscopic imaging based on second-harmonic generation. In their report, they further proposed that other nonlinear optical effects, such as two-photon fluorescence, could also be applied. However, the developments in the field of nonlinear optical stalled due to a lack of a suitable laser source. This obstacle was removed with the advent of femtosecond lasers in the 1980s. In 1990, the seminal study of Denk, Strickler, and Webb on two-photon laser scanning fluorescence microscopy was published in Science. Their paper clearly demonstrated the capability of two-photon excitation microscopy for biology, and it served to convince a wide audience of scientists of the potential capability of the technique.     

Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation

A technique for both the measurement and the control of the pulse width at the focal point of a high-NA lens, based on two-photon absorption interferometric autocorrelation, is presented. The technique is applied to measuring the pulse broadening induced on a pulse propagating through a high-NA lens system for several objectives. It is known that the pulse width may increase by up to ?50% of its original value by propagation through an objective which has wide field compensation for spherical and chromatic aberrations. The two-photon absorption autocorrelation technique allows adjustment of the actual pulse width in the focus of a high-NA lens through pre-chirp compensation. The pulse width is shown to be almost independent of penetration depth into the sample, while the amplitude of the autocorrelation signal shows a strong decrease with depth. The ability to both measure and control the actual pulse width under strong focusing conditions is of direct importance to, among others, two-photon absorption imaging approaches.     

纳米半导体复合薄膜的非线性光学性质及其在激光器中的应用

采用射频磁控溅射技术制备了Ge掺二氧化硅（Ge-SiO₂）和Ge,Al共掺二氧化硅（Ge/Al-SiO₂）两种复合薄膜,并进行了热退火处理形成了纳米Ge镶嵌结构。通过紫外-可见吸收谱测量,确定了两种薄膜中纳米Ge的光学带隙,并采用皮秒激光Z-扫描技术研究了薄膜的非线性光学性质。测试结果显示,在1 064 nm激发下得到的Ge-SiO₂和Ge/Al-SiO₂薄膜的非线性吸收系数分别为-1.23×10^-7 m/V和4.35×10^-8 m/W,前者为饱和吸收,而后者为双光子吸收。把两种薄膜作为可饱和吸收体均可实现1.06 μm激光的被动调Q和被动锁模运转。与Ge-SiO₂薄膜比较,采用Ge/Al-SiO2薄膜可以获得较窄的调Q脉冲和锁模脉冲。最后,理论分析和实验比较了两种薄膜实现被动调Q和锁模的机理。     

光脉冲在半导体光放大器中传输的计算机模拟

提出了一个用于描述脉冲在半导体光放大器(SOA)中传播的模型,此模型主要考虑了半导体光放大器内载流子加热(CH)、光谱烧孔(SHB)、双光子吸收(TPA)和非线性折射率(NR)这些非线性效应以及群速色散和ASE噪声对放大器性能的影响.介绍了基于此模型编制的SOA模拟软件,软件可以模拟SOA的输入-输出过程,即给定输入端的脉冲复振幅即可求出输出端的脉冲复振幅.软件由几个功能模块组成,这些功能模块可以复用于更大的模拟系统.     

Two-photon image correlation spectroscopy and image cross-correlation spectroscopy

We introduce two-photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two-photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two-photon ICS, we measured diffusion coefficients as large as 10⁻⁸ cm² s⁻¹ that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions. We also show the sensitivity of the method for measuring microscopic flow using analogous test samples. We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal-to-noise in relatively short time periods and discuss situations when these methods represent improvements over non-imaging fluorescence correlation spectroscopy. We present the first demonstration of two-photon image cross-correlation spectroscopy where we simultaneously excite (via two-photon absorption) non-identical fluorophores with a single pulsed laser. We also demonstrate cellular application of two-photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells.     

Highly efficient upconverters for multiphoton fluorescence microscopy

A new generation of efficient two-photon absorbing fluorescent molecules has been developed and used effectively for two-photon laser scanning microscopy. Several examples of the use of these new fluorophores have been presented. In addition, issues relating to the two-photon absorption cross-section, excitation power, sample properties and resolution in two-photon laser scanning microscopy are discussed.     

A simple twist for signal enhancement in non-linear optical microscopy

We describe a very simple but elegant approach to two-photon fluorescence signal enhancement by intensity modulation with immediate application in two-photon laser-scanning fluorescence microscopy. This method of enhancement shows potential application in any microscopic technique that result from non-linear photon absorption and plays a pivotal role in live cell imaging.     

Cell-permeant cytoplasmic blue fluorophores optimized for in vivo two-photon microscopy with low-power excitation

Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity.     

LaPO4纳米粒子在铝合金表面的摩擦学性能

在TritonX-100/C10H21OH/H2O微乳液中合成了LaPO4纳米粒子,其粒径约5 nm。用高速环块摩擦磨损试验机考察了LaPO4纳米粒子在铝合金表面的摩擦学性能,并用X射线光电子能谱对其摩擦化学作用机制进行了研究。研究结果表明,LaPO4纳米粒子能提高微乳液的润滑性能,其在摩擦过程中,发生了摩擦化学反应,在摩擦表面生成了由LaPO4、La2O3、A lPO4等组成的化学反应膜。同时,微乳液中的有机分子吸附在铝合金表面构成了有机吸附膜,也起到了减摩抗磨的作用。     

基于太赫兹波谱的液体极性测量

太赫兹波能与极性液体的氢键发生强烈的共振吸收作用,分子极性越大,吸收作用越强。基于此特性,在0.2～1.0 THz波段利用太赫兹时域光谱技术对甲醇、乙醇、正丙醇、正丁醇四种一元醇液体的极性进行检测研究。根据测量得到的四种液体太赫兹波谱数据,得出了液体的极性与其太赫兹波谱的关系式,再利用此关系式求得四种液体的计算值。通过对四种液体极性的标准值和计算值比对,验证了所得液体极性求取公式的有效性。研究结果表明,四种不同液体的时域光谱因分子的极性差异有显著的不同,所用的液体极性测量方法能够对液体的极性进行快速有效的检测与鉴别,可为其他液体极性的检测提供参考。     

Numerical analysis of entropy generation in nanofluid flow over a transparent plate in porous medium in presence of solar radiation,viscous dissipation and variable magnetic field

The entropy generation of magneto-hydrodynamic mixed convection flow of nanofluid over a nonlinear stretching inclined transparent plate embedded in a porous medium due to solar radiation is investigated numerically. The nanofluid is made of Cu nanoparticles with water as the base fluid. The two-dimensional governing equations, in presence of the effects of viscous dissipation, variable magnetic field and solar radiation are transformed by similarity method to two coupled nonlinear ODEs and then solved using the numerical implicit Keller-Box method. The effects of various parameters such as nanoparticle volume fraction, magnetic parameter, porosity, effective extinction coefficient of porous medium, diameter of porous medium solid particles and Eckert, Brinkman and Hartman numbers is investigated on velocity, temperature and entropy generation number profiles. The results reveal that near to the plate surface the increase of nanoparticle volume fraction, porosity and porous medium geometric parameter cause the entropy generation number to increase, but far enough from the plate surface the increase of nanoparticle volume fraction, porosity and porous medium geometric parameter cause the entropy generation number to decrease. Also the entropy generation number increases with the increase of Brinkman number and Hartman number, and this increase is dominant near the plate surface. Closer to the plate surface the reduction of Eckert number causes the increase of entropy generation number, but the entropy generation number increases with the increase of Reynolds number.     

Effect of a confocal pinhole in two-photon microscopy.

We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large.     

CsPbBr3纳米片的三光子非线性吸收性质研究

为了研究不同层数纳米片在近红外二区的非线性光学吸收性质的变化,采用配体辅助再沉淀方法制备了不同层数(3~5层)的CsPbBr₃纳米片。以中心波长为1030 nm、脉宽为6 ps、重复频率为25 kHz的激光作为激发光源,利用Z?扫描技术研究了CsPbBr₃纳米片的非线性三光子吸收光学性质。研究结果表明:CsPbBr₃纳米片的非线性三光子吸收截面随层数减小而增大,量子限域效应增强,三层纳米片的三光子吸收截面高达4.1×10^(?71 )cm⁶s²photon^?2。CsPbBr₃纳米片在近红外二区具有优良的非线性光学吸收性质,可应用于多光子激发荧光成像领域。     

Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers

The spectral properties of one-photon, two-photon excited autofluorescence and second harmonic generation (SHG) from ex vivo human skin induced by a femtosecond (fs) laser and three visible lasers in backscattering geometry are systematically investigated. Our experimental results indicate that peak position of autofluorescence spectra from the dermis and epidermis shift toward long wavelengths, and the fluorescent intensity decreases when the excitation wavelength increases due to an effect of the excitation wavelength on autofluorescence signals. However, the intensity of the SHG signal in collagen has the maximal value of 800 nm excitation wavelength. This may be the result that the energy of the SHG signal is in resonance with an electronic absorption band. The two-photon excited autofluorescence and SHG intensity all obey a quadratical dependence on the excitation power. Compared with the two-photon excited fluorescence and SHG, the one-photon excited fluorescence in the dermis and epidermis exhibits different spectral characteristics. The investigation of the spectral characteristics of autofluorescence and SHG from ex vivo human skin can provide new insights into morphologic structures and biochemical components of tissues, which are vital for improving the application of laser-induced autofluorescence and SHG spectroscopy technique for noninvasive in vivo tissue diagnostics.     

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use requires optical fibers to conduct light into tight space, where free-space delivery is difficult. The delivery of high-peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this article, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these fibers is also provided.     

Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photoactivation. Two-photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser. The efficiency and accuracy of fluorescence photobleaching induced by two-photon absorption were determined using fluorescein-containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by ≈40%. Under these conditions the spatial accuracy of photobleaching was 0.5±0.1 μm in the lateral ( x y ) and 3.5±0.5 μm in the axial ( z ) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by ≈30%. The method was applied to write complex three-dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying large transmembrane pores and packed into three-dimensional arrays. Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored. The results suggest that two-photon scanning microphotolysis provides new possibilities for the optical analysis and manipulation of both technical and biological microsystems.     

Cr:Forsterite-laser-based fiber-optic nonlinear endoscope with higher efficiencies

We demonstrate a beam-scanning nonlinear light endoscope based on a flexible fiber bundle. Excited with a femtosecond Cr:Forsterite laser, the degradation in multiphoton multiharmonic excitation efficiency due to the pulse-broadening effect is significantly reduced without utilizing any external devices. The system resolution has been characterized to be 5.4 microm in the two-photon fluorescence endoscope, limited by the sampling theory. Finally, several image examples have been given.     

Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.     

Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz

We show the viability of high-resolution two-photon fluorescence imaging of fixed and live cells by exciting the fluorophores with a train of near-infrared pulses with duration in the picosecond range. This is exemplified with a compact, diode-pumped Nd:YVO₄ laser, emitting trains of 7-ps pulses at a wavelength of 1064 nm, with a repetition rate of 200 MHz at two separate outputs. Incoherent combination of the outputs enabled two-photon excitation with a repetition rate of 400 MHz. For a numerical aperture of 1.4 (oil), we used an average illumination power of up to 20–40 mW at the sample. The pulses were coupled into a beam scanning microscope, either directly or through a single mode glass fibre. Compared with standard femtosecond titanium–sapphire excitation conditions, our experiments were performed with a 2.5 or 5 times higher repetition rate, 30–70 times longer pulses and 10–35 times lower pulse peak intensity. The experiments indicate the possibility of significantly relaxing the temporal pulse width constraints for a series of applications.