Two-color two-photon 4Pi fluorescence microscopy

In 4Pi fluorescence microscopy the point-spread function is composed of a strong central lobe accompanied by interference sidelobes that produce artifacts in the image. We propose to combine two-color two-photon fluorescence microscopy and 4Pi fluorescence microscopy to overcome this sidelobe problem. Simulation results show that a single sharp fluorescence spot can be produced by use of two excitation wavelengths of 400 and 800 nm and detected at 350-nm emission wavelength.     

Out-of-focus fluorescence collection in two-photon microscopy of scattering media

Imaging depth in two-photon microscopy is ultimately limited by the out-of-focus fluorescence background which is collected indistinctly with the useful in-focus signal in scattering samples. We report on a complete Monte Carlo analysis of the collection process in two-photon imaging of turbid media. Epifluorescence collection efficiency of the microscope is shown to vary significantly from point to point inside the scattering medium, to the detriment of the in-focus signal and in favor to the background, lowering the signal-to-background ratio in the image. Moreover we found that this ratio is almost independent of the collecting path field of view for situations where the background overcomes the signal. Assuming that the out-of-focus background can be subtracted to the image, the signal-to-noise ratio in two-photon microscopy is forecast to benefit from enlarging the collection field of view, with a gain roughly proportional to this enlargement for deep imaging. Asymptotic behaviors of the Monte Carlo simulations are quantitatively interpreted from ballistic and diffusive approximations.     

Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy

We demonstrate selective two-photon excited fluorescence microscopy with shaped pulses produced with a simple yet efficient scheme based on dispersive optical components. The pulse train from a broadband oscillator is split into two subtrains that are sent through different amounts of glass. Beam recombination results in pulse-shape switching at a rate of 150MHz. Time-resolved photon counting detection then provides two simultaneous images resulting from selective two-photon excitation, as demonstrated in a live embryo. Although less versatile than programmable pulse-shaping devices, this novel arrangement significantly improves the performance of selective microscopy using broadband shaped pulses while simplifying the experimental setup.     

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.     

双光子荧光与相干反斯托克斯拉曼散射显微成像技术的实验研究

双光子荧光与相干反斯托克斯拉曼散射同属于三阶非线性效应,二者之间的差异与联系是一个值得研究的问题.本文基于自行搭建的超连续谱近红外宽带相干反斯托克斯拉曼散射显微成像系统进行光谱成像,同时通过理论与实验对比分析了双光子荧光与相干反斯托克斯拉曼散射图像存在差异的原因.结果表明,具有亚微米以上横向分辨率的相干反斯托克斯拉曼散射成像系统,可以使用较大尺寸的荧光珠进行双光子荧光成像,通过解卷积得到双光子荧光成像的系统分辨率,并将它近似等效于相干反斯托克斯拉曼散射成像系统的当下分辨率.如果需要得到相干反斯托克斯拉曼散射成像系准确的分辨率结果,就必须使用尺寸比相干反斯托克斯拉曼散射成像系统实际分辨率小的球形样品进行实验测量.     

Visualization of meiotic events in intact living anthers by means of two-photon microscopy

In this paper we describe the application of two-photon microscopy (2 PM) to the study of meiosis in plants. Fresh, unfixed anthers of Agapanthus umbelatus were briefly incubated on a minimal medium containing the DNA fluorophore DAPI. DAPI incorporation took place in about 30 min and nuclei and other DNA-containing organelles kept their fluorescence for more than 24 h. Using PM it was possible to optically section the whole, unfixed anthers to a depth of approximately 200 microm. This was up to the mid sagital section and into the sporogenic tissue. Several meiotic figures were observed with unparalleled resolution. Sequences of nuclear dynamics and division were occasionally observed in the surrounding tissues and epidermal layer of cells. However we could not optimize the procedures up to the level of observing the dynamics of division on the meiotic nuclei as well. We hypothesize that either (1) meiotic cells are sensitive to the reasonably high excitation levels of infrared light needed to attain such penetration in the tissue, or (2) that our incubation procedures are not sufficiently non-invasive for meiosis to remain unperturbed. To the best of our knowledge this is the first report on direct observation of living meiotic cells in plants and establishes the potential of 2 PM for intact organ research.     

The fluorescence spectrum of a three-level atom in two-photon resonance with the laser field

The influence of the two-photon resonant interaction on the fluoresence spectrum of a three-level atom is studied. The levels in questions are the 1S, 2P and 3S states of a two-electron atom. It is shown, that in the case of exact two-photon resonance between the 1S–3S transition and the laser field, and when the 2P state is out of resonance, the fluorescence spectrum contains 4 lines. The properties of these lines are discussed.     

一种使用高灵敏度荧光显微镜系统进行活细胞研究的方法

利用显微镜进行细胞观察和实验，一般的做法都是将细胞做成切片，细胞难免会受到损伤，从而影响实验及观察结果。为了使细胞研究更为准确，便于临床应用，开发、研制活细胞的观察工具是必由之路。以高灵敏度荧光显微镜为基础，合理设计了一种新型暗场照明器。这种新型暗场照明器的工作距离可达３００ｍｍ，完全可以满足为活细胞滴加试剂的需要，已采得经过荧光染色的活细胞图像。这一设计解决了一个非常实际的问题，为活细胞的医学研究奠定了坚实的基础。     

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.     

Pulse characterization during femtosecond laser filamentation in air by two-photon fluorescence measurement

This work reports a temporal characteristic of a laser pulse undergoing filamentation in air. The diagnostic method is based on two-photon fluorescence measurement. The results show that the pulse duration, chirp rate and beam radius could be simultaneously quantitatively retrieved. This simple technique would be useful in practice to trace the underlying dynamics of filamentation in air.     

Plasmonic-enhanced two-photon fluorescence with single gold nanoshell

Single gold nanoshell with mutilpolar plasmon resonances is proposed to enhance two-photon fluorescence efficiently.The single emitter single nanoshell configuration is studied systematically by employing the finite-difference time-domain method.The emitter located inside or outside the nanoshell at various positions leads to a significantly different enhancement effect.The fluorescent emitter placed outside the nanoshell can achieve large fluorescence intensity given that both the position and orientation of the emission dipole are optimally controlled.In contrast,for the case of the emitter placed inside the nanoshell,it can experience substantial two-photon fluorescence enhancement without strict requirements upon the position and dipole orientations.Metallic nanoshell encapsulating many fluorescent emitters should be a promising nanocomposite configuration for bright two-photon fluorescence label.The results provide a comprehensive understanding about the plasmonic-enhanced two-photon fluorescence behaviors,and the nanocomposite configuration has great potential for optical detecting,imaging and sensing in biological applications.     

On nonclassical effects in two-photon optical bistability and two-photon laser

We analyse the fluctuations at steady state in the good quality cavity case in two-photon lasers with running wave and two-photon absorptive optical bistability in a ring cavity. In the case of two-photon laser we do not find any nonclassical effect, contrary to expectations. On the other hand, in two-photon optical bistability we find squeezing and antibunching in the low transmission branch. The maximum reduction of fluctuations that can be obtained is slightly larger than by a factor of two.     

Temperature imaging with single- and dual-wavelength acetone planar laser-induced fluorescence

Two sensitive techniques for temperature imaging by use of acetone planar laser-induced fluorescence, applicable at temperatures up to 1000 K, are introduced and demonstrated. Photophysics data on the wavelength-dependent temperature variation of acetone fluorescence permit the implementation of a single-wavelength technique in environments with constant pressure and constant acetone mole fraction, and a dual-wavelength method can be applied in flows with mixing and (or) chemical reaction. Preliminary imaging results are presented for acetone-air flow over a heated cylinder (single-wavelength strategy) and for a heated laminar jet (dual-wavelength strategy).     

双光子成像观测拟南芥细胞骨架及根尖生长过程

细胞骨架在植物细胞及其运动、发育变化等过程中起着重要作用。本文利用MS培养基在无菌条件下培养转染GFP(绿色荧光蛋白)的拟南芥,使其经过从种子萌发、植株生长直至开花结果的完整生命周期;在此过程中,利用适合对较大较厚样品进行活体四维成像的双光子激光扫描显微术,观测活的拟南芥种子、根尖、导管和根毛等器官内的细胞骨架形态,以及拟南芥根尖生长发育的动态过程,量化计算出根尖的生长速度。用低浓度(10-10mol.L-1)吲哚-3-乙酸刺激拟南芥,观测到根尖生长速度比正常情况下提高约3.8倍。     

Quantum beats in two-photon resonance fluorescence

The two-photon resonance fluorescence spectrum of a three-level atom is shown to consist of the low frequency modes in addition to the high frequency ones in the limit of high photon densities. The spectral function for the low frequency modes consists of two lorentzian lines describing: the peak occuring at the renormalized beat frequency Δ₊ and that of the zero-photon excitation at the frequency Δ_-, where Δ_±=Δ-3Ω²/2ω_a±Ω²u/2ω_a, u²=1+(2Δω_a/Ω ²)². Here, 2Δ is the energy splitting between the two excited states, ω_a is the photon energy of the pump field and Ω is the Rabi frequency. The peak at the renormalized beat frequency Δ₊ occurs provided that the condition (2Δω_a/Ω²)² > 1 is satisfied. The two-photon laser spectroscopy is expected to be a useful tool for the observation of the low frequency modes in question.     

Improvement of two-photon laser induced fluorescence measurements of H- and O-atoms in premixed methane/air flames

3 photodissociation is shown to be an important photolytic source of H atoms in the reaction zone of the methane flames. At 226 nm, an efficient energy transfer between O(³P) and N₂ is established from the observation of O-resonant emissions from the second positive system of N₂. The subsequent rate of O(³P) depletion appears to be essentially “controlled” by the O(³P) concentration and is probably only minimally temperature dependent. Finally quenching rate coefficients for O(³P) by water and nitrogen are deduced from quenching rates measurements performed in CH₄/O₂/N₂ and CH₄/O₂/Ar flames. Received: 7 November 1996/Revised version: 28 January 1997     

Resolution enhancement of digital laser scanning fluorescence microscopy with a dual-lens optical pickup head

The resolution of the cell fluorescence image captured by a digital laser scanning microscopy with a modified dual-lens BD-ROM optical pickup head is enhanced by image registration and double sample frequency. A dual objective lens of red (655 nm) and blue (405 or 488 nm) laser sources with numerical apertures of 0.6 and 0.85 is used for sample focusing and position tracking and cell fluorescence image capturing, respectively. The image registration and capturing frequency are based on the address-coded patterns of a sample slide. The address-coded patterns are designed as a string of binary code, which comprises a plurality of base-straight lands and grooves and data-straight grooves. The widths of the base-straight lands, base-straight grooves, and data-straight grooves are 0.38, 0.38, and 0.76 μm, respectively. The numbers of sample signals in the x-direction are measured at every intersection point by intersecting the base intensity of the push–pull signal of the address-coded patterns, which has a minimum spacing of 0.38 μm. After taking a double sample frequency, the resolution of the measured cell fluorescence image is enhanced from 0.38 μm to the diffraction limit of the objective lens.     

Instability in degenerate two-photon running wave laser with detuning

The previous stability analysis of the degenerate two-photon running wave laser is extended to the inclusion of detuning between frequencies of cavity and atoms. We derive the analytical equation for the critical pumping and prove analytically that for the special case ofr (/) being unity, there is no Hopf bifurcation instability for the bad cavity. The good cavity case is analysed numerically. The role played by detuning is to raise the critical pumping. In the case ofk (or <K) where there is no Hopf bifurcation instability for the perfect tuning case, the large detuning can give rise to self-pulsing instability.     

Hyperspectral in vivo two-photon microscopy of intrinsic contrast

In vivo two-photon imaging of intrinsic contrast can provide valuable information about structural tissue elements such as collagen and elastin and fluorescent metabolites such as nicotinamide adenine dinucleotide. Yet low signal and overlapping emission spectra can make it difficult to identify and delineate these species in vivo. We present a novel approach that combines excitation scanning with spectrally resolved emission two-photon microscopy, allowing distinct structures to be delineated based on their characteristic spectral fingerprints. The amounts of intrinsic fluorophores present in each voxel can also be evaluated. We demonstrate our method using in vivo imaging of nude mouse skin.