长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。     

Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination

We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.     

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.     

Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes

The influence of the pulse length, tau , of ultrashort laser pulses at 780 and 920 nm on cell vitality and cellular reproduction has been studied. A total of 2400 nonlabeled cells were exposed to a highly focused scanning beam from a mode-locked 80-MHz Ti:sapphire laser with 60-micros pixel dwell time. For the same pulse energy, destructive effects were more pronounced for shorter pulses. The damage behavior was found to follow approximately a P(2)/tau dependence (P , mean power), indicating that cell destruction is likely based on a two-photon excitation process rather than a one- or a three-photon event. Therefore, femtosecond as well as picosecond pulses provide approximately the same relative optical window for safe two-photon fluorescence microscopy.     

DHL细胞中5-ALA代谢PpIX的双光子荧光光谱

双光子激发生物组织荧光,激发光仅作用于焦点区域,对生物样品的光漂白性和光毒性都很小,因而双光子荧光显微技术已成为细胞生物学研究的一种新技术。文章采用波长为820 nm飞秒激光激发孵育有5-ALA的DHL细胞,在激光扫描显微镜的Lambda模式中获得单个DHL细胞的双光子荧光光谱,并测量DHL细胞内积聚的卟啉九(PpIX)特征荧光值。获得了浓度分别为2, 4和10 mmol·L-1的5-ALA溶液中,细胞代谢的PpIX含量随孵育时间的变化情况。DHL细胞内积聚的PpIX处于动态变化过程,并呈现出两阶段性的特点：细胞内积聚的PpIX含量随着孵育时间增长而增加,在3 h附近达到最大值,随后随着孵育时间增长反而下降。结果表明,基于激光扫描显微的双光子荧光光谱可成为DHL细胞等白血病细胞摄取5-ALA并生成PpIX的动力学研究的有效方法。     

Improved depth resolution in video-rate line-scanning multiphoton microscopy using temporal focusing

By introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain video-rate images with depth resolution similar to point-by-point scanning multiphoton microscopy while mechanically scanning in only one dimension. This is achieved by temporal focusing of the illumination pulse: The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration (and highest peak intensity) at the focal plane before stretching again beyond it. This method is applied to produce, in a simple and scalable setup, video-rate two-photon excitation fluorescence images of Drosophila egg chambers with nearly 100,000 effective pixels and 1.5 microm depth resolution.     

Tunable, low-repetition-rate, cost-efficient femtosecond Ti:sapphire laser for nonlinear microscopy

We report on a broadly tunable, long-cavity Ti:sapphire laser oscillator being mode-locked in the net negative intracavity dispersion regime by Kerr-lens mode-locking, delivering τ _FWHM<300 fs pulses at 22 MHz repetition rate. The wavelength of the laser can be tuned over a 170 nm wide range between 712 nm and 882 nm. Having a typical pump power of 2.6 W, the maximum pulse peak power is 60 kW. Comparison of the reported laser with a standard, 76 MHz Ti:sapphire oscillator regarding two-photon excitation efficiency in a laser scanning microscope shows that the 22 MHz laser generates the same fluorescence signal at considerably, 1.82 times lower average power, which is expected to result in a reduced photothermal damage probability of biological samples. This fact along with the broad tunability and a low pump power requirement makes this cost-effective laser an ideal light source for nonlinear microscopy.     

Wavelength dependent damage in biological multi-photon confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. Thus induced two-photon absorption and the accompanied multi-photon absorption/ionization not only cause photo-bleaching but also cell damage in the vicinity of the focal point. In this paper, we study the wavelength dependent cell damage induced by high intensity femtosecond near infrared lasers. The study was performed with a Ti:sapphire laser and a Cr:forsterite laser. With a longer output wavelength from a Cr:forsterite laser, multi-photon absorption and auto-fluorescence were found to be significantly suppressed and the destructive plasma formation was found to be greatly reduced. Sustained multi-photon spectra can be observed in most plant specimens with a tightly focused Cr:forsterite laser beam under long term irradiation with more than 100 mW laser average power. In contrast, multi-photon absorption induced destructive plasma formation were frequently observed with a tightly focused Ti:sapphire laser beam within seconds with more than 10 mW laser average power.     

Effects of absorption saturation in colloidal quantum dots as fluorophores for multiphoton fluorescence microscopy

It is shown that colloidal semiconductor nanocrystals (quantum dots), which are promising fluorophores for multiphoton fluorescence microscopy, exhibit a two-photon absorption saturation effect at moderate powers (not exceeding 10 mW) of femtosecond pumping radiation. An analytical expression for the power of two-photon fluorescence of quantum dots as a function of the average pumping power is obtained. With this expression, the deviation of the found dependence from the quadratic law is explained by two factors, i.e., a large two-photon absorption cross section of quantum dots and their slow (compared to the typical pumping pulse-repetition period) relaxation to an unexcited state. Using an LSM 510 Carl Zeiss laser scanning microscope equipped with a Ti:Sa tunable femtosecond laser, a series of model experiments is performed to reveal the saturation effect in a solution of commercially available quantum dots. Good agreement is obtained between the measured dependence of the power of two-photon fluorescence on the average pump power and the theoretical calculation results. It is also experimentally demonstrated that, under fluorescence saturation conditions, the spatial resolution of the method of multiphoton fluorescence microscopy is lost; this effect is analyzed numerically.     

Photolytic-interference-free, femtosecond two-photon fluorescence imaging of atomic hydrogen

We discuss photolytic-interference-free, high-repetition-rate imaging of reaction intermediates in flames and plasmas using femtosecond (fs) multiphoton excitation. The high peak power of fs pulses enables efficient nonlinear excitation, while the low energy nearly eliminates interfering single-photon photodissociation processes. We demonstrate proof-of-principle, interference-free, two-photon laser-induced fluorescence line imaging of atomic hydrogen in hydrocarbon flames and discuss the method's implications for certain other atomic and molecular species.     

Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator

In wide-field fluorescence microscopy, illuminating the specimen with evanescent standing waves increases lateral resolution more than twofold. We report a versatile setup for standing-wave illumination in total internal reflection fluorescence microscopy. An adjustable diffraction grating written on a phase-only spatial light modulator controls the illumination field. Selecting appropriate diffraction orders and displaying a sheared (tilted) diffraction grating allows one to tune the penetration depth in very fine steps. The setup achieves 91 nm lateral resolution for green emission.     

Application of a femtosecond self-sustaining mode-locked Ti:sapphire laser to the field of laser scanning confocal microscopy

Developments in ultrafast Ti:sapphire laser technology can be applied in the investigation of nonlinear optical processes. We describe the application of a self-sustaining femtosecond Ti:sapphire laser as an illumination source in the field of confocal laser scanning fluorescence microscopy (LSM). We present spectra for various fluorescent stains under two-photon excitation and present LSM images of stained samples under mode-locked illumination. The potential for such a system as a non-destructive technique for studying live cells in biomedical research is discussed.     

Resolution enhancement in a light-sheet-based microscope (SPIM)

Light-sheet-based microscopy [single-plane illumination microscope (SPIM)] performs very well at low numerical apertures. It complements conventional (FM), confocal (CFM), and two-photon fluorescence microscopy (2hnu-FM) currently used in modern life sciences. Lateral and axial SPIM point spread function (PSF) extents are measured by using fluorescent beads to determine the 3D resolution. The results are compared with values derived from an analytical theory and numerical simulations. The discrepancies are found to be less than 5%. The axial extent of a SPIM-PSF (10x/0.3 W) is approximately 5.7 microm. This value is almost a factor of 2 smaller than in CFM, more than 2.5 times smaller than in FM, and more than three times smaller than in 2hnu-FM. SPIM outperforms 2hnu-FM and FM, while CFM has a better axial resolution at NAs above 0.8.     

Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation

The main advantage of two-photon fluorescence confocal microscopy is the low absorption obtained with live tissues at the wavelengths of operation. However, the resolution of two-photon fluorescence confocal microscopes is lower than in the case of one-photon excitation. The 4Pi microscope type C working in two-photon regime, in which the excitation beams are coherently superimposed and, simultaneously, the emitted beams are also coherently added, has shown to be a good solution for increasing the resolution along the optical axis and for reducing the amplitude of the side lobes of the point spread function. However, the resolution in the transverse plane is poorer than in the case of one-photon excitation due to the larger wavelength involved in the two-photon fluorescence process. In this paper we show that a particular arrangement of the 4Pi microscope, referenced as 4Pi′ microscope, is a solution for obtaining a lateral resolution in the two-photon regime similar or even better to that obtained with 4Pi microscopes working in the one-photon excitation regime.     

双氰基荧光染料的合成、光学性质及其生物成像

设计合成了一种A-π-D-π-A型的双光子荧光染料3,6-双(4-乙烯基苯腈)-9-乙基咔唑,测试了其在二氯甲烷(DCM)、乙酸乙酯(EA)、乙醇(Et OH)、乙腈(ACN)、二甲亚砜(DMSO)和磷酸缓冲盐溶液(PBS)等不同溶剂中的紫外吸收光谱、单光子及双光子荧光光谱。化合物3,6-双(4-乙烯基苯腈)-9-乙基咔唑在紫外吸收光谱中存在两个相似的特征吸收带并呈现出复杂的溶剂化效应,在DMSO中具有最大荧光量子产率(86.02%),其相应的活性吸收截面为12.56 GM。在双光子荧光成像方面,染料分子具有优良的细胞膜通透性并且在双光子荧光显微镜下呈现出明亮的绿色荧光,表现出较好的双光子荧光成像性能。这些数据表明,化合物3,6-双(4-乙烯基苯腈)-9-乙基咔唑可用作一种较为理想的双光子荧光标记染料。     

Compact two-photon fluorescence microscope based on a single-mode fiber coupler

We present a two-photon fluorescence microscope based on a three-port single-mode optical fiber coupler. It is found that the coupler behaves as a low-pass filter that can deliver an ultrashort-pulsed laser beam of as much as 150 mW of power in the wavelength range from 770 to 870 nm as well as collect a two-photon fluorescence signal in the visible range. As a result of using the fiber coupler, the new two-photon imaging system exhibts a number of advantages, including a compact arrangement, freedom from vibration from lasers and electronic devices, self-alignment, reduction of multiple scattering, and an enhanced optical sectioning effect. The effectiveness of the new instrument is demonstrated with a set of three-dimensional images of biological samples. This instrument may make two-photon fluorescence endoscopy possible for in vivo medical applications.     

三苯甲烷染料的表面发光

用同步泵浦染料激光器与时间相关单光于计数技术相结合,测量了孔雀绿和结晶紫水溶液的表面发光。在激发波长为608nm时,除了观测到304nm二次谐波外,还观测到了由双光于吸收所产生的荧光。     

压缩真空库场中二能级原子的非简并双光子荧光谱

利用投影算符技巧从刘维方程出发导出了二能级原子约化密度算符主方程，研究了压缩真空库场中二能级原子的非简并双光子荧光谱特性.发现与真空库或热库中双光子荧光不同，在非简并双光子情况中存在一种新型振荡现象，它具有上下两个阈值，在上下阈值之间原子发生振荡，在两阈值之外原子将不发生振荡.在两阈值之间原子的非简并双光子荧光谱呈现正峰结构，在两阈值之外双光子荧光谱具有负的峰结构.光谱形状强烈地依赖于双光子自然线宽，但光谱是非相敏性的，这是未曾预料到的. 关键词：     

Beam damage by the induced electric field in transmission electron microscopy

Electric fields can be induced by electron irradiation of insulating thin film materials. In this work, the electric fields under a broad beam illumination in transmission electron microscopy (TEM) are analyzed for insulating samples. Some damage phenomena observed can be interpreted by the mechanism of damage by the induced electric field (DIEF). For broad-beam illumination in an ultra-thin specimen, the electric field near the center of the illumination may not be strong, but at the periphery of the illumination the electric field can be significant. Therefore, damage may be easily observed in these regions rather than at the center of the illumination. For a beam which is broad compared to the specimen thickness, e.g. 100 ∼ 1000 nm, a strong electric field pointing inward into the specimen near the surface region may result in cation diffusion into the specimen and/or anion diffusion out to the surface region. Meanwhile, a strong electric field perpendicular to the beam direction near the edge of the illumination may attract anions into the illuminated region, but eject cations to the periphery. For a wedge-shaped specimen, the electric field points inward into thicker region, driving cations toward the thicker region, while attracting anions to the edge region. On the sharp edge, a strong electric field pointing outward may be responsible for the edge-smoothing effect observed in insulating materials.     

Lateral resolution improvement in two-photon excitation microscopy by aperture engineering

A technique for resolution improvement in two-photon excitation (2PE) fluorescence microscopy based on radially-symmetric annular binary filter (consist of central circular aperture and a concentric peripheral annulus) is proposed. Resolution improvement is achieved by engineering the aperture of the objective lens in a way so as to enhance high spatial frequencies. The structure of the electromagnetic field in the regions of focus and nearby regions are determined. The central lobe of the time-averaged electric energy density is considerably reduced for both linearly- and circularly-polarized illuminated light. An impressive combined comparative percentage improvement of 40% and 53.71% both at low (α = 30) and high (α = 60) aperture angle is obtained for linearly-polarized light. Proposed aperture engineering technique complements conventional, confocal, two-photon fluorescence microscopy, and may facilitate working at low-to-medium magnifications and large free-working distances.