飞秒激光声光扫描双光子显微镜的理论研究进展

本刊讯 现有的商品化双光子显微镜受限于其较低的成像速度（数帧／秒）,而不能应用于观测亚毫秒以内的快速生物网络动态活动。采用声光偏转器对飞秒激光进行随机扫描,可将随机扫描速度提高至10微秒／像素,在此基础上建立的快速随机扫描双光子显微成像装置,可实现kHz量级的成像速度,并可进一步提高。然而,用声光偏转器扫描飞秒激光存在严重的色散效应。其时间色散使得脉冲展宽,     

衍射透镜补偿声光偏转器扫描飞秒激光的色散

在飞秒激光随机扫描双光子显微成像系统中使用宽带二维声光偏转器扫描飞秒激光,可以增大扫描角度至74 mrad,增大双光子显微成像范围。但宽带二维声光偏转器在大角度扫描时引入的色散较大,造成成像范围边缘的光斑严重畸变,边缘光斑直径达2.3 μm,影响边缘视场的成像质量。为了提高成像质量,设计了一种新的色散补偿方法,基于衍射透镜组成的开普勒望远系统,可以同时补偿不同扫描角度的不同色散。经过色散补偿后成像边缘的光斑直径小于1 μm,使系统获得大范围扫描成像的同时,所有扫描角度的色散都能够得到很好的补偿,在整个视场范围内光斑直径小于1 μm,实现更均匀的荧光激发,均匀成像。     

基于电动可调焦透镜的大范围快速光片显微成像

搭建了一种基于电动可调焦透镜(electrically tunable lens)的大范围快速光片荧光显微成像系统.通过引入电动可调焦透镜与一维振镜以实现成像物平面和光片位置的快速移动,再结合高速s CMOS完成快速光片荧光显微成像.另外实验中通过改善光路与提升动态成像质量,实现了大范围扫描并减少了伪像.最终对成像性能进行测试,本系统的纵向分辨率和横向分辨率分别达到约5.5μm和约0.7μm,单幅图像稳定成像的速度约为275 frames/s,成像深度可超过138μm,能满足对具有一定尺寸的生物样本进行实时清晰成像的需求.     

用于双光子激发荧光寿命显微成像的高重复频率皮秒扫描相机

建立了一台基于新研制的高重复频率皮秒扫描相机的双光子激发荧光寿命显微成像系统,重点介绍所研制的高重复频率皮秒扫描相机。为了在高时间分辨力的同时扩大时间测量范围,实现大面积两维空间高时间分辨取样测量,从而提高采样速率和更有效地发挥扫描相机的作用,设计和研制了一种大面积、高时间分辨力扫描变像管和一种重复频率高达1MHz的斜坡电压扫描电路。基于上述关键部件所研制的扫描相机具有重复频率高、扫描速度可调、时间分辨力高、工作面积大、非线性低、触发晃动小等优点。用钛宝石飞秒激光器作为激光脉冲源,通过脉冲提取器将76MHz的高重复频率降低为1MHz,采用可调延时器和标准具对扫描相机的时间分辨力、扫描速度和非线性进行标定。该系统的时间分辨力达到6.5ps,非线性为2.60%,可测量的时间范围从十几皮秒到几十纳秒。测量了若丹明6G和香豆素314两种标准荧光染料的荧光寿命,取得了与参考文献一致的实验结果。     

共焦扫描荧光显微镜的信噪比与测量精度

激光扫描共焦显微术和多光子显微术等新的显微成像技术可以对厚的生物样品实现光学断层成像 ,因而在生物医学诊断领域具有重要的应用前境。在Fried的一维分辨度理论的基础上 ,系统地讨论了运用共焦扫描荧光显微术在进行光学断层成像时 ,其光学断层平面分辨度与信噪比之间的定量关系 ,建立了实际显微成像系统平面测量精度的定量计算方法。所得出的结果对于选择共焦扫描显微成像系统的最佳参数及评价所设计的显微成像系统的性能具有重要的意义。     

混浊介质多光子激发显微成像的快速模型

将二维点扩散函数（PSF）和蒙特卡罗方法相结合,引入了一种研究混浊介质显微成像的快速仿真模型,将该模型用于混浊介质的多光子激发（MPE）显微成像研究,极大地提高了模拟效率,与直接蒙特卡罗方法计算结果的比较证实了该方法的正确性和有效性,此外,一个计算实例说明了混混介质的多光子激发显微成像具有比共焦荧光显微成像更优的横向分辨率。     

基于液体变焦透镜和振镜的三维光片显微成像系统

搭建了一种基于液体变焦透镜和振镜的三维光片显微成像系统,设计了振镜、液体变焦透镜、相机的同步控制采集成像系统,通过调谐振镜和液体变焦透镜,使得光片激发样品和成像同步,获得样品不同切面的图像堆栈并实现样品的三维重建。当采用数值孔径为0.3、放大倍率为10的成像物镜时,该系统的轴向扫描范围为507μm,横向视场达到1970μm×1300μm,横向分辨率为1.32μm,轴向分辨率可达12.75μm。在轴向扫描过程中,系统的放大倍率保持恒定,可以用于对一定尺寸生物样品的成像实验和相关研究,并通过对斑马鱼胚胎进行成像验证所提系统对厚生物样品成像的可行性。     

微扫描显微热成像系统的降采样高分辨力图像重建算法

提出了一种基于降采样模型的显微热图像高分辨力重建算法,该算法通过与三次样条插值放大得到的像素点相比较以补偿微扫描产生的系统误差。仿真和实验结果表明,提出的方法减少了由于微扫描误差所带来的图像重建质量下降,提高了光学微扫描显微热成像系统的空间分辨力,具有较强的实用价值。提出的方法还可以应用到其他光电成像系统中以提高系统空间分辨力。     

基于声光可调谐滤光器的显微光谱成像技术

为了解决传统声光可调谐滤光器(AOTF)成像模糊的缺点,设计了一种新的AOTF。该器件通过在传统的AOTF的出射孔后面放置一个自行设计的等边色散棱镜来实现对衍射光的色散展宽进行补偿,明显地提高了成像的对比度和空间分辨率。将此器件附加在传统光学显微镜上,获得了一种新型的光谱显微成像仪器。其光谱分辨率在575nm波长处为4.2nm、成像空间分辨率为2μm、图像采集速度为毫秒量级。为基于AOTF的光谱成像技术在生物医学等领域的更广泛的应用奠定了基础。     

硅基半导体芯片激光红外显微成像系统研究

为了解决可见光显微成像技术无法实现硅基芯片内部结构观测的问题,根据1 064nm的红外激光对硅材料具有一定穿透深度的特性,设计了一种硅基半导体芯片激光红外显微成像系统.该系统采用数值孔径为0.42的长工作距显微物镜,通过音圈电机振动多模石英光纤消除散斑噪音,由系统观察CD-RW盘片道间距,表明系统分辨率可达到1.6μm,接近理论值,实现了对芯片厚度为70μm的静态随机存储器内部结构显微成像的观测.     

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 μm × 136 μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.     

Compensation-free, all-fiber-optic, two-photon endomicroscopy at 1.55 μm

We present an all-fiber-optic scanning multiphoton endomicroscope with 1.55 μm excitation without the need for prechirping femtosecond pulses before the endomicroscope. The system consists of a 1.55 μm femtosecond fiber laser, a customized double-clad fiber for light delivery and fluorescence collection, and a piezoelectric scan head. We demonstrate two-photon imaging of cultured cells and mouse tissue, both labeled with indocyanine green. Free-space multiphoton imaging with near-IR emission has previously shown benefits in reduced background fluorescence and lower attenuation for the fluorescence emission. For fiber-optic multiphoton imaging there is the additional advantage of using the soliton effect at the telecommunication wavelengths (1.3-1.6 μm) in fibers, permitting dispersion-compensation-free, small-footprint systems. We expect these advantages will help transition multiphoton endomicroscopy to the clinic.     

时域两维荧光寿命显微测量研究

提出了一种新的时域两维荧光寿命显微测量技术,建立了一套荧光寿命成像显微系统,介绍了这种测量技术的数据处理方法。用标准样品对该系统进行了测试,实验表明,该系统的时间分辨率为2ps,在放大倍率为100倍的情况下,该系统的空间分辨率为8um。如果在现有的设备下采用更细的网格板和微位移系统,那么该系统的空间分辨率可小于1um.     

Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy

We investigate a novel concept to efficiently generate multiphoton induced fluorescence from organic molecules. The concept is based on frustrating the energy transfer between a fluorescent donor and one or more acceptors in conjugated molecules. The nonlinearity is not based on higher order molecular susceptibilities but entirely on their linear properties. Therefore, in contrast to nonresonant multiphoton absorption, this method does not require high local intensities. Likewise, the production of visible fluorescence does not require an infrared excitation wavelength. Hence, when applied to scanning microscopy this property is predicted to increase spatial resolution. Instead of the ∼10 GW/cm² required in non-resonant multiphoton excitation, focal intensities ∼10 MW/cm² are expected to produce an equally strong nonlinear signal. The predicted resolution is up to 30% greater than that of an ideal confocal microscope operating at the same fluorescence wavelength. The resolution improvement over non-resonant two-photon absorption microscopes is about two-fold in all directions.     

Improving signal levels in intravital multiphoton microscopy using an objective correction collar

Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point-spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreases the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.     

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.     

Multifocal multiphoton microscopy

We present a real-time, direct-view multiphoton excitation fluorescence microscope that provides three-dimensional imaging at high resolution. Using a rotating microlens disk, we split the near-infrared light of a mode-locked titanium-sapphire laser into an array of beams that are transformed into an array of high-aperture foci at the object. We typically scan at 225 frames per second and image the fluorescence with a camera that reads out the images at video rate. For 1.4 aperture oil and 1.2 water immersion lenses at 780-nm excitation we obtained axial resolutions of 0.84 and 1.4mum , respectively, which are similar to that of a single-beam two-photon microscope. Compared with the latter setup, our system represents a 40-100-fold increase in efficiency, or imaging speed. Moreover, it permits the observation with the eye of high-resolution two-photon images of (live) samples.     

Resolution improvement by nonconfocal theta microscopy

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.     

Multiphoton dynamics of qutrits in the ultrastrong coupling regime with a quantized photonic field

Multiphoton resonant excitation of a three-state quantum system (a qutrit) with a single-mode photonic field is considered in the ultrastrong coupling regime, when the qutrit–photonic field coupling rate is comparable to appreciable fractions of the photon frequency. For ultrastrong couplings, the obtained solutions of the Schrödinger equation that reveal multiphoton Rabi oscillations in qutrits with the interference effects leading to the collapse and revival of atomic excitation probabilities at the direct multiphoton resonant transitions.     

Multiphoton pi pulses

Multiphoton excitation in a two-level system is exceedingly weak because of small multiphoton coupling strengths, large ac Stark shifts, and ionization. I will discuss a three-level system in which the ac Stark shift is greatly reduced and the multiphoton coupling strength is greatly enhanced over a two-level system, to such an extent that multiphoton pi pulses can be produced. I will also present two-electron calculations in a model potential, including ionization that shows a 12-photon pi pulse driven with 800-nm photons. This three-level configuration may provide the basis for an amplifying medium in the vacuum ultraviolet, as well as multiphoton adiabatic passage and innershell ionization.