Simultaneous Spatial and Temporal Focusing in Nonlinear Microscopy

Simultaneous spatial and temporal focusing (SSTF), when combined with nonlinear microscopy, can improve the axial excitation confinement of wide-field and line-scanning imaging. Because two-photon excited fluorescence depends inversely on the pulse width of the excitation beam, SSTF decreases the background excitation of the sample outside of the focal volume by broadening the pulse width everywhere but at the geometric focus of the objective lens. This review theoretically describes the beam propagation within the sample using Fresnel diffraction in the frequency domain, deriving an analytical expression for the pulse evolution. SSTF can scan the temporal focal plane axially by adjusting the GVD in the excitation beam path. We theoretically define the axial confinement for line-scanning SSTF imaging using a time-domain understanding and conclude that line-scanning SSTF is similar to the temporally-decorrelated multifocal multiphoton imaging technique. Recent experiments on the temporal focusing effect and its axial confinement, as well as the axial scanning of the temporal focus by tuning the GVD, are presented. We further discuss this technique for axial-scanning multiphoton fluorescence fiber probes without any moving parts at the distal end. The temporal focusing effect in SSTF essentially replaces the focusing of one spatial dimension in conventional wide-field and line-scanning imaging. Although the best axial confinement achieved by SSTF cannot surpass that of a regular point-scanning system, this trade-off between spatial and temporal focusing can provide significant advantages in applications such as high-speed imaging and remote axial scanning in an endoscopic fiber probe.     

Increasing the imaging depth of coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.     

光学微操纵过程的轴平面显微成像技术

光学俘获技术利用光与物质相互作用产生的光势阱效应来实现对微粒的操控,已经成功应用于生物医学、材料科学等交叉领域.在对微粒进行三维俘获时,传统的宽场光学显微技术只能观测到某一平面内微粒的横向运动,对微粒沿轴向运动的观测受到很大限制.本文将轴平面显微成像技术引入光学微粒操控研究中,利用45?倾斜的反射镜把微粒的轴向运动信息转换到横向平面进行观测,与传统宽场显微成像技术相结合,实现了对二氧化硅小球俘获过程横向和轴向运动的同步观测.该成像方法无需扫描和数据重构,具有实时快速等优点,在新型光束光镊、厚样品三维观测和成像等领域具有潜在的应用价值.     

In situ measurement of three-dimensional ion densities in focused femtosecond pulses

We image spatial distributions of Xeq+ ions in the focus of a laser beam of ultrashort, intense pulses in all three dimensions, with a resolution of approximately 3 microm and approximately 12 microm in the two transverse directions. This allows for studying ionization processes without spatially averaging ion yields. Our in situ ion imaging is also useful to analyze focal intensity profiles and to investigate the transverse modal purity of tightly focused beams of complex light. As an example, the intensity profile of a Hermite-Gaussian beam mode HG1,0 recorded with ions is found to be in good agreement with optical images.     

Extended depth of focus in tomographic phase microscopy using a propagation algorithm

Tomographic phase microscopy is a laser interferometry technique in which a 3D refractive index map of a biological sample is constructed from quantitative phase images collected at a set of illumination angles. Although the resulting tomographic images provide valuable information, their resolution declines at axial distances beyond about 1 microm from the focal plane. We describe an improved 3D reconstruction algorithm in which the field at the focal plane is numerically propagated to depths throughout the sample. Diffraction is thus incorporated, extending the depth of focus to more than 10 mum. Tomograms with improved focal depth are demonstrated for single HT29 cells.     

Improved in vivo photoacoustic microscopy based on a virtual-detector concept

Recently an in vivo high-resolution backward-mode photoacoustic microscope was developed that shows potential for applications in dermatology and related cancer research. However, the limited depth of focus of the large-numerical-aperture (NA) ultrasonic lens employed in this system causes the image quality to deteriorate significantly in the out-of-focus region. To solve this problem, we devised and explored, for the first time to our knowledge, a virtual-detector-based synthetic-aperture focusing technique, combined with coherence weighting, for photoacoustic microscopy with such a large-NA transducer. Images of phantoms show that the proposed technique improves the -6 dB lateral resolution from 49-379 to 46-53 microm and increases the signal-to-noise ratio by up to 29 dB, depending on the distance from the ultrasonic focal point. In vivo experiments show that the technique also provides a clearer representation of the vascular distribution in the rat's scalp.     

Beam-quality measurement of a focused high-order harmonic beam

Using a multilayer spherical mirror, we focus the high-order harmonic radiation produced near 55 nm by the nonlinear interaction of an intense femtosecond laser pulse and a xenon gas jet. The focused XUV beam is characterized by a knife-edge technique in the focal region and by far-field imaging. We show that good-quality beams, nearly two times diffraction limited, can be generated, a conclusion that is at variance with recent predictions of harmonic phase-front distortion. Spot sizes close to 10 microm are obtained, resulting in a high XUV intensity. Increasing the gas density and the length of the generating medium results in a large increase in the divergence in and degradation of the beam quality.     

Optical tweezers for confocal microscopy

In confocal laser scanning microscopes (CLSMs), lasers can be used for image formation as well as tools for the manipulation of microscopic objects. In the latter case, in addition to the imaging lasers, the light of an extra laser has to be focused into the object plane of the CLSM, for example as optical tweezers. Imaging as well as trapping by optical tweezers can be done using the same objective lens. In this case, z-sectioning for 3D imaging shifts the optical tweezers with the focal plane of the objective along the optical axis, so that a trapped object remains positioned in the focal plane. Consequently, 3D imaging of trapped objects is impossible without further measures. We present an experimental set-up keeping the axial trapping position of the optical tweezers at its intended position whilst the focal plane can be axially shifted over a distance of about 15 μm. It is based on fast-moving correctional optics synchronized with the objective movement. First examples of application are the 3D imaging of chloroplasts of Elodea densa (Canadian waterweed) in a vigorous cytoplasmic streaming and the displacement of zymogen granules in pancreatic cancer cells (AR42 J). Received: 24 March 2000 / Revised version: 23 June 2000 / Published online: 11 October 2000     

Automatic alignment of a Kirkpatrick-Baez active optic by use of a soft-x-ray Hartmann wavefront sensor

We present what we believe to be the first automatic alignment of a synchrotron beamline by the Hartmann technique. Experiments were performed, in the soft-x-ray range (E=3 keV, lambda=0.414 nm), by using a four-actuator Kirkpatrick-Baez (KB) active optic. A system imaging the KB focal spot and a soft-x-ray Hartmann wavefront sensor were used alternatively to control the KB optic. The beam corrected with the help of the imaging system was used to calibrate the wavefront sensor. With both closed loops, we focused the beam into a 6.8 microm x 9 microm FWHM focal spot.     

Molecularly sensitive optical coherence tomography

Molecular contrast in optical coherence tomography (OCT) is demonstrated by use of coherent anti-Stokes Raman scattering (CARS) for molecular sensitivity. Femtosecond laser pulses are focused into a sample by use of a low-numerical-aperture lens to generate CARS photons, and the backreflected CARS signal is interferometrically measured. With the chemical selectivity provided by CARS and the advanced imaging capabilities of OCT, this technique may be useful for molecular contrast imaging in biological tissues. CARS can be generated and interferometrically measured over at least 600 microm of the depth of field of a low-numerical-aperture objective.     

Automatic compensation of laser beam focusing parameters for flying optics

Flying optics technologies are used for a range of applications such as a large workpiece processing and laser texturing. It is essential to compensate the variations of laser beam focus parameters while the focus head is moving. A flying optics automatic compensation approach is proposed to achieve invariable laser focal size, focus depth and focus position through computer controlled objective lens and focus lens position. A simple mechanical control method is also presented for the realization of constant beam parameters for flying optics. Numerical simulation is illustrated for a CO₂ laser texturing application. The flying optics parameters compensation is simple and easy to control.     

Doppler optical coherence tomography with a micro-electro-mechanical membrane mirror for high-speed dynamic focus tracking

An elliptical microelectromechanical system (MEMS) membrane mirror is electrostatically actuated to dynamically adjust the optical beam focus and track the axial scanning of the coherence gate in a Doppler optical coherence tomography (DOCT) system at 8 kHz. The MEMS mirror is designed to maintain a constant numerical aperture of approximately 0.13 and a spot size of approximately 6.7 microm over an imaging depth of 1mm in water, which improves imaging performance in resolving microspheres in gel samples and Doppler shift estimation precision in a flow phantom. The mirror's small size (1.4 mm x 1 mm) will allow integration with endoscopic MEMS-DOCT for in vivo applications.     

A brief review on mass/optical spectrometry for imaging analysis of biological samples

Imaging analysis, especially bioimaging analysis, has been a hot research topic in recent years. There are numerous imaging analysis techniques for diverse applications of a wide spectrum of samples, with their unique advantages and disadvantages, and there are several related reviews published yearly. Among them, imaging mass spectrometry (IMS) is a relatively novel analytical technique for studying the distribution of molecular or ionic species at the level of tissue, cell, or subcellular, with its main feature of combining mass spectra for molecular identification and image visualization for quick and convenient analysis. The IMS does not require chemical labeling or complex sample preparation. This review, therefore, mainly focuses on the popular emerging IMS technique, including related ionization techniques in connection with their IMS applications, and some unique optical imaging techniques such as chemiluminescence imaging and dual-modal bioimaging for biological sample analysis, with 105 related recent references.     

Generation of tightly focused twin Bessel beams using circular Dammann gratings under radial polarization incidence

Generation of longitudinally polarized focusing twin Bessel beams in focal region of a high numerical aperture (NA) objective is described based on circular Dammann gratings for radially polarized Bessel–Gauss input fields. Numerical simulations show that, under focusing of an objective of NA=0.95, the depth of focus (DOF) of the focused twin Bessel beams can reach as long as tens or even ～10² of wavelengths while its average transverse spot over the whole range of the DOF is kept subdiffration-limited. At the same time, the longitudinal polarization purity in focus volume is higher than 90% for the central lobe. Therefore, this tightly focused non-diffracting field should be of great interest for applications in numerous areas, such as particle acceleration and manipulation, micromachining, second-harmonic generation, Raman spectroscopy, etc.     

Tight focusing of a double-ring-shaped, azimuthally polarized beam

We study the focusing properties of a double-ring-shaped azimuthally polarized beam through an annular high NA objective lens. It is shown that a subwavelength focal hole (～0.5λ) with a quite long depth of focus (～26λ) is achieved near the focus. This kind of nondiffracting focal hole is called dark channel, which may have applications in atom optical experiments, such as with atomic lenses, atom traps, and atom switches.     

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.     

White light micrograting multiplexing for high density data storage

Recording of multiplexed microholographic gratings with improved recording volume for high density optical data storage is proposed and demonstrated. By using a hybrid diffractive-refractive objective lens with extended depth of focus, we have achieved a recording beam size of approximately 1 microm and a focal depth of 20 microm. Multiple gratings corresponding to spectral lines within the 400-650 nm spectral band have been successfully multiplexed in a single recording spot or pit of size 1.25 microm on a DuPont photopolymer film using a white light source along with narrowband filters or dispersion elements, thus demonstrating the storage of multiple bits in a single pit. Simultaneous readout of multiple bits in a single storage pit is accomplished with a microspectrometer-type readout head using a white light source.     

基于相位展开及拼接算法的一种高精度大量程宽光谱干涉显微术

宽光谱干涉显微术广泛应用于高精密检测领域,它测量样品形貌通常采用垂直扫描干涉术对亚微米至毫米级特征进行测量,以及相移干涉术对纳米级特征进行测量。其中,相移干涉术精度可达纳米级,但量程有限,高度变化对应的相位需限制在区间内。采用包裹相位展开算法可以扩展相移干涉术的量程,也仅适用于平滑表面,当高度起伏超出焦深或者光源相干长度的限定范围时,干涉条纹模糊或对比度丧失,所解算的结果将产生较大误差甚至错误。提出一种基于相位展开及拼接算法的高精度、大量程宽光谱干涉显微测量方法,以干涉条纹调制度量化条纹质量,条纹对比度高、成像清晰的区域对应调制度较高,定义当前焦面条纹调制度高于阈值的区域为理想区域,定义焦面条纹调制度低于阈值的区域为问题区域。以相位展开算法获得理想区域中的样品相位分布,问题区域的包裹相位不进行展开。使用微位移结构纵向移动物镜焦平面,选择合理的步长,使相邻焦面位置理想区域展开后的真实相位保持部分区域重合,根据重合区域的相位值均差可以实现不同焦面位置的高精度相位拼接,最终获得扩展量程的高精度真实相位结果,进而可以恢复样品完整的表面形貌分布。该算法通过对理想区域的筛选,避免了相位在问题区域展...     

双焦距立体视觉中的光学成像模型

成像物镜具有两个独立的焦距,并分别对空间物体成像,这种系统被称为双焦成像系统。双焦成像系统从单一角度记录三维场景中各物点的图像,它构成了单目立体视觉的基本光学成像模型。由于焦距为f1和f2的物镜所成的像的矢量值存在差别,即视差,并且视差的大小与物点的深度存在着定量关系,因此可利用双焦成像的视差特征来恢复场景的深度信息。叙述了双焦成像的立体视觉原理和系统的改进方案,并根据双焦成像的深度算法对已知深度的物方平面进行了深度测量。试验结果表明,深度恢复的相对误差约为-0.14%,测量结果的方差为0.97mm。     

Strong two-photon absorption at telecommunications wavelengths in nickel bis(dithiolene) complexes

The two-photon absorption spectrum of a nickel bis(dithiolene) complex with extended conjugation and pi-donor substitution is measured by using Z-scan and pump-probe techniques with femtosecond pulses over the spectral range from 1.20 to 1.58 microm, which includes much of the telecommunications range. The peak two-photon cross section of over 5000 GM (1 GM = 10(-50) cm4 s photon(-1) molecule(-1)) occurs at approximately 1.24 microm, with significant two-photon absorption (>440 GM) throughout the spectral range examined.