High-speed high-resolution laser diode-based photoacoustic microscopy for in vivo microvasculature imaging

Laser diodes(LDs)have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy(PAM).However,the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously.In this paper,we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD,operating at a pulsed mode,with a repetition rate of 30 kHz,as an excitation source.A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio.By optimizing the optical system,a high lateral resolution of 4.8μm has been achieved.In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.

Low-cost photoacoustic imaging systems based on laser diode and light-emitting diode excitation

Photoacoustic imaging,an emerging biomedical imaging modality,holds great promise for preclinical and clinical researches.It combines the high optical contrast and high ultrasound resolution by converting laser excitation into ultrasonic emission.In order to generate photoacoustic signal e±-ciently,bulky Q-switched solid-state laser systems are most commonly used as excitation sources and hence limit its commercialization.As an alternative,the miniaturized semiconductor laser system has the advantages of being inexpensive,compact,and robust,which makes a signi¯cant e®ect on production-forming design.It is also desirable to obtain a wavelength in a wide range from visible to nearinfrared spectrum for multispectral applications.Focussing on practical aspect,this paper reviews the state-of-the-art developments of low-cost photoacoustic system with laser diode and light-emitting diode excitation source and highlights a few representative installations in the past decade.

Fiber laser technologies for photoacoustic microscopy

Fiber laser technology has experienced a rapid growth over the past decade owing to increased applications in precision measurement and optical testing,medical care,and industrial applications,including laser welding,cleaning,and manufacturing.A fiber laser can output laser pulses with high energy,a high repetition rate,a controllable wavelength,low noise,and good beam quality,making it applicable in photoacoustic imaging.Herein,recent developments in fiber-laser-based photoacoustic microscopy(PAM)are reviewed.Multispectral PAM can be used to image oxygen saturation or lipid-rich biological tissues by applying a Q-switched fiber laser,a stimulated Raman scattering-based laser source,or a fiber-based supercontinuum source for photoacoustic excitation.PAM can also incorporate a single-mode fiber laser cavity as a high-sensitivity ultrasound sensor by measuring the acoustically induced lasing-frequency shift.Because of their small size and high flexibility,compact head-mounted,wearable,or hand-held imaging modalities and better photoacoustic endoscopes can be enabled using fiber-laser-based PAM.

Ultrafast dynamics observation during femtosecond laser-material interaction

Femtosecond laser technology has attracted significant attention from the viewpoints of fundamental and application;especially femtosecond laser processing materials present the unique mechanism of laser-material interaction.Under the extreme nonequilibrium conditions imposed by femtosecond laser irradiation,many fundamental questions concerning the physical origin of the material removal process remain unanswered.In this review,cutting-edge ultrafast dynamic observation techniques for investigating the fundamental questions,including timeresolved pump-probe shadowgraphy,ultrafast continuous optical imaging,and four-dimensional ultrafast scanning electron microscopy,are comprehensively surveyed.Each technique is described in depth,beginning with its basic principle,followed by a description of its representative applications in laser-material interaction and its strengths and limitations.The consideration of temporal and spatial resolutions and panoramic measurement at different scales are two major challenges.Hence,the prospects for technical advancement in this field are discussed finally.

Advances in Probing Wood-Coating Interface by Microscopy: A Review

Surface coatings provide protection to wood products against weathering and other deteriorating factors, such as moisture uptake and microbial invasion. The effectiveness of coatings depends on many factors, including how well the applied coatings adhere to the wood surface. Coating adhesion to wood involves both chemical and physical interactions between the coating and wood tissues in contact, and the particular focus of this mini-review will be on the advances being made in understanding the physical aspects of the interaction by probing wood-coating interface using novel and high resolution imaging techniques, including confocal laser scanning microscopy (CLSM), SEM-backscattered electron imaging and correlative microscopy employing light, confocal and scanning electron microscopy.

Direct imaging of the near field and dynamics of surface plasmon resonance on gold nanostructures using photoemission electron microscopy

Localized surface plasmon resonance(LSPR)can be supported by metallic nanoparticles and engineered nanostructures.An understanding of the spatially resolved near-field properties and dynamics of LSPR is important,but remains experimentally challenging.We report experimental studies toward this aim using photoemission electron microscopy(PEEM)with high spatial resolution of sub-10 nm.Various engineered gold nanostructure arrays(such as rods,nanodisk-like particles and dimers)are investigated via PEEM using near-infrared(NIR)femtosecond laser pulses as the excitation source.When the LSPR wavelengths overlap the spectrum of the femtosecond pulses,the LSPR is efficiently excited and promotes multiphoton photoemission,which is correlated with the local intensity of the metallic nanoparticles in the near field.Thus,the local field distribution of the LSPR on different Au nanostructures can be directly explored and discussed using the PEEM images.In addition,the dynamics of the LSPR is studied by combining interferometric time-resolved pump-probe technique and PEEM.Detailed information on the oscillation and dephasing of the LSPR field can be obtained.The results identify PEEM as a powerful tool for accessing the near-field mapping and dynamic properties of plasmonic nanostructures.

Molecular vibrational imaging by stimulated Raman scattering microscopy: principles and applications [Invited]

The recent development of stimulated Raman scattering(SRS) microscopy allows for highly sensitive biological imaging with molecular vibrational contrast, opening up a variety of applications including label-free imaging,metabolic imaging, and super-multiplex imaging. This paper introduces the principle of SRS microscopy and the methods of multicolor SRS imaging and describes an overview of biomedical applications.

Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser

Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning.However,most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach.In this report,we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm,which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs.The two excitation wavelengths of 1055 and 1240 nm,along with the effective two-color excitation wavelength of 1140 nm,provide an almost complete coverage of fluorophores excitable within the range of 1000–1300 nm.When compared with 1055 nm excitation,two-color excitation at 1140 nm offers a 90%increase in signal for many far-red emitting fluorescent proteins(for example,tdKatushka2).We demonstrate multicolor imaging of tdKatushka2 and Hoechst 33342 via simultaneous two-color two-photon,and two-color three-photon microscopy in engineered 3D multicellular spheroids.We further discuss potential benefits and applications for two-color three-photon excitation.In addition,we show that this laser system is capable of in vivo imaging in mouse cortex to nearly 1 mm in depth with two-color excitation.

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分.采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400～ 700 nm）内的光谱带宽,其最小光谱带宽优于5 nm.用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm.在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor（R） 488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分.实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围.

激光扫描共聚焦显微镜的基本原理及其使用技巧

介绍了激光扫描共聚焦显微镜(laser scanning confocal microscopy,LSCM)的原理和构造,分析了LSCM的优点和缺点,并以花粉为例,详细介绍了共聚焦针孔直径、光电倍增管检测器增益、激光强度、扫描速度、扫描方式和Z轴步距等重要参数设置对共聚焦成像的不同影响。探讨了正确使用LSCM的方法与技巧,如获取高质量的图像、图像保存及图像处理,以便为科技人员利用LSCM开展更多植物学与环境科学相关的研究提供参考。

蔡司LSM 7DUO NLO激光共聚焦显微镜的应用和管理

介绍了蔡司LSM7DUO NLO激光扫描共聚焦显微镜三个组成部分单光子、双光子和快速扫描的硬件配置和软件功能以及成像和图像分析的应用,例如单、双光子图像叠加,多视野拼接拍摄、三维重构等功能在生命科学研究中的应用。介绍管理该仪器的一些方法和心得,保障仪器为教学和科研工作提供更好的服务。

ZEISS LSM 780激光扫描共聚焦显微镜的三维成像及分析

针对ZEISS LSM 780激光扫描共聚焦显微镜的三维图像获取步骤做了系统的介绍,详述了光学切片的设置和获取过程,后期应用Zen2011 black和blue软件进行三维重构的渲染及图像分析、数据采集。列举出三维成像中常见问题及解决办法,使仪器为教学和科研工作提供更优质的服务。

多孔介质微观孔隙结构三维成像技术

详细介绍目前国际上最先进的多孔介质微观结构三维成像技术,包括系列切片技术、聚焦离子束技术(FIB)、激光共聚焦扫描显微镜、Micro-CT技术,分析了各自的优越性及局限性,探讨了Micro-CT技术的应用现状及其在地质学领域的应用前景。分析结果表明,Micro-CT及今后的Nano-CT技术是一种无损伤的3D成像技术,不但可获得足够分辨率的多孔介质微观孔隙结构3D图像,而且还可以现场实时检测孔隙中流体的渗流状态,在多孔介质孔隙结构及渗流机理研究中具有重要的应用价值。该技术必定成为今后的主流技术,不仅将在石油工程、水文工程等领域发挥重要的作用,而且还将广泛应用于岩石学、矿物学、古生物学、材料学、资源科学及环境科学等领域。该技术的研究和应用在我国还处在起步阶段,应加大设备引进和研究力度。

激光扫描共聚焦显微镜实验技术与应用

介绍了激光扫描共聚焦显微镜的工作原理、常规样品制备要求,以及图像获取的基本方法,包括光路设置及光强度调节、针孔的调节、增益值的调节、透射光路及DIC设置等.在此基础上,进一步介绍了共聚焦在生物学研究领域上的应用技巧,如多通道荧光采集、多层扫描及三维构建、荧光共定位及强度分析、时间序列扫描及光漂白技术,为快速掌握共聚焦的操作技巧提供有力的技术参考.

CLSM技术在纤维表面形态和纸张结构研究中的应用

采用共聚焦激光扫描显微镜(CLSM),可以在不损伤样品的前提下获得样品的三维图像,具有较高的横向(XY向)分辨率和信噪比。综述了CLSM的工作原理,重点讨论了其在纤维表面分析(纤维形态和表面的木素)、纤维形变性研究(柔韧性和压溃性能)以及纸张结构观察(表面结构和内部结构)中的应用。

大肠杆菌长时间观察的方法

细菌个体微小,在液体中运动性强,且有的细菌有鞭毛、菌毛等结构,增加其运动能力,因此对感兴趣的细菌长时间观察很有难度。本文采用激光扫描共聚焦显微镜,比较多聚赖氨酸或低熔点琼脂糖处理后,大肠杆菌长时间观察的效果。结果发现在多聚赖氨酸包被的培养皿上加入含1.5%低熔点琼脂糖的LB培养基、室温培养或者在多聚赖氨酸包被的培养皿上加入含6%低熔点琼脂糖的LB培养基37℃培养,大肠杆菌均可在聚焦面贴壁分裂生长。连续观察2 h以上大肠杆菌没有漂离视野,实现了显微镜下清晰呈现大肠杆菌分裂生长过程的目的。

金纳米棒标记HepG2人肝癌细胞的荧光成像及其AFM探测

金纳米棒具有独特的光学性质,在生物医学领域有着广泛而重要的应用前景。本文制备了长径比为8:1的金纳米棒,其在480 nm波长激发下,在560 nm和707 nm波长处有两个荧光发射峰。基于金纳米棒的荧光性质,将其标记于HepG2人肝癌细胞表面,利用激光扫描共聚焦显微镜对标记后的细胞进行荧光成像。在488 nm激发下,获得了绿色和红色两种颜色的荧光图像,并进一步用高分辨率的原子力显微镜探测了GNRs标记后细胞形貌及机械性质的变化。

计算机三维物体实体组建方法的研究

在计算机绘图和三维图像显示,例如激光扫描共焦显微图像、CT图像、MRI图像等的绘制和显示中,三维物体的组建是必不可少的。目前,三维物体的组建都是基于二维物体组建的基础之上。二维物体组建是基于连通性进行二维连通区域检出的,因而三维物体的组建速度比较慢。为此,文章提出一种新方法:围线积分法区域标号和链接表法三维叠片,用于三维图像中三维物体实体的组建。这种方法的基本原理是在三维二值图像中,先采用围线积分法区域标号,组建二维物体切片,再采用链接表法三维叠片,由二维物体切片组建三维实体。这种方法的优点是组建速度比较快。

油炸藕片含油量快速预测及微观结构的三维重建

以油炸藕片为研究对象,首先利用高光谱图像技术(hyperspectral imaging technology,HSIT)结合联合区间偏最小二乘(synergy interval partial least squares,si PLS)和净分析物法(net analyze signal,NAS)提取油炸藕片的光谱信息,建立快速预测模型,并计算每个像素点的油含量值,将其表面油分布可视化;然后利用激光共聚焦显微镜(confocal laser scanning microscopy,CLSM)观察油在细胞中的分布,并扫描藕片Z轴方向序列图片进行微观结构的三维重建。结果显示,优选4个子区间,依据NAS法挖掘同油相关的NAS信号NAsk,建模后其预测集相关系数和均方根误差分别为0.819和0.682 mg/g,表明HSIT可快速预测油炸藕片含油量并可视化表面油分布。经CLSM扫描并图像三维重建后观察,油主要分布在细胞表面、边缘、细胞间隙以及破碎细胞内,且细胞的形状、大小均发生了很大的变化,可观察其内部油分布。结果表明,该研究可为油分布提供有效手段,并依据微观结构的变化为减少含油量提供科学的理论依据。

豚鼠耳蜗内毛细胞、外毛细胞及外指细胞钙成像

用ｆｌｕｏ－３／ｆｕｒａ－ｒｅｄ荧光比值作细胞内游离钙指标，用激光扫描共聚焦显微镜测定豚鼠耳蜗分离的内、外毛细胞及外指细胞的游离钙，并利用钙成像技术进行细胞形态计量。单个外毛细胞呈试管状。内毛细胞呈烧瓶状，有明显的颈部。外指细胞分为体部及指状突部。毛细胞的胞内游离钙浓度明显高于外指细胞。毛细胞的胞质、胞核及表皮板的游离钙浓度不同。外指细胞的细胞体与指状突的游离钙浓度也不同。本研究认为，有无颈部为区别分离的内、外毛细胞的重要标准，外指细胞可凭借其特异的指状突结构以资识别。内耳毛细胞及外指细胞胞内游离钙分布的不均一性可能与各部位生理功能不同有关。