小型化望远式激光共焦扫描显微内窥镜

研制了一种激光共焦扫描显微内窥镜,采用望远式显微内窥光学系统,同时实现长距离的图像中继传输、远心f-theta光学扫描和显微内窥成像功能.二维共焦扫描由双振镜实现,低噪音扫描控制信号由嵌入式系统产生.为实现便携式应用,激光共焦扫描显微内窥镜采用小型化设计方案.首先,体内的显微内窥成像光学系统,外径尺寸为8 mm,工作长度为250.3 mm,可通过标准腹腔镜手术孔进行体内显微内窥成像;其次,采用3 mm通光孔径的小尺寸平面反射镜实现体外共焦扫描,摆动频率为100 Hz,实现快速共焦扫描;最后,激光控制和荧光探测仅通过电缆和光纤与共焦扫描显微内窥镜前端连接,减小了显微内窥镜的前端尺寸和重量.通过实验验证,本系统的成像视场为φ 600 μm,光学分辨率为2.2 μm,可采用手持式或者其他方式工作,进行体内组织的共焦扫描成像,实现微创、在体的荧光显微内窥术.     

An integrated phase-resolved fluorescence imaging system with sub-nanosecond lifetime resolution

In this paper, a novel configuration of an integrated phase-resolved (PR) fingerprint fluorescence imaging system is proposed and implemented. In this integrated PR imaging system, a current modulated 402 nm dual diode laser is proposed to be the light source, to obtain both high laser power and easy modulability. To estimate the lifetime resolution of this PR imaging system, a novel method of using distance-selective suppression of fluorescence signals from two identical fluorescing samples is proposed. Detailed theoretical and experimental analyses are presented. The experimental results demonstrate that this integrated PR imaging system has a lifetime resolution of 0.1 ns. Fingerprint detection experiments are also carried out using this system with latent fingerprints deposited on substrates of aluminum foil and currency.     

基于激光共焦扫描术的亚心形扁藻显微图像与光谱

采用激光共聚焦扫描显微技术,针对亚心形扁藻开展了研究.从获得的488 nm Ar+激光单光子激发的亚心形扁藻自体荧光光谱与图像,可知细胞内有一杯状叶绿体物质,其荧光峰值为682 nm,对应叶绿体发出的红色荧光.在单通道模式下,获得800 nm fs激光双光子激发的扁藻自体荧光光谱与图像,可知每个杯状叶绿体的内部有一个自体荧光更强的圆形物质.在双通道模式下,可分别获得小圆形物质的自体荧光图像,杯状叶绿体自体荧光图像,以及两个通道图像的叠加.进一步获得了双光子藻细胞荧光图的6个主要的荧光峰.采用单光子激光激发可获得亚心形扁藻叶绿体自体荧光图像及其荧光光谱,而双光子激光激发荧光光谱的多通道以及Lambda模式下采集光谱信号与图像,不仅可观察到亚心形扁藻的内部形态结构,还可能从双光子激发荧光图中研究分析亚心形扁藻生化物质的存在,灵敏度较单光子激发高.激光扫描共聚焦显微技术,特别是双光子荧光与图像技术可为海藻的检测与研究提供一种快速、实时、有效、简便的方法.     

Two-Color Two-Photon Fluorescence Laser Scanning Microscopy

We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.     

激光扫描实时共聚焦显微成像系统设计

共聚焦扫描显微镜已成为生物医学和材料科学领域研究中非常有价值的一种工具.本文给出了一种反射型激光扫描共聚焦显微成像系统的系统结构和具体设计.采用多面体转镜进行水平扫描,摆镜进行垂直扫描.利用商品透镜设计了光学扫描中继系统,采用光电倍增管作为激发出的荧光探测器,同时给出了数据采集和扫描同步控制系统的组成与设计.利用CODE V优化光学扫描系统以获得尽可能小的扫描光斑尺寸和较大的视场,并综合考虑了采样频率、扫描速度和探测器对整个系统性能的影响,从而给出了该型共聚焦显微成像系统的相互匹配的设计参量.分析结果表明:共聚焦扫描系统设计合理可行|从光学扫描系统到PMT探测单元的各项技术指标得到优化,满足实时探测的要求|该系统具有适应性强,易升级,低成本的技术特点,同时可达到同类商品的技术性能.     

Peak Multiphoton Excitation of mCherry Using an Optical Parametric Oscillator (OPO)

mCherry is a red fluorescent protein which is bright, photostable, and has a low molecular weight. It is an attractive choice for multiphoton fluorescence imaging; however, the multiphoton excitation spectrum of mCherry is not known. In this paper we report the two photon excitation spectrum of mCherry measured up to 1190 nm in the near infrared (NIR) region. Skin tissues of transgenic mice that express mCherry were used in the experiments. mCherry in the tissues was excited with a Titanium:Sapphire laser and an optical parametric oscillator pumped by the Titanium:Sapphire laser. We found that the peak excitation of mCherry occurs at 1160 nm.     

Guiding synchrotron X‐ray diffraction by multimodal video‐rate protein crystal imaging

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X‐ray diffraction. Using the synchronous digitization instrument, second‐harmonic generation, two‐photon‐excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video‐rate (15 frames s^?1). A simple change in the incident wavelength enabled simultaneous imaging by two‐photon‐excited ultraviolet fluorescence, one‐photon‐excited visible fluorescence and laser transmittance. Development of an analytical model for the signal‐to‐noise enhancement afforded by synchronous digitization suggests a 15.6‐fold improvement over previous photon‐counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. These capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension.     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

激光扫描实时共聚焦显微成像系统设计

共聚焦扫描显微镜已成为生物医学和材料科学领域研究中非常有价值的一种工具.本文给出了一种反射型激光扫描共聚焦显微成像系统的系统结构和具体设计.采用多面体转镜进行水平扫描,摆镜进行垂直扫描.利用商品透镜设计了光学扫描中继系统,采用光电倍增管作为激发出的荧光探测器,同时给出了数据采集和扫描同步控制系统的组成与设计.利用COD...     

Near-infrared laser scanning confocal microscopy and its application in bioimaging

Near-infrared (NIR) fluorescence imaging is an important imaging technology in deep-tissue biomedical imaging and related researches, due to the low absorption and scattering of NIR excitation and/or emission in biological tissues. Laser scanning confocal microscopy (LSCM) plays a significant role in the family of fluorescence microscopy. Due to the introduction of pinhole, it can provide images with optical sectioning, high signal-to-noise ratio and better spatial resolution. In this study, in order to combine the advantages of these two techniques, we set up a fluorescence microscopic imaging system, which can be named as NIR-LSCM. The system was based on a commercially available confocal microscope, utilizing a NIR laser for excitation and a NIR sensitive detector for signal collection. In addition, NIR fluorescent nanoparticles (NPs) were prepared, and utilized for fluorescence imaging of the ear and brain of living mice based on the NIR-LSCM system. The structure of blood vessels at certain depth could be visualized clearly, because of the high-resolution and large-depth imaging capability of NIR-LSCM.     

Diffuse optical imaging and spectroscopy,in vivo

Based on photon migration the new goal of diffuse optical imaging is to reveal optical contrasts in the depth of biological tissues. We discuss first the origin of contrast mechanism (absorption, fluorescence and scattering) used on diffuse optical imaging and spectroscopy. Then, various experimental approaches are described based on CW, pulsed and modulated light excitation and detection. Theoretical models which provide solutions for direct and inverse problems are presented using random walk theory. Finally two studies on breast imaging and on the use of fluorescence exogeneous markers are discussed in detail.     

Backward enhanced emission from multiphoton processes in aerosols

We have investigated, both theoretically and experimentally, multiphoton-induced processes in aerosol particles using femtosecond laser pulses. More specifically, we have demonstrated that both multiphoton (1, 2 and 3 photon)-induced fluorescence (MPEF) and laser-induced breakdown (LIB) emissions are strongly enhanced in the backward direction. The backward enhancement increases from 1.8 to 35 (emission ratio between the backward direction and 90°) with increasing non-linear process order n. Application to non-linear lidar of biological aerosols is discussed. Received: 24 April 2002 / Revised version: 3 June 2002 / Published online: 2 September 2002 RID="*" ID="*"Corresponding author. Fax: +33-472/431507, E-mail: wolf@lasim.univ-lyon1.fr     

Dual-color prism-type TIRFM system for direct detection of single-biomolecules on nanoarray biochips

This study examined the applicability of a prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) system for the simultaneous detection of nano biomolecules on nanoarray biochips at the single-molecule level. The dual-color TIRFM system with two individual laser beams and a high-sensitivity camera was used for the simultaneous dual-color detection of two different nano beads (i.e. 20 nm yellow–green and crimson fluorescent FluoSpheres beads), and single-protein molecules labeled with different fluorescent dyes (i.e. actin from rabbit muscle conjugated with Alexa Fluor^® 488 and Alexa Fluor^® 633 goat anti-rabbit IgG) without a time-delay and the need to move the sample. When this system was applied to two different single-protein molecules labeled with different fluorescent dyes on the GPTS/CHI/GA-modified glass nanoarray chip, the full images of the biomolecules at the single-molecule level were obtained simultaneously in two different colors using a Dual-View™. The dual-color TIRFM system is quite suitable for the biological imaging at the single-molecule level on nanoarray biochips. This study provides a benchmark for directly monitoring the interactions and detecting the colocalization of two different nano biomolecules, and can be applied to the development of a nanoarray biochip at the single-molecule level.     

Fluorescence lifetime imaging with a low-repetition-rate passively mode-locked diode-pumped Nd:YVO4 oscillator

We report a wide-field fluorescence lifetime imaging microscope based on a low-repetition-rate (3.7-MHz) passively mode-locked diode-pumped laser source. This inexpensive and compact laser source operating in the visible and UV range can excite a wide range of fluorophores of biological interest. We demonstrate that the power of this laser source is highly sufficient for studying biological systems with low quantum yields (autofluorescence of tissues and stained living cells). The maximum measurable lifetime is also strongly increased with this laser source, as fluorescence intensity measurement can occur 250 ns after the excitation pulse.     

Biochip fluorescence detection system with spatial and spectral separation

A novel optical arrangement for fluorescence detection that employs spatial separation as well as spectral filter to increase the signal to noise ratio is proposed. Using a prism and two mirrors, the elliptical laser beam of a laser diode, as an excitation light, is homogenized and the transmitted excitation light is separated from the fluorescence not to reach the collecting optics. Uncooled CCD can capture the fluorescence image of up to 40 fluorescently-labeled protein patterns without scanning or mechanical translation. This paper presents the simulation, construction and measurement results of the developed optical system. The measurements show that the combination of prism and mirrors converts the excitation light from the laser diode to uniform illumination on the specimen, and provides the separation between excitation and fluorescence light to give high signal to noise ratio. It is also possible to assay various protein concentrations ranging from 1000 to 10 ng/ml reliably. We believe that the proposed fluorescence detection system can be used to build a commercially valuable, low cost, hand-held or miniature fast detection device for point-of-care applications.     

In vivo imaging of cell morphology and cellular processes in Caenorhabditis elegans, using non-linear phenomena

We present the detailed imaging of structures and processes of the nematode Caenorhabditis elegans (C. elegans) using non-linear microscopy. Complementary information about the anatomy of the nematode was collected by implementing a combination of two photon excitation fluorescence (TPEF), second and third harmonic generation (SHG and THG) image contrast modes on the same microscope. Three-dimensional (3D) reconstructions of TPEF, SHG and THG images were also performed. Moreover, THG imaging technique has been tested as a potential, novel, non-destructive diagnostic tool for monitoring cellular processes in vivo, such as neuronal degeneration.     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

Photostability comparison of CdTe and CdSe/CdS/ZnS quantum dots in living cells under single and two-photon excitations

The photostability is an outstanding feature of quantum dots (QDs) used as fluorescence probes in biological staining and cell imaging. To find out the related factors in the QD photostability, the photobleaching of naked CdTe QDs and BSA coated CdSe/CdS/ZnS QDs in human hepatocellular carcinoma (QGY) cells and human nasopharynx carcinoma (KB) cells were studied under single photon excitation (SPE) and two-photon excitation (TPE). In these two cell lines the cellular QDs were irradiated by a 405 nm continuous wave laser for SPE or an 800 nm femto-second (fs) laser for TPE. The QD photobleaching with the irradiation time was found to fit a biexponential decay. The fast decay plays a dominant role in the bleaching course and thus can be used as the parameter to quantitatively evaluate the QD photostability. The TPE decreased the QD photobleaching as compared to SPE. The BSA coated core/shell QDs had improved the photostability up to 4-5 times than the naked QDs due to the shielding effect of the QD shell. Therefore, it is better to use core/shell structured QDs as the fluorescence probe combining with a TPE manner for those long-term monitoring studies.     

毛细管电泳——多光子激发荧光检测分析生物胺

构建了毛细管电泳－连续光多光子激发荧光检测系统并应用于生物分析.对几种常见生物胺,如腐胺,尸胺,组胺,精胺,亚精胺和苯乙胺的分析结果表明,该系统具有分离效率高、质量检测灵敏度高和低消耗等特点,适于复杂体系如生物样品的测量.定量分析显示,生物胺的检测限在nM级,线性范围超过2个数量级,进一步将多光子激发荧光检测与传统单光子激发荧光检测结果对比,发现前者还具有改善分离选择性的优势.     

3D photon counting imaging at 1550 nm with 1.5-GHz sine-wave-gated InGaAs/InP GAPD

We demonstrated a 3D laser imaging system at 1550 nm with a 1.5-GHz sine-wave gated Geiger-mode InGaAs/InP avalanche photodiode (APD). An optical fiber bundle with 100 individual fiber outputs was implemented at the focal plane of the telescope, providing a 2.5-mrad imaging view. The system used single-pixel near-infrared single-photon detector to measure photons at fiber outputs instead of a photon counting array. The 1.5-GHz gated Geiger-mode InGaAs/InP APD with a timing jitter of 290 ps was operated in quasi-continuous mode with detection efficiency of ∼4.3%. We achieved higher than 6-cm surface-to-surface resolution at single-photon level, showing a potential of low-energy and eye-safe laser imaging system for long-distance measurements.