大视场线扫描共聚焦全息显微成像

传统显微成像一般记录样本的强度信息,对于半透明或相位组织成像对比度较差.为实现相位组织非荧光标记成像,采用线扫描共聚焦全息成像方法,在线扫描共聚焦成像的基础上增加一路参考光,在共聚焦狭缝处形成离轴像面数字全息,通过控制样本的移动实现对样本的扫描,将获得的干涉线合成为二维全息图,通过频域滤波的方式获得振幅与相位分布,采用...     

时间延迟积分荧光显微成像平场校正技术

时间延迟积分(Time Delay Integration,TDI)图像传感器具有高速、高灵敏度等特点,广泛应用于高通量、大视场的荧光显微成像系统中.显微物镜视场内响应均匀是精确获取荧光能量分布的基础,为提高系统成像质量和测量准确度,研究了适用于TDI荧光显微成像系统的平场校正或响应非均匀性校正方法.根据TDI荧光成像...     

基于线扫描方式的激光共焦显微镜的研究

激光共焦扫描显微镜(LCSM)是一种新型光学显微镜,它能够对活体组织的深度结构进行清晰的二维或三维成像.由于传统共焦扫描显微镜采用单点扫描方式,成像速度慢,无法满足活体组织实时成像的要求.介绍一种以线扫描方式代替点扫描方式的激光共焦扫描显微镜.线扫描方式不但简化了扫描机构的设计,而且提高了扫描速度,使活体组织的实时成像成为可能.     

生物芯片的一次成像检测技术

现在生物芯片(微阵列)的检测一般是基于扫描成像分析技术，这种方法在需要平行快速检测的应用中成像速度受到限制。我们设计搭建了一套检测生物芯片一次成像装置，其原理示意图如图1所示。     

激光共焦高速扫描显微成像的高帧速重构算法

针对激光共焦扫描显微镜的往复式逐行扫描成像方式带来的帧图像数据分割难的问题,在分析系统扫描方式、振镜的实际运动方式与理论运动方式差异的基础上,利用相邻两帧图像相似性大的特点,提出了一套完整的高帧速重构算法。该算法通过连续帧特征区域差分的方式实现了一维信号序列的自适应分割,即实现了对一维信号序列进行动态排列及分割成二维阵列图像数据,从而重构出多帧高精度图像。实验表明,该算法的成像误差低于1.6%,适用于成像速度高达300帧/s的激光共焦扫描显微成像。     

双侧远心线阵视觉系统的动态扫描成像标定

目前对于由双侧远心镜头和线阵相机组成的成像系统标定方法的研究较少,尤其是考虑动态扫描情况下的双侧远心线阵成像系统标定.文中首先对线阵相机扫描方向做类投影变换处理,进而结合仿射成像建立了双侧远心线阵成像系统的几何成像模型;之后,分析了影响成像效果的各类畸变因素,包括扫描方向速度波动导致的畸变以及成像镜头畸变;接着,给出了...     

生物芯片荧光检测光学系统综述

对生物芯片上生物反应信息的检测是生物芯片技术的重要组成部分。对以荧光物质作为示踪物的微阵列生物芯片和悬浮式生物芯片检测系统中的光学部分进行了综合分析。     

基于VC++的纳米分辨远场光学共焦成像系统的扫描控制

为降低纳米分辨远场光学共焦成像系统的出错率和提高此系统的实验效率,设计了纳米分辨远场光学共焦成像系统的扫描控制子系统。纳米分辨远场光学共焦成像系统的主要组成器件为纳米移动平台,被测物体置于此平台上随平台移动而运动。如何使被测物体作有规律的运动是实验能否成功的关键。采用VC++开发平台设计的扫描自动控制子系统即为解决这一问题而开发。子系统使用方便、界面友好。     

基于共焦显微条件下线结构光共焦显微术的研究

美国的Minsky在20世纪50年代末提出共焦显微术的概念以来,尽管共焦显微术可以获得更高的分辨率,但不仅成像速度慢,而且需要使用光电增强器对采集光点信号进行增强,导致制造成本过高,所以除一些成像质量要求极高的显微系统外,其他的显微系统很少应用。讨论了线结构光共焦显微术的原理、优缺点以及影响性能的几个重要因素,并介绍其最新的研究状况。     

生物芯片及其荧光信号检测

系统介绍了生物芯片的概念和制造方法 ,重点讨论了生物芯片的荧光检测方法 ,并对不同的检测方法进行了对比和分析。总体来说 ,激光共聚焦芯片扫描仪的荧光检测灵敏度和扫描分辨力较高 ,而 CCD芯片扫描仪的荧光检测灵敏度和扫描分辨力较低 ,但 CCD芯片扫描仪的检测速度较快 ,成本也较低     

Three-dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three-dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.     

Chromatic two-photon excitation fluorescence imaging

We report on a chromatic axial scanning method for two-photon excitation fluorescence imaging. Effective axial scanning is achieved by incorporating a Fresnel lens in the system, which has large chromatic aberration and can therefore focus the excitation beam to different axial positions depending on its wavelength. We experimentally demonstrated this technique and used it to image the cross-section of fluorescent microspheres.     

荧光成像系统对比度分析与成像仿真

目前荧光成像技术在生物医学领域得到越来越广泛的应用。为了缩短产品设计和开发周期,且能更直观地反映系统的成像效果,根据各模块光谱特性曲线,提出了荧光成像链路模型。利用该模型,对荧光成像系统的对比度进行了分析,验证了滤光片光密度(OD)值的合理范围是在5~7之间。最后以荧光显微镜为例,对系统成像过程进行了仿真。结果表明,各模块不匹配也会对系统对比度产生影响,仿真图像能够直观反映出系统的匹配程度和成像效果,并与实际系统测试结果相吻合,证明了该链路模型仿真的可行性和有效性。     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Digital scanned laser light-sheet fluorescence lifetime microscopy with wide-field time-gated imaging

We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.     

Time-domain whole-field fluorescence lifetime imaging with optical sectioning

A whole-field time-domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode-locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time-gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time-gated images, 2-D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real-world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all-solid-state diode-pumped laser technology. Whole-field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC–BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to α 3.0 ns. Control experiments using fluorescein conjugated poly- l -lysine and poly- d -lysine demonstrated that the increase in fluorescence parameters observed with FITC–BSA were due to intracellular proteolysis since addition of the inert d -isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC–dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.     

Navigating transdermal diffusion with multiphoton fluorescence lifetime imaging

We demonstrate the potential of fluorescence lifetime imaging by time-correlated single-photon counting as a method for monitoring the transdermal diffusion pathway and diffusion rate of pharmaceuticals in human skin. The current application relies on observing subtle changes in the fluorescence lifetime of the intrinsic fluorophores present in the intracellular region between corneocytes of the stratum corneum. We have comprehensively characterized the measured fluorescence lifetimes from intracorneocyte junctions in three skin section types (dermatomed skin, epidermal membranes and stratum corneum) revealing statistically significant differences of the short lifetime component between each of the types, which we attribute to the sample preparation and imaging method. We show using epidermal membrane sections that application of a drug/solvent formulation consisting of ethinyl estradiol and spectroscopic grade ethanol to the surface gives rise to a slight but statistically significant shortening of the fluorescence lifetime of the long-lived emitting species present in the sample, from approximately 2.8 ns to 2.5 ns. The method may be useful for future studies where the kinetics and pathways of a variety of applied formulations could be investigated.