Microlens arrays for confocal microscopy

A new; high resolution measuring system based on confocal microscopy has been developed for the evaluation of microlens arrays; in particular for applications in confocal microscopy itself. Lenslet arrays for parallel scanning and processing in confocal microscopy were designed as phase-matched Fresnel lenslets and fabricated by direct laser writing. Replica arrays were produced by ultraviolet embossing and hot embossing techniques. Fabricated arrays with a numerical aperture of 0.28 exhibited near diffraction limited performance and a focal length standard deviation of 120 nm in a nominal value of 250 μm. The technique developed represents a convenient and powerful technique for the characterization of lenslet arrays in general.     

共焦显微扫描探测技术的发展

传统的共焦显微探测多是采用单点机械扫描方式完成的,存在着扫描效率低的缺点,针对于此,一种多光束共焦显微系统成为当前的研究热点,通过不同方式对光束进行分割,使单点检测变为多路并行检测,提高了测量速度,减少了扫描过程中光源和振动噪声的影响,实现多光束的同步测量。介绍了几种典型的单光束和多光束共焦显微测量系统的原理、研究进展、发展方向及作者在该方面的研究。     

Polarized confocal theta microscopy

We propose a comprehensive treatment of theta microscopy based on dipole emission, which better describes fluorescence emission than the isotropic emission model, as fluorescence emission is often polarized. Formulas describing the point spread function for polarized confocal fluorescence theta microscopy are given. Examples are given and some advantages of polarized theta fluorescence microscopy are presented. To cite this article: O. Haeberlé et al., C. R. Physique 3 (2002) 1445–1450.     

共聚焦显微镜激光高速扫描控制系统设计及实现

 在传统光学扫描方法基础上,有效地将检流式与共振式光学扫描振镜结合起来,提出一种速度可达4 M/s采样率的高速激光扫描方法,并基于单片机系统设计搭建了系统控制硬件平台,编写了上位机端和下位机端应用软件。实验结果表明:该控制系统扫描速度快,性能稳定可靠,能够应用于共聚焦显微镜,实现实时扫描成像 。     

Photobleaching, Mobility, and Compartmentalisation: Inferences in Fluorescence Correlation Spectroscopy

In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter P(B) = sigma(B)P/D (where sigma(B) is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when P(B) < 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like sigma(B)P/R2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.     

Fluorescence confocal polarizing microscopy: Three-dimensional imaging of the director

Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by fluorescence confocal polarizing microscopy (FCPM). The technique employs the property of LC to orient the fluorescent dye molecules of anisometric shape, added in small quantities to the LC. In LC, smooth director deformations do not alter mass density of the material. Thus the density of dye is also uniform across the sample, except, perhaps, near the surfaces or at the cores of topological defects. In polarized light, the measured fluorescence signal is determined by the spatial orientation of the molecules rather than by dye concentration (as in regular biological samples stained with tissue-specific dyes). The contrast is enhanced when both excitation and detection of fluorescence light are performed in polarized light. This short review describes the essence of FCPM technique and illustrates some of its applications, including imaging of Frederiks electric-field induced effect in a nematic LC and defects such as dislocations in cholesteric LCs.     

微液膜动力学特性与稳定性实验研究

气-液两相流设备的性能受限于临界热流密度,开展流动微液膜动力学特性及其稳定性的相关研究是深入理解沸腾危机及临界热流密度机理的关键。采用光学玻璃制成的矩形通道作为实验段,使用微流量齿轮泵驱动去离子水,使其在实验通道入口处与在其上部流动的压缩空气接触形成同向流动的分层流。利用共轭光学探测器对流动微液膜的厚度进行了测量,利用高速摄像机对气-液两相分层流波动特性进行了可视化观测。研究表明,在绝热情况下,当液速一定时,液膜的平均厚度随着气速增加而减小,当气速增加到某一阈值时会导致液膜破裂。     

Dual-axes differential confocal microscopy with high axial resolution and long working distance

We discover that the slight transverse offset of a point detector results in a shift of the axial intensity response curve in a dual-axes confocal microscopy (DCM). Based on this, we propose a new dual-axes differential confocal microscopy (DDCM) with high axial resolution and long working distance, in which two point detectors are placed symmetrically about the collection axis. And a signal is obtained through the differential subtraction of two signals received simultaneously by the two point detectors. Theoretical analyses and preliminary experiments indicate that DDCM is feasible and suitable for the high precision tracing measurement of microstructures and surface contours.     

Fibre-optic double-pass confocal microscopy

This paper reports on the construction and characterisation of a novel double-pass confocal scanning microscope that uses a four-port fibre coupler. The new imaging system is self-aligned, compact, vibration-free, and purely coherent. Compared with fibre-optic transmission- and reflection-mode confocal systems, the fibre-optic double-pass confocal imaging system exhibits higher resolution.     

Characterization of a confocal microscope for time-resolved photon counting fluorescence

A confocal microscope setup is developed for time-resolved fluorescence measurements. It is added to a traditional cuvette time-resolved setup, with a pumped Ti-Sa light source. The temporal resolution of 37 ps (FWHM) is not degraded, in comparison with the cuvette setup also described. These setups allow both decay lifetime and anisotropy relaxation time determination. Fluorescence correlation spectroscopy (FCS) is used to determine the observation point size. When associated with the calcium probe calcium green, calcium concentration in single cells can be determined in 10 ms by simultaneous acquisition of early and late fluorescence photons.     

Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

ItisreportedrecentlythatnonlinearopticalphenomenonofSHGandTHGhasbeenobservedinmanybiologicaltissues[16].SHGandTHGhavebeenusedtoperformthethree-dimensionalimaginginlivingtissuesandhaveattractedmuchattentionrecently.TherearemanyadvantagesofusingSHGandTHGtoperformthethree-dimensionalimaginginlivingtissues,suchasnoninvasiveandnophotobleaching,inadditiontotheimagingpropertiesofmulti-photonfluorescenceimaging[7—9].Firstly,unlikeinthesingle-andmulti-photonfluorescenceprocesses,onlyvirtualstat…     

Hybrid Rayleigh,Raman and two‐photon excited fluorescence spectral confocal microscopy of living cells

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low‐wavenumber‐resolution Raman imaging, Rayleigh scatter imaging and two‐photon fluorescence (TPE) spectral imaging, fast ‘amplitude‐only’ TPE‐fluorescence imaging and high‐spectral‐resolution Raman imaging. This multi‐dimensional fluorescence–Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman ‘chemical’ imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence–Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of one‐dimensional spherically aberrated point spread function in depth profiling by confocal Raman microscopy

We present a simple experiment that allows the complete and direct characterization of the point spread function (PSF) in refraction‐aberrated depth profiling experiments with confocal Raman microscopy. We used a wedge‐shaped solid polymer film to induce refraction aberrations on the response of an infinitesimally thin Raman scatterer, represented by a polished silicon wafer. The system, with the film pasted on top of the Si wafer, was probed by a depth slicing technique under a dry‐optics configuration. Post‐acquisition processing of the Si and polymer intensity maps allowed the reconstruction of the axial PSF spatially resolved each 1 µm or less in the z‐axis and for virtually continuous values of focusing depth. In agreement with theory, we found that PSF broadens asymmetrically with focusing depth, with a marked shift in the focus point. From the shape of PSF, we obtained values of depth resolution within the film that confirm that axial discrimination is not drastically deteriorated, as suggested by previous works, and that confocal aperture effectively reduces the collection volume even under severe refraction interference. Copyright © 2013 John Wiley & Sons, Ltd.     

Antifading embedding media in confocal immunoflourescence microscopy

Fading or bleaching of fluorescence intensity during continuous illumination of stained objects is a serious problem in fluorescence microscopy. Fluorescence intensity as well as bleaching characteristics of dyes are dependent primarily upon physical parameters such as molecular constants (absorption rate and quantum efficiency), excitation energy and brightness (causes photon saturation), and environmental parameters (pH, ions, binding to proteins, etc.) that can strongly influence the properties of fluorochrome molecules. We have studied the effect of various antifading reagents on the behavior of the common dyes fluorescein isothiocyanate (FITC) and phycoerythrin (PE) using immunofluorescent-stained living cells in suspension or membrane-permeabilized dried cells as test systems. As expected, fading cannot be completely eliminated but may be reduced to varying degrees. In our hands, the most efficient antifading reagent for FITC isn-propyl gallate (NPG) dissolved in glycerol. No additive was found to retard fading, but complete dehydration of the cell suspension reduces this effect.     

Fast mapping of inhomogeneities in the popular metallic perovskite Nb:SrTiO3 by confocal Raman microscopy

Confocal Raman microscopy was applied in order to investigate the homogeneity of donor doping in Nb:SrTiO₃ single crystals. Measurements of local Raman spectra revealed a systematic relation between the intensity of the Raman signal and the donor content of the crystals. We successfully elaborated a correspondence between the electronic structure and the intensity of the Raman lines using a crystal with macroscopic inhomogeneity as a demonstration sample. By mapping the distribution of the intensity of the Raman signal, we identified a characteristic inhomogeneous structure related to the presence of clusters with sizes of 5 µm to 20 µm, indicating inhomogeneous donor distribution caused by flaws introduced during crystal growth. Hence, we propose confocal Raman microscopy as a convenient technique for investigating the homogeneity and quality of doped perovskite surfaces, which are needed for various technological applications. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

超分辨率活体人眼视网膜共焦扫描成像系统

活体人眼共焦扫描成像系统的分辨率受到人眼像差、数值孔径和探测针孔尺度的限制,本文设计了一套超分辨活体人眼视网膜共焦扫描系统,采用自适应光学技术探测并校正人眼像差,结合光学超分辨技术提高系统分辨率,补偿有限尺度针孔对分辨率的影响,并获得活体人眼的实时、高分辨图像. 关键词： 超分辨 共焦扫描光学显微术 眼科光学 自适应光学     

双Toraldo光瞳的共焦系统

Toraldo光瞳是超分辨中研究最多的光瞳之一.不过，由于实际中在制作精确度以及制造效率上的限制，它的应用受到了很大程度的制约.提出了双Toraldo光瞳的共焦系统，它能够用两个相对容易制作的Toraldo光瞳实现多区复杂Toraldo光瞳的功能. 关键词： 超分辨 Toraldo光瞳 共焦成像     

位相型光瞳滤波器对共焦系统离焦工作状态横向分辨特性的影响

分析了共焦显微系统(CMS)离焦状态工作时分辨特性的变化,指出了离焦状态是导致CMS横向分辨特性变差的主要影响因素之一。提出并采用了位相型超分辨光瞳滤波技术来改善CMS工作在离焦状态下的横向分辨特性,分析了超分辨光瞳滤波器位置变化对CMS横向分辨特性的影响。理论分析和实验验证表明,位相型超分辨光瞳滤波技术的引入,显著抑制了CMS工作在离焦状态时艾里斑旁瓣的增长,克服了CMS因工作在离焦状态而引起的横向分辨特性下降的缺点。     

一种提高共聚焦显微镜信噪比算法的研究

基于共焦显微镜的成像特点,建立了Kalman滤波算法的理论模型,把Kalman滤波方法引入到系统中,提出一种基于图像像素的Kalman滤波算法,并实现了实时化的Kalman滤波器。实验结果表明:该算法能够有效地提高共焦显微镜信噪比,但是以牺牲时间为代价,提高系统分辨率的根本方法还是要着重考虑优化成像系统光路和探测电路。     

Assessment of murine neuroblastoma (N1E-115) resting membrane potential by confocal microscopy

Digital imaging (confocal microscopy) and a slow potentiometric dye (tetramethylrhodamine methyl ester) were used to assess the resting membrane potential (V _m) of murine neuroblastoma cells (N1E-115). The averageV _m was found to be –64.0±2.0 mV. The difference between this and the previously reported higher values was attributed to the use of glass microelectrode techniques that probably caused mechanical injury to the cell membranes: Digital imaging of N1E-115V _m was found to be sensitive, reproducible, fast, and simple.