A combined light sheet fluorescence and differential interference contrast microscope for live imaging of multicellular specimens

We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three‐dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.     

远场均匀照明的LED排列设计

为了实现远场的照度均匀性,在已设计的LED平面排列组合的基础上,提出了LED白光照明单元的球面排列设计方法。相比平面的排列,把LED照明单元组合排列在半球形表面,在被照接收面上产生更宽角度的照度分布。并对几种不同排列的LED照明单元进行分析,建立了光学模型和设计方程式,模拟出远场的照度分布情况。结果表明在极限角度内LED球形排列具有均匀广泛的远场照度分布。     

Selectable light‐sheet uniformity using tuned axial scanning

Light‐sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three‐dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light‐sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade‐off between light‐sheet thickness and area over which this thickness is preserved. Recently, an increase in light‐sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z‐scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light‐sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.     

基于Web的协同设计构模系统

提出了一种建立基于 Web的协同设计系统的模块化方法。系统的设计模块结构包括中心设计模块和学科设计子模块。中心设计模块就是设计小组依据用户对产品性能的需求而建立的设计项目管理和控制模块 ;学科设计子模块则是与产品设计项目相关的各学科领域在 Web上建立的分布式模块。它们分别封装了各个学科专业领域的设计知识、应用程序和工具软件等。系统框架为设计模块提供一致性的设计表征语言 ,协同、协调工具及模块间信息交互机制 ,依靠框架支持的各设计模块通过 Web联系在一起就构成了一个开放的、协同的集成设计系统。本文还以毛化钢板的功能形貌设计为例介绍了协同设计系统的多学科设计模块结构的构造方法     

Image scanning microscopy: an overview

For almost a century, the resolution of optical microscopy was thought to be limited by Abbé’s law describing the diffraction limit of light. At the turn of the millennium, aided by new technologies and fluorophores, the field of optical microscopy finally surpassed the diffraction barrier: a milestone achievement that has been recognized by the 2014 Nobel Prize in Chemistry. Many super‐resolution methods rely on the unique photophysical properties of the fluorophores to improve resolution, posing significant limitations on biological imaging, such as multicoloured staining, live‐cell imaging and imaging thick specimens. Structured Illumination Microscopy (SIM) is one branch of super‐resolution microscopy that requires no such special properties of the applied fluorophores, making it more versatile than other techniques. Since its introduction in biological imaging, SIM has proven to be a popular tool in the biologist's arsenal for following biological interaction and probing structures of nanometre scale. SIM continues to see much advancement in design and implementation, including the development of Image Scanning Microscopy (ISM), which uses patterned excitation via either predefined arrays or raster‐scanned single point‐spread functions (PSF). This review aims to give a brief overview of the SIM and ISM processes and subsequent developments in the image reconstruction process. Drawing from this, and incorporating more recent achievements in light shaping (i.e. pattern scanning and super‐resolution beam shaping), this study also intends to suggest potential future directions for this ever‐expanding field.     

Sample holder for axial rotation of specimens in 3D microscopy

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three‐dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three‐dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three‐dimensional specimens is essential. Fluorescence z‐projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.     

MRT letter: Experimental verification of vectorial theory to determine field at the geometrical focus of a cylindrical lens

We provide experimental evidence supporting the vectorial theory for determining electric field at and near the geometrical focus of a cylindrical lens. This theory provides precise distribution of field and its polarization effects. Experimental results show a close match (≈ 95% using χ²‐test) with the simulation results (obtained using vectorial theory). Light‐sheet generated both at low and high NA cylindrical lens shows the importance of vectorial theory for further development of light‐sheet techniques. Potential applications are in planar imaging systems (such as, SPIM, IML‐SPIM, imaging cytometry) and spectroscopy. Microsc. Res. Tech. 77:105–109, 2014. © 2014 Wiley Periodicals, Inc.     

Two‐dimensional profiling of carriers in terahertz quantum cascade lasers using calibrated scanning spreading resistance microscopy and scanning capacitance microscopy

The distribution of charge carriers inside the active region of a terahertz (THz) quantum cascade laser (QCL) has been measured with scanning spreading resistance microscopy (SSRM) and scanning capacitance microscopy (SCM). Individual quantum well‐barrier modules with a 35.7‐nm single module thickness in the active region of the device have been resolved for the first time using high‐resolution SSRM and SCM techniques at room temperature. SSRM and SCM measurements on the quantum well‐barrier structure were calibrated utilizing known GaAs dopant staircase samples. Doping concentrations derived from SSRM and SCM measurements were found to be in quantitative agreement with the designed average doping values of the n‐type active region in the terahertz quantum cascade laser. The secondary ion mass spectroscopy provides a partial picture of internal device parameters, and we have demonstrated with our results the efficacy of uniting calibrated SSRM and SCM to delineate quantitatively the transverse cross‐sectional structure of complex two‐dimensional terahertz quantum cascade laser devices.     

Light sheet based imaging flow cytometry on a microfluidic platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts‐off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state‐of‐art techniques the proposed technique shows marked improvement. Using fluorescently‐coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on‐the‐go. Overall, the proposed system is cost‐effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101–1107, 2013. © 2013 Wiley Periodicals, Inc.