新型可控焦距液体透镜阵列

为了提高成像品质,提出了一种新型液体透镜阵列镜头。先将油滴沉入弹性体底部形成具有可控焦距的单个液体平凸透镜,再通过唯一平台实验液体透镜阵列,制作液体透镜阵列。通过控制滴入硅胶基底的硅油液体体积大小,控制单个微透镜的折射面曲率和透镜焦距。通过位移平台,控制阵列中微透镜的排布和个数。透镜阵列制作方法简单且成本低,透镜抗挤压能力强,焦距可控,阵列规整,在制造过程中,可以控制镜头光圈和初始焦距,因此是一种实用性较高的透镜阵列。     

Remote focusing in confocal microscopy by means of a modified Alvarez lens

Alvarez lenses are actuated lens‐pairs which allow one to tune the optical power by mechanical displacement of subelements. Here, we show that a recently realized modified Alvarez lens design which does not require mechanical actuation can be integrated into a confocal microscope. Instead of mechanically moving them, the sublenses are imaged onto each other in a 4f‐configuration, where the lateral image shift leading to a change in optical power is created by a galvo‐mirror. The avoidance of mechanical lens shifts leads to a large speed gain for axial (and hence also 3D) image scans compared to classical Alvarez lenses. We demonstrate that the suggested operation principle is compatible with confocal microscopy. In order to optimize the system, we have drawn advantage of the flexibility a liquid‐crystal spatial light modulator offers for the implementation. For given specifications, dedicated diffractive optical elements or freeform elements can be used in combination with resonant galvo‐scanners or acousto‐optic beam deflectors, to achieve even faster z‐scans than reported here, reaching video rate.     

An open‐source deconvolution software package for 3‐D quantitative fluorescence microscopy imaging

Deconvolution techniques have been widely used for restoring the 3‐D quantitative information of an unknown specimen observed using a wide‐field fluorescence microscope. Deconv , an open‐source deconvolution software package, was developed for 3‐D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3‐D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications.     

一款条码扫描成像镜头的多方案设计和选择

设计了一种扫描成像镜头,镜头焦距为7.9mm,视场角为52°,采用CMOS线性传感器接收。为了使镜头成本更低,同时保证好的成像性能,引入了塑料非球面,设计了一个含有高次塑料非球面镜片和一个玻璃球面镜片(1G1P)的结构。相对传统三片式玻璃球面镜片(3P)的结构来讲,1G1P结构的光学性能更好地满足了技术指标要求,成本为3G结构的50%,推广应用前景好。     

Label‐free imaging characteristics of colonic mucinous adenocarcinoma using multiphoton microscopy

Colorectal carcinoma (CRC) has high mortality and increased incidence rates. An early detection of CRC is very important. Multiphoton microscopy (MPM) with high resolution and high sensitivity is used to effectively distinguish the microstructure changes of normal and mucinous adenocarcinoma slices of ex vivo human colonic tissues. In mucinous adenocarcinoma mucosa, the glands are distorted and elongated, the gland cavity is indistinct, and the mesh collagen fibers are diminished. In the submucosa, the collagens are seriously disordered, elongated, pushed aside, and sparsely visible, the content of elastic fibers is also broken and almost disappearing. Many cancer cells, some in cavity‐like shape full of mucus surrounded by some collagen fibers, occupied the submucosa, which are comparable to hematoxylin‐eosin (HE) stained images. Second harmonic generation and two‐photon excitation fluorescence (SHG/TPEF) intensity ratio can be used further to quantitatively evaluate normality and abnormality. The fast Fourier transform (FFT) images show that the normal collagen fibrils are dense and in random order, and the cancerous collagen is certainly organized. The exploratory results show that it has potential for the development of multiphoton mini‐endoscopy in real‐time early diagnosis of CRC. SCANNING 35: 277‐282, 2013. © 2012 Wiley Periodicals, Inc.     

Time‐lapse microscopy using smartphone with augmented reality markers

A prototype system that replaces the conventional time‐lapse imaging in microscopic inspection for use with smartphones is presented. Existing time‐lapse imaging requires a video data feed between a microscope and a computer that varies depending on the type of image grabber. Even with proper hardware setups, a series of tedious and repetitive tasks is still required to relocate to the region‐of‐interest (ROI) of the specimens. In order to simplify the system and improve the efficiency of time‐lapse imaging tasks, a smartphone‐based platform utilizing microscopic augmented reality (μ‐AR) markers is proposed. To evaluate the feasibility and efficiency of the proposed system, a user test was designed and performed, measuring the elapse time for a trial of the task starting from the execution of the application software to the completion of restoring and imaging of an ROI saved in advance. The results of the user test showed that the average elapse time was 65.3 ± 15.2 s with 6.86 ± 3.61 μm of position error and 0.08 ± 0.40 degrees of angle error. This indicates that the time‐lapse imaging task was accomplished rapidly with a high level of accuracy. Thus, simplification of both the system and the task was achieved via our proposed system. Microsc. Res. Tech. 77:243–249, 2014. © 2014 Wiley Periodicals, Inc.     

Three‐dimensional imaging of porous media using confocal laser scanning microscopy

In the last decade, imaging techniques capable of reconstructing three‐dimensional (3‐D) pore‐scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO₂ storage potential. CLSM has a unique capability of producing 3‐D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z‐dimension) that can be imaged in porous materials. In this study, we introduce a ‘grind and slice’ technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3‐D confocal volumetric data into pores and grains. Finally, we use the resulting 3‐D pore‐scale binarized confocal data obtained to quantitatively determine petrophysical pore‐scale properties such as total porosity, macro‐ and microporosity and single‐phase permeability using lattice Boltzmann (LB) simulations, validated by experiments.     

Advanced spinning disk‐TIRF microscopy for faster imaging of the cell interior and the plasma membrane

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high‐resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high‐resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD‐TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD‐TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup.     

Real‐time three‐dimensional imaging of cell division by differential interference contrast microscopy

Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub‐diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer‐controlled vertical scanning of the microscope stage.     

Application of sensitive,high‐resolution imaging at a commercial lab‐based X‐ray micro‐CT system using propagation‐based phase retrieval

Several dedicated commercial lab‐based micro‐computed tomography (μCT) systems exist, which provide high‐resolution images of samples, with the capability to also deliver in‐line phase contrast. X‐ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single‐distance phase‐contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high‐quality images, measured without the use of a synchrotron X‐ray source, demonstrate that highly sensitive, micrometre‐resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.     

Fluorescence lifetime imaging microscopy using near-infrared contrast agents

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.     

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

We developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.     

Construction and evaluation of a wavelet-based focus measure for microscopy imaging

Microscopy imaging can not achieve both high resolution and wide image space simultaneously. Autofocusing is of fundamental importance to automated micromanipulation. This article proposes a new wavelet-based focus measure, which is defined as a ratio of high frequency coefficients and low frequency coefficients. 8 series of 49 microscope images each acquired under five magnifications are used to comprehensively compare the performance of our focus measure with the classic and popular focus measures, including Normalized Variance, Entropy, Energy Laplace and wavelet-based high frequency focus measures. The robustness of these focus measures is evaluated using noisy image sequences corrupted by Gaussian white noise with standard deviations (STD) 5 and 15. An evaluation methodology is proposed, based on which these 5 focus measures are ranked. Experimental results show that the proposed focus measure can provide significantly the best overall performance and robustness. This focus measure can be widely applied to the automated biological and biomedical applications.     

Frequency domain lifetime and spectral imaging microscopy

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.     

Second‐harmonic imaging microscopy for identifying colorectal intraepithelial neoplasia

In this study, second‐harmonic imaging microscopy was used to monitor precancerous colorectal lesions at different stages. It was found that the morphology of glands and lamina propria in mucosa changes with the progression of colorectal diseases from normal to low‐grade intraepithelial neoplasia to high‐grade intraepithelial neoplasia and this microscopy has the ability of direct visualization of these warning symptoms. Furthermore, two morphologic variables were quantified to determine the changes of glands and collagen in lamina propria during the development of colorectal intraepithelial neoplasia. These results suggest that second‐harmonic imaging microscopy has the potential in label‐freely and effectively distinguishing between normal and precancerous colorectal tissues, and will be helpful for early diagnosis and treatment of colorectal diseases.     

High‐contrast optical microscopy of graphene sheets

In the way of making graphene an industry‐friendly material, it must be mass‐produced with high‐quality and reduced cost over large areas. Assisted by machine‐learning techniques, rapid, nondestructive and accurate determination of large graphene sheets on SiO₂/Si substrates has been made possible in recent years by the optical microscopy method. Optimization of the substrate to achieve the maximum contrast can further extend the application of the optical microscopy method for quality control of the mass‐produced graphene. Graphene/n₂/n₃three‐layer structures, where n₂ and n₃ are refractive indices, are routinely used for identifying the number of graphene layers by optical reflection microscopy. In this paper, two analytical equations are derived that can be easily used for high‐contrast optical imaging of graphene sheets without any need to resort to the cumbersome numerical methods. One of the equations is derived for choosing the best material with refractive index n₂ that when coated on a substrate with refractive index n₃, maximizes the optical contrast. The other equation is derived for finding the best thickness of the SiO₂ layer in graphene/SiO₂/Si structures, which are in common use for fabrication of graphene‐based devices. The results are implemented in a MATLAB GUI, see Supporting Information, to assist the users in using the equations.     

High‐resolution,high‐throughput imaging with a multibeam scanning electron microscope

Electron–electron interactions and detector bandwidth limit the maximal imaging speed of single‐beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.     

Orientation independent retardation imaging using quantitative polarized phase microscopy

Simultaneous optical phase and retardation measurement of a birefringent specimen is demonstrated independently of a priori knowledge of the optic axis orientation. The two‐dimensional retardation distribution in both magnitude and angle of the fast axis orientation is uniquely determined from transverse phase images recorded with a bright field transmission microscope using light polarized at a minimum of three different polarization orientations. This approach opens a new possibility for stain‐free phase and orientation‐independent retardation characterization of samples using only one polarizer without needing other additional optical elements traditionally used in polarimetric measurements. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.