Predictive-focus illumination for reducing photodamage in live-cell microscopy

Due to photobleaching and phototoxicity induced by high-intensity excitation light, the number of fluorescence images that can be obtained in live cells is always limited. This limitation becomes particularly prominent in multidimensional recordings when multiple Z-planes are captured at every time point. Here we present a simple technique, termed predictive-focus illumination (PFI), which helps to minimize cells' exposure to light by decreasing the number of Z-planes that need to be captured in live-cell 3D time-lapse recordings. PFI utilizes computer tracking to predict positions of objects of interest (OOIs) and restricts image acquisition to small dynamic Z-regions centred on each OOI. Importantly, PFI does not require hardware modifications and it can be easily implemented on standard wide-field and spinning-disc confocal microscopes.     

The Large-Scale Digital Cell Analysis System: an open system for nonperturbing live cell imaging

The Large-Scale Digital Cell Analysis System (LSDCAS) was designed to provide a highly extensible open source live cell imaging system. Analysis of cell growth data has demonstrated a lack of perturbation in cells imaged using LSDCAS, through reference to cell growth data from cells growing in CO₂ incubators. LSDCAS consists of data acquisition, data management and data analysis software, and is currently a Core research facility at the Holden Comprehensive Cancer Center at the University of Iowa. Using LSDCAS analysis software, this report and others show that although phase-contrast imaging has no apparent effect on cell growth kinetics and viability, fluorescent image acquisition in the cell lines tested caused a measurable level of growth perturbation using LSDCAS. This report describes the current design of the system, reasons for the implemented design, and details its basic functionality. The LSDCAS software runs on the GNU/Linux operating system, and provides easy to use, graphical programs for data acquisition and quantitative analysis of cells imaged with phase-contrast or fluorescence microscopy (alone or in combination), and complete source code is freely available under the terms of the GNU Public Software License at the project website ( http://lsdcas.engineering.uiowa.edu ).     

A Bayesian reconstruction method for micro-rotation imaging in light microscopy

The authors present a three-dimensional (3D) reconstruction algorithm and reconstruction-based deblurring method for light microscopy using a micro-rotation device. In contrast to conventional 3D optical imaging where the focal plane is shifted along the optical axis, micro-rotation imaging employs dielectric fields to rotate the object inside a fixed optical set-up. To address this entirely new 3D-imaging modality, the authors present a reconstruction algorithm based on Bayesian inversion theory and use the total variation function as a structure prior. The spectral properties of the reconstruction by simulations that illustrate the strengths and the weaknesses of the micro-rotation approach, compared with conventional 3D optical imaging, were studied. The reconstruction from real data sets shows that this method is promising for 3D reconstruction and offers itself as a deblurring method using a reconstruction-based procedure for removing out-of-focus light from the micro-rotation image series.     

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.     

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.     

Configurations of the Re‐scan Confocal Microscope (RCM) for biomedical applications

The new high‐sensitive and high‐resolution technique, Re‐scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re‐scan detection unit. The re‐scan unit includes a pair of re‐scanning mirrors that project the emission light onto a camera in a scanning manner. The signal‐to‐noise ratio of Re‐scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re‐scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re‐scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re‐scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re‐scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio‐metric (e.g. pH and intracellular Ca²⁺ measurements) and FRAP imaging.     

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.     

三维培养结肠癌细胞的微流控芯片荧光成像研究

改良了一种微流控芯片,可用于对结肠癌细胞进行三维培养并实现实时荧光成像。在结肠癌细胞内植入内源性的红色荧光蛋白,使用激光共聚焦显微镜对芯片中三维培养的细胞进行成像。通过细胞内部红色荧光蛋白的表达,可以观测到细胞的生长状态,实现对细胞的实时监测和高分辨率荧光成像。同时,通过免疫荧光染色来表征反映细胞活性的特征蛋白,其荧光强度和蛋白表达呈正相关。研究结果提示,细胞活性相关蛋白的表达受到微环境的影响,其在芯片三维培养中的活性强于二维培养,表明芯片内环境更加接近真实的人体微环境。该方法为进一步探究肿瘤细胞转移机制及相关药物的筛选研究提供了一种新的技术手段及实验平台。     

Optical complexities of living cytoplasm--implications for live cell imaging and photo-micromanipulation techniques

The ability to manipulate the intracellular environment within living cells and to monitor the cytosolic chemical changes which occur during cell stimulation has lead to major advances in our understanding of how cells read and respond to their environment. Perhaps the most powerful suite of techniques for achieving these dual objectives is based on the use of light (photons). Because cells are 'transparent', light has been used to both interrogate and manipulate the chemistry inside living cells, exploiting technical advances in both the physical and biochemical sciences. However, cells are neither transparent nor homogeneous with respect to their optical properties. The interface between light and the living cell cytoplasm thus represent an important, yet largely ignored, interface. There has been no review of the optical properties of cytoplasm and little discussion about how the optical properties of living cytoplasm influence the outcome of such measurements and manipulations. In this short review, we discuss the importance of understanding the optical properties of cytoplasm for such techniques and how imperfections in experimental interpretation can arise.     

抗高过载微流体惯性开关

基于微连通器结构,提出了一种使用盐水(Na Cl溶液)作为工作流体且具有高的抗过载能力的微流体惯性开关。分析了液滴的分离机理,设计了开关的流道结构。然后,对开关进行了理论分析,建立了开关模型。最后,利用流体动力学仿真和样机实验相结合的方法,对开关结构和功能进行了验证。验证结果显示:在幅值为30 000 g阶跃型加速度作用下,开关的工作流体仍未发生分离,加速度的幅值与开关响应时间相关。另外,开关样机能够使盐水液面形成高度差,样机的静态加速度阈值为134.6 g~152.3 g,非常接近其理论计算的加速度阈值142.7 g。得到的结果表明,采用的微连通器结构能够极大地增强微流体惯性开关的抗液体分离能力,能够对加速度幅值进行区分,并实现闭锁功能,同时显示了高的抗过载能力。     

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.     

Immersion oil for high‐resolution live‐cell imaging at 37°C: optical and physical characteristics

The use of normal immersion oil, developed for 23°C, at 37°C greatly compromises both axial resolution and signal intensity. We developed and characterized an immersion oil for optimal performance in live‐cell imaging at 37°C. We quantify the improvements in resolution and intensity obtained when using the new oil instead of its standard 23°C counterparts.     

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.     

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.     

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.     

新型惯性式微驱动器及其在微纳米定位中的应用

研制了一种基于压电陶瓷的惯性式微驱动器,其驱动电压直接由电子线路产生。介绍了微驱动器的工作原理和结构设计,测试了微驱动器的位移特性和动态响应性能。结果表明,在电压幅值20V~350V、频率20H z~125H z范围内,微驱动器可产生平稳和均匀的运动,平均步长在60nm~1502nm范围内连续可调,最大运动速度可达120μm/s。且驱动行程不受限制。给出了微驱动器在扫描隧道显微镜定位机构中的应用实例。     

Comparative evaluation of performance measures for shading correction in time‐lapse fluorescence microscopy

Time‐lapse fluorescence microscopy is a valuable technology in cell biology, but it suffers from the inherent problem of intensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artefacts. This will lead to inaccurate estimates of single‐cell features such as average and total intensity. Numerous shading correction methods have been proposed to remove this effect. In order to compare the performance of different methods, many quantitative performance measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. In this paper, the state‐of‐the‐art shading correction methods and performance evaluation methods are reviewed. We implement 10 popular shading correction methods on two artificial datasets and four real ones. In order to make an objective comparison between those methods, we employ a number of quantitative performance measures. Extensive validation demonstrates that the coefficient of joint variation (CJV) is the most applicable measure in time‐lapse fluorescence images. Based on this measure, we have proposed a novel shading correction method that performs better compared to well‐established methods for a range of real data tested.     

A confident scale-space shape representation framework for cell migration detection

Automated segmentation of time-lapse images is a method to facilitate the understanding of the intricate biological progression, e.g. cancer cell migration. To address this problem, we introduce a shape representation enhancement over popular snake models in the context of confident scale-space such that a higher level of interpretation can hopefully be achieved. Our proposed system consists of a hierarchical analytic framework including feedback loops, self-adaptive and demand-adaptive adjustment, incorporating a steerable boundary detail term constraint based on multiscale B-spline interpolation. To minimize the noise interference inherited from microscopy acquisition, the coarse boundary derived from the initial segmentation with refined watershed line is coupled with microscopy compensation using the mean shift filtering. A progressive approximation is applied to achieve represented as a balance between a relief function of watershed algorithm and local minima concerning multiscale optimality, convergence and robust constraints. Experimental results show that the proposed method overcomes problems with spurious branches, arbitrary gaps, low contrast boundaries and low signal-to-noise ratio. The proposed system has the potential to serve as an automated data processing tool for cell migration applications.     

A novel substrate for multisensor hyperspectral imaging

The quality of chemical imaging, especially multisensor hyperspectral imaging, strongly depends on sample preparation techniques and instrumental infrastructure but also on the choice of an appropriate imaging substrate. To optimize the combined imaging of Raman microspectroscopy, scanning‐electron microscopy and energy‐dispersive X‐ray spectroscopy, a novel substrate was developed based on sputtering of highly purified aluminium onto classical microscope slides. The novel aluminium substrate overcomes several disadvantages of classical substrates like impurities of the substrate material and contamination of the surface as well as surface roughness and homogeneity. Therefore, it provides excellent conditions for various hyperspectral imaging techniques and enables high‐quality multisensor hyperspectral chemical imaging at submicron lateral resolutions.