Microscope alignment using real-time Imaging FCS

Modern electron-multiplying charge-coupled device (EMCCD) and scientific complementary metal-oxide semiconductor (sCMOS) cameras read out fluorescence data with single-molecule sensitivity at thousands of frames per second. Exploiting these capabilities in full requires data evaluation in real time. The direct camera-read-out tool presented here allows access to the data while the camera is recording. This provides simplified and accurate alignment procedures for total internal reflection fluorescence microscopy (TIRFM) and single-plane illumination microscopy (SPIM), and simplifies and accelerates fluorescence experiments. The tool handles a range of widely used EMCCD and sCMOS cameras and uses imaging fluorescence correlation spectroscopy for its evaluation. It is easily extendable to other camera models and other techniques and is a base for automated TIRFM and SPIM data acquisition.     

Video rate bioluminescence imaging of secretory proteins in living cells: localization, secretory frequency, and quantification

We have developed a method of video rate bioluminescence imaging to investigate protein secretion from a single mammalian cell and analyzed the localization, secretory frequency, and quantification of secreted protein. By detecting the luminescence signals from the Gaussia luciferase (GLase) reaction using a high-speed electron-multiplying charge-coupled device (EM-CCD) camera, video rate imaging was performed with a time resolution within 500 ms/image over 30 min in living cells. As a model study, we applied the method to visualize the glucose-stimulated insulin secretion from clustered pancreatic MIN6 β cells using the fused protein of GLase with preproinsulin. High-quality video images clearly showed that the glucose-stimulated insulin secretion from the clustered MIN6 β cells oscillated within a period of a few minutes over 10 min. In addition, the glibenclamide-induced insulin secretion from the clustered MIN6 β cells was visualized, suggesting that bioluminescence video rate imaging is a useful method for validating drug action in living cells.     

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.     

Imaging Spatial Variations of Optical Bandgaps in Perovskite Solar Cells

A fast, nondestructive, camera‐based method to capture optical bandgap images of perovskite solar cells (PSCs) with micrometer‐scale spatial resolution is developed. This imaging technique utilizes well‐defined and relatively symmetrical band‐to‐band luminescence spectra emitted from perovskite materials, whose spectral peak locations coincide with absorption thresholds and thus represent their optical bandgaps. The technique is employed to capture relative variations in optical bandgaps across various PSCs, and to resolve optical bandgap inhomogeneity within the same device due to material degradation and impurities. Degradation and impurities are found to both cause optical bandgap shifts inside the materials. The results are confirmed with micro‐photoluminescence spectroscopy scans. The excellent agreement between the two techniques opens opportunities for this imaging concept to become a quantified, high spatial resolution, large‐area characterization tool of PSCs. This development continues to strengthen the high value of luminescence imaging for the research and development of this photovoltaic technology.     

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass.     

A reconfigurable real-time compressive-sampling camera for biological applications

Many applications in biology, such as long-term functional imaging of neural and cardiac systems, require continuous high-speed imaging. This is typically not possible, however, using commercially available systems. The frame rate and the recording time of high-speed cameras are limited by the digitization rate and the capacity of on-camera memory. Further restrictions are often imposed by the limited bandwidth of the data link to the host computer. Even if the system bandwidth is not a limiting factor, continuous high-speed acquisition results in very large volumes of data that are difficult to handle, particularly when real-time analysis is required. In response to this issue many cameras allow a predetermined, rectangular region of interest (ROI) to be sampled, however this approach lacks flexibility and is blind to the image region outside of the ROI. We have addressed this problem by building a camera system using a randomly-addressable CMOS sensor. The camera has a low bandwidth, but is able to capture continuous high-speed images of an arbitrarily defined ROI, using most of the available bandwidth, while simultaneously acquiring low-speed, full frame images using the remaining bandwidth. In addition, the camera is able to use the full-frame information to recalculate the positions of targets and update the high-speed ROIs without interrupting acquisition. In this way the camera is capable of imaging moving targets at high-speed while simultaneously imaging the whole frame at a lower speed. We have used this camera system to monitor the heartbeat and blood cell flow of a water flea (Daphnia) at frame rates in excess of 1500 fps.     

A new setup for in vivo fluorescence imaging of photosynthetic activity

Here, we describe a new imaging setup able to assess in vivo photosynthetic activity. The system specifically measures time-resolved chlorophyll fluorescence in response to light. It is composed of a fast digital camera equipped with a wide-angle lens for the analysis of samples up to 10 × 10 cm, i.e. entire plants or petri dishes. In the choice of CCD, we have opted for a 12-bits high frame rate [150 fps (frames per second)] at the expense of definition (640 × 480 pixels). Although the choice of digital camera is always a compromise between these two related features, we have designed a flexible system allowing the fast sampling of images (down to 100 μs) with a maximum spatial resolution. This image readout system, synchronized with actinic light and saturating pulses, allows a precise determination of F ₀ and F _M, which is required to monitor PSII activity. This new imaging system, together with image processing techniques, is useful to investigate the heterogeneity of photosynthetic activity within leaves or to screen large numbers of unicellular algal mutant colonies to identify those with subtle changes in photosynthetic electron flow.     

基于空间频域滤波高动态光学投影层析的斑马鱼三维成像研究

本文提出了一种基于空间频率滤波的多曝光融合的高动态投影层析三维成像方法,实现了活体斑马鱼（17 mm × 4 mm,最大厚度为2.33 mm,最小厚度为0.29 mm）的三维结构成像. 通过相机采用不同曝光时间记录系列吸收图像,将每张图像取变换到频域去除低频后,将各张滤波后叠加并逆傅里叶变换回空域,对变换后的图像进行归一化处理,最终获得高动态图像. 在每个投影角度获得这种高动态吸收投影图像,进行滤波反投影算法重建,获得高动态的整条斑马鱼三维结构信息. 实验成像结果表明,这种空间频率滤波多曝光融合的高动态光学投影层析三维成像研究,可以获得复杂结构更丰富的空间信息,对斑马鱼等模式生物早期胚胎生长发育进程进行监测和定量评估有一定的应用前景.     

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.     

A 15.6 frames per second 1‐megapixel multiple exposure laser speckle contrast imaging setup

A multiple exposure laser speckle contrast imaging (MELSCI) setup for visualizing blood perfusion was developed using a field programmable gate array (FPGA), connected to a 1000 frames per second (fps) 1‐megapixel camera sensor. Multiple exposure time images at 1, 2, 4, 8, 16, 32 and 64 milliseconds were calculated by cumulative summation of 64 consecutive snapshot images. The local contrast was calculated for all exposure times using regions of 4 × 4 pixels. Averaging of multiple contrast images from the 64‐millisecond acquisition was done to improve the signal‐to‐noise ratio. The results show that with an effective implementation of the algorithm on an FPGA, contrast images at all exposure times can be calculated in only 28 milliseconds. The algorithm was applied to data recorded during a 5 minutes finger occlusion. Expected contrast changes were found during occlusion and the following hyperemia in the occluded finger, while unprovoked fingers showed constant contrast during the experiment. The developed setup is capable of massive data processing on an FPGA that enables processing of MELSCI data in 15.6 fps (1000/64 milliseconds). It also leads to improved frame rates, enhanced image quality and enables the calculation of improved microcirculatory perfusion estimates compared to single exposure time systems.      

动脉粥样硬化斑块中脂肪沉积的扫频光热波成像

动脉粥样硬化和易损斑块破裂在全球范围内具有最高的死亡率,超过传染病和癌症导致的死亡率的总和。动脉粥样硬化斑块是由一层很薄的”纤维帽”和导致血栓形成的脂质核心构成。光热波成像是基于对被目标发色团（本文中指脂肪沉积）吸收的光信号强度进行周期调制,从而实现对目标发色团释放的热（红外）信号的调制。这里,我们利用光热波成像来检测来自兔子动脉硬化模型的粥样硬化斑块中脂肪沉积的三维分布。波长为1210纳米的激光被用来靶向检测脂肪。动脉粥样硬化斑块组织在0．1到5赫兹连续扫频的激光的激发下发出光热波,光热波传播到样品表面形成红外辐射温度并被红外相机以25．6帧／秒的速度接收并录制20秒。红外相机上的每一个像素（总共256~256像素）在进行时域傅里叶变换以后得到强度和相位的频域光热波图像。某一特定频率的强度和相位光热波图像对应着脂肪沉积在动脉粥样硬化斑块样品中的横向和纵向分布。对强度和相位光热波图像的分析指出：光热波成像能够用来检测脂肪在动脉粥样硬化斑块中的三维分布,并且脂肪的分布和动脉粥样硬化斑块的形状特征有着紧密联系。     

Imaging technologies for the detection of multiple stains in proteomics

Laser-based scanners and charge-coupled device (CCD) camera systems are evolving to have greater functional capabilities for capturing images from a range of staining technologies used in gel electrophoresis and electroblotting. Digitizing Coomassie Brilliant Blue (CBB) stained gels and silver stained gels has now become possible using a laser-based gel scanner, the FLA-5000 fluorescent image analyzer system. Also, a simultaneous dual fluorescent imaging function has been incorporated into the FLA-5000 system, utilizing dichroic mirrors with both the optical system and the emission filter. In the workflow of routine proteomics research, the relationship between SYPRO dye staining and fluorescent detection using the FLA-5000 system have become symbiotic. Additionally in many cases, subsequent staining of the gel with CBB is useful for future research, and thus imaging instruments should be able to handle both staining formats. Digitizing the CBB stained gel can now be easily performed by the FLA-5000 fluorescent image analyzer system using a fluorescent board as an epi-illumination background. A cooled CCD camera system has the potential of imaging not only chemiluminescent membranes but also digitizing molecular weight markers and fluorescent detection of SYPRO dye-stained gels. With Multi Gauge software version 2.0 it is now a simple task to combine two images into one, as commonly required in dual detection experiments. The LAS-3000 system was designed to capture chemiluminescent images and to digitize the images automatically. Thus, new capabilities added to gel imaging systems make them capable of detecting and displaying multiple signals more conveniently.     

Imaging polarimetry for high throughput chiral screening

Imaging polarimetry was demonstrated as a highly parallel method of determining optical rotation of biochemical samples. The imaging polarimeter utilized a bright, uniform light source wavelength-filtered to near the sodium D line, a sample array flanked by inlet and analyzing polarizers, and a CCD camera fitted with an equal-perspective telecentric lens. The prototype apparatus was demonstrated to have an optical resolution better than 0.08 degrees. The potential for high throughput screening was demonstrated by imaging chiral solutions in 1536-well microtiter plates and by real-time monitoring of 30 simultaneous chiral enzymatic reactions. Improvements in polarizer and CCD technology may broadly expand the technique's applicability to fields such as directed evolution and combinatorial chemistry, where screening throughput is currently limiting for chiral applications.     

Video-rate bioluminescence imaging of matrix metalloproteinase-2 secreted from a migrating cell

Background

Matrix metalloproteinase-2 (MMP-2) plays an important role in cancer progression and metastasis. MMP-2 is secreted as a pro-enzyme, which is activated by the membrane-bound proteins, and the polarized distribution of secretory and the membrane-associated MMP-2 has been investigated. However, the real-time visualizations of both MMP-2 secretion from the front edge of a migration cell and its distribution on the cell surface have not been reported.

Methodology/Principal Findings

The method of video-rate bioluminescence imaging was applied to visualize exocytosis of MMP-2 from a living cell using Gaussia luciferase (GLase) as a reporter. The luminescence signals of GLase were detected by a high speed electron-multiplying charge-coupled device camera (EM-CCD camera) with a time resolution within 500 ms per image. The fusion protein of MMP-2 to GLase was expressed in a HeLa cell and exocytosis of MMP-2 was detected in a few seconds along the leading edge of a migrating HeLa cell. The membrane-associated MMP-2 was observed at the specific sites on the bottom side of the cells, suggesting that the sites of MMP-2 secretion are different from that of MMP-2 binding.

Conclusions

We were the first to successfully demonstrate secretory dynamics of MMP-2 and the specific sites for polarized distribution of MMP-2 on the cell surface. The video-rate bioluminescence imaging using GLase is a useful method to investigate distribution and dynamics of secreted proteins on the whole surface of polarized cells in real time.     

Functional Lung Imaging during HFV in Preterm Rabbits

Although high frequency ventilation (HFV) is an effective mode of ventilation, there is limited information available in regard to lung dynamics during HFV. To improve the knowledge of lung function during HFV we have developed a novel lung imaging and analysis technique. The technique can determine complex lung motion information in vivo with a temporal resolution capable of observing HFV dynamics. Using high-speed synchrotron based phase contrast X-ray imaging and cross-correlation analysis, this method is capable of recording data in more than 60 independent regions across a preterm rabbit lung in excess of 300 frames per second (fps). This technique is utilised to determine regional intra-breath lung mechanics of preterm rabbit pups during HFV. Whilst ventilated at fixed pressures, each animal was ventilated at frequencies of 1, 3, 5 and 10 Hz. A 50% decrease in delivered tidal volume was measured at 10 Hz compared to 1 Hz, yet at the higher frequency a 500% increase in minute activity was measured. Additionally, HFV induced greater homogeneity of lung expansion activity suggesting this ventilation strategy potentially minimizes tissue damage and improves gas mixing. The development of this technique permits greater insight and further research into lung mechanics and may have implications for the improvement of ventilation strategies used to support severe pulmonary trauma and disease.     

多光谱成像技术在生物医学中的应用进展

多光谱成像(multispectral imaging,MSI)技术在生物医学可视化方面是一种新技术,它结合了两个已建立的光学模块:成像学和光谱学。它的原理是基于液晶可调谐滤光片,从可见光到近红外波长(400-970nm)区域获取多光谱图像。自从MSI系统加上显微镜商品化以来,MSI已经成为一种快速发展的领域,可应用于细胞生物学、临床前药物开发和临床病理学等。国外已有大量关于MSI在生物医学中应用的研究报道,但国内报道少见。本文主要对多光谱成像的基本原理,近三年内该技术在生物医学领域的应用进展作一简要综述。     

PALM成像中激光强度和定位精度相关性的研究

近十年来,基于单分子定位的PALM成像技术快速发展,将显微镜的分辨率提高到了2-25nm。本文发现PALM成像过程中采用的激发光强度与成像的定位精度之间有密切的联系。我们分别选择了PALM成像使用的光激活荧光蛋白、光转换荧光蛋白和光开关荧光蛋白中最常用的荧光蛋白进行验证。实验发现伴随激光强度的增加,大部分荧光蛋白的光子数先升高然后趋于饱和,背景噪声几乎线性升高。进一步分析发现荧光蛋白的定位误差随着激光强度增强先降低后升高,因此选用合适的激光强度在PALM成像实验中至关重要。如何提高PALM成像的分辨率一直是科学家研究的热点,本研究内容可以指导研究人员在PALM成像中选用合适的激发光强度,从而得到高分辨率的图像。     

Imaging technique implemented in CellTracks system

BACKGROUND: We developed the CellTracks cell analysis system that, similar to flow cytometry, yields multiparameter information by which the cells can be differentiated. We describe the implementation of a laser scanning imaging method in the system. Image analysis of the cells improves the specificity of cell classification, especially in cases where the particular cells are found relatively infrequently and one has to discriminate between artifacts and real events. METHODS: Fluorescent images of immunomagnetically labeled and aligned cells are obtained by passing the cells through a laser focus. The laser focus is smaller than the objects and subsequent frames captured by a regular surveillance CCD camera with a frame grabber board represent different parts of the cells. Complete images of the cells are constructed by shifting each image with respect to each other and adding individual pixel values. RESULTS: The power of combining a fluorescent image with multiparametric data is demonstrated by imaging fluorescent and magnetically labeled beads and cells. The image gives additional information about the dye distribution across the objects. Changes in dye distribution as a function of time were observed in leukocytes labeled with the red fluorescent label, Oxazine750, which are imaged at different time intervals. CONCLUSIONS: An imaging technique implemented in the CellTracks system provides high-resolution fluorescent images of events previously identified by the system. The images of the fluorescent cells enhance the ability to classify rare events.     

利用非侵入性光学成像技术监测在体肿瘤的生长

利用电穿孔技术将gfp表达质粒转染于SP2／0细胞,通过G418筛选获得稳定表达GFP的小鼠骨髓瘤细胞株SP2／0—GFP细胞。将稳定的转化细胞移植于同系BALB／c小鼠后腿、耳部皮下及腹腔成瘤,每隔一天对小鼠肿瘤成像,跟踪肿瘤的生长过程。结果表明,探测到的GFP荧光强度和发光范围与肿瘤的生长或消亡相关,从而证明可以利用GFP作为肿瘤细胞的标记,结合光学成像技术,对肿瘤生长过程实时追踪。     

Hyperpolarized 129Xe MR Imaging of Alveolar Gas Uptake in Humans

Background

One of the central physiological functions of the lungs is to transfer inhaled gases from the alveoli to pulmonary capillary blood. However, current measures of alveolar gas uptake provide only global information and thus lack the sensitivity and specificity needed to account for regional variations in gas exchange.

Methods and Principal Findings

Here we exploit the solubility, high magnetic resonance (MR) signal intensity, and large chemical shift of hyperpolarized (HP) ¹²⁹Xe to probe the regional uptake of alveolar gases by directly imaging HP ¹²⁹Xe dissolved in the gas exchange tissues and pulmonary capillary blood of human subjects. The resulting single breath-hold, three-dimensional MR images are optimized using millisecond repetition times and high flip angle radio-frequency pulses, because the dissolved HP ¹²⁹Xe magnetization is rapidly replenished by diffusive exchange with alveolar ¹²⁹Xe. The dissolved HP ¹²⁹Xe MR images display significant, directional heterogeneity, with increased signal intensity observed from the gravity-dependent portions of the lungs.

Conclusions

The features observed in dissolved-phase ¹²⁹Xe MR images are consistent with gravity-dependent lung deformation, which produces increased ventilation, reduced alveolar size (i.e., higher surface-to-volume ratios), higher tissue densities, and increased perfusion in the dependent portions of the lungs. Thus, these results suggest that dissolved HP ¹²⁹Xe imaging reports on pulmonary function at a fundamental level.