Erratum: High speed sCMOS‐based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

In the article by M.B. Sikkel et al. (doi: 10.1002/jbio.201500193), published in J. Biophotonics 9 , 311–323 (2016), an error occurred in the computer code that was used to generate Figure 3. This erratum is published to correct Figure 3, the calculated value of t_geom and the experimentally determined value of t_optics in the text of the article.     

Broad frequency sensitivity and complex neural coding in the larval zebrafish auditory system

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活细胞钙动态的共聚焦扫描显微镜检测技术

共聚焦激光扫描显微镜（Confocal Laser Scarming Microscope,CLSM）广泛应用于活细胞内钙敏感探针标记的钙水平的动态测量。较之传统的显微镜CLSM在钙成像分析上有着不可比拟的优越性,但也存在一些缺陷,近些年陆续出现了一些针对这些缺陷的改善措施,如比率法、葡聚糖探针及其他一些新技术与共聚焦显微镜的联合应用等,并且出现了诸如双光子显微镜等新型激光共聚焦显微镜。随着共聚焦钙成像技术的不断发展进步,其今后的应用前景将会越越广阔。     

Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems.     

Behavioral state-dependent modulation of insulin-producing cells in Drosophila

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Characterization of the Life Cycle and Heteromeric Nature of the Macronucleus of the Ciliate Chilodonella uncinata Using Fluorescence Microscopy

Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon indicate the presence of a heteromeric macronucleus, marked by separate DNA‐rich and DNA‐poor regions. Here, we study the life cycle of C. uncinata using confocal laser scanning microscopy with 4′,6‐diamidino‐2‐phenylindole staining, which allows us to differentiate nuclear dynamics of the micronucleus and the macronucleus during life‐cycle stages. We photo‐documented various stages and confirmed aspects of the development of the new macronucleus previously characterized by electron microscopy. We further reveal the heteromeric structure of the macronucleus with Z‐stacks and three‐dimensional (3D) reconstructions. We find no evidence for the presence of an endosome at the center of the macronucleus during vegetative growth. In addition to illustrating the life cycle of this ciliate, the approaches developed for this study will enable additional comparative analyses of nuclear dynamics using fluorescence microscopy.     

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant’s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed ‘residence time’) of the dot-like structures. The use of VAEM is discussed in the context of this example.     

AFM探测缺铁性贫血病人的红细胞

在纳米量级上探测红细胞生理病理特性对于揭示疾病的起源、早期诊断和有效的治疗是十分重要的。疾病可以从分子水平上扰乱红细胞的形貌和功能。缺铁性贫血病人的红细胞的形貌具有严重的表面畸形。通过高分辨率的原子力显微镜成像研究了健康人和缺铁性贫血病人红细胞的整个形貌和表面膜的差异。结果表明,红细胞的形貌参数（例如细胞的峰、谷、峰谷差、表面起伏和标准方差）可以探测健康和病理的红细胞。因此,红细胞的形貌信息可望成为诊断健康和疾病,以及评估治疗效果的重要指标。     

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.     

A Paradoxical Kind of Sleep in Drosophila melanogaster

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Color Processing in the Early Visual System of Drosophila

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Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system

Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human OR 17-209 or OR 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each OR was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional ORs. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.     

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state¹ and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish^2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.     

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.     

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.     

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins^1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N₂; <2 ppm O₂) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.     

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.     

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4'',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.