Name: applications of spatio-temporal mapping and particle analysis techniques to quantify intracellular ca2+ signaling in situ
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ at JoVE.com

Funding was provided by the NIDDK, via P01 DK41315.

All animals used and the protocols carried out in this study were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno.
1. Generation of KitGCaMP6f Mice
<ol>
	<li>Cross Ai95 (RCL-GCaMP6f)-D (GCaMP6f mice) and c-Kit+/Cre-ERT2 (Kit-Cre mice) to generate ICC specific GCaMP6f expressing animals (Kit-Cre-GCaMP6f mice). ...

Ca2+ imaging of specific types of cells within intact tissues or within networks of cells often reveals complex patterns of Ca2+ transients. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events and kinetics of these events as possible. STM and PTCL analysis provide an opportunity to maximize the amount of quantitative data yielded from recordings of this type.
The narrow, spindle shaped morpholo...

Ca2+ is a ubiquitous intracellular messenger which controls a wide range of cellular processes, such as muscle contraction1,2, metabolism3, cell proliferation3,4,5, stimulation of neurotransmitter release at nerve terminals6,7, and activation of transcription factors in the nucleus.7 Intracellular Ca2+ signals often take the form of transient elevations in cytosolic Ca2+, and these can be spontaneous or arise from agonist stimulation depending on the cell type8. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity, and amplitude can provide the biological information required for intracellular signalling5,7,9. Cytoplasmic Ca2+ signals can result from the influx of Ca2+ from the extracellular space or via Ca2+ release from the endoplasmic reticulum (ER) via Ca2+ release channels such as ryanodine receptors (RyRs) and inositol-tri-phosphate receptors (IP3Rs)10. RyRs and IP3Rs may both contribute to the generation of Ca2+ signals and the concerted opening of these channels, which combined with various Ca2+ influx mechanisms can result in a myriad of Ca2+ signalling patterns that are shaped by the numbers and open probability of Ca2+ influx channels, the expression profile of Ca2+ release channels, the proximity between Ca2+ influx and release channels, and expression and distribution of Ca2+ reuptake and extrusion proteins. Ca2+ signals may take the form of uniform, long lasting, high intensity global oscillations that may last for several seconds or even minutes, propagating intracellular and intercellular Ca2+ waves that may cross intracellular distances over 100 &#181;m10,11,12,13,14,15,16, or more brief, spatially localized events such as Ca2+ sparks and Ca2+ puffs that occur on tens of millisecond timescales and spread less than 5 &#181;m17,18,19,20.
Fluorescent microscopy has been used widely to monitor Ca2+ signalling in isolated and cultured cells and in intact tissues. Traditionally, these experiments involved the use of fluorescent Ca2+ indicators, ratiometric and non-ratiometric dyes such as Fura2, Fluo3/4 or Rhod2, among others 21,22,23. These indicators were designed to be permeable to cell membranes and then become trapped in cells by the cleavage of an ester group via endogenous esterases. Binding of Ca2+ to the high affinity indicators caused changes in fluorescence when the cells and tissues were illuminated by appropriate wavelengths of light. The use of cell permeable Ca2+ indicators greatly enhanced our understanding of Ca2+ signaling in living cells and permitted spatial resolution and quantification of these signals that was not possible by assaying Ca2+ signals through other means, such as electrophysiology. However, traditional Ca2+ indicators have several limitations, such as photobleaching that occurs over extended recording periods24. While newer Ca2+ indicator dyes such as Cal520/590 have greatly improved signal to noise ratios and the ability to detect local Ca2+ signals25, issues with photobleaching can still remain a concern for some investigators26,27,28. Precipitous photobleaching also restricts the magnification, rate of image acquisition, and resolution that can be used for recordings, as increased objective power and higher rates of image acquisition require increased excitation light intensity that increases photobleaching.
These limitations of traditional Ca2+ indicator dyes are exacerbated when recording Ca2+ signals in situ, for example when recording intracellular Ca2+ signals from intact tissues. Due to the problems above, visualization of Ca2+ signals in situ using cell permeable Ca2+ indicators has been limited to low power magnification and reduced rates of image capture, constraining the ability of investigators to record and quantify temporally or spatially restricted subcellular Ca2+ signals. Thus, it has been difficult to capture, analyze, and appreciate the spatial and temporal complexity of Ca2+ signals, which can be important in the generation of desired biological responses, as outlined above. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from selected regions of interest (ROI) within a field of view (FOV). The arbitrary choice of the number, size and position of ROIs, dependent on the whim of the researcher, can severely bias the results obtained. As well as inherent bias with ROI analysis, this approach ignores much of the important signaling information contained in the FOV, as dynamic Ca2+ events within an arbitrarily chosen ROI are selected for analysis. Furthermore, analysis of ROIs fails to provide information about the spatial characteristics of the Ca2+ signals observed. For example, it may not be possible to distinguish between a rise in Ca2+ resulting from a propagating Ca2+ wave and a highly localized Ca2+ release event from tabulations of Ca2+ signals within an ROI.
The advent of genetically encoded Ca2+ indicators (GECIs) has radically changed how Ca2+ imaging can be performed in situ29,30,31,32,33. There are several advantages to using GECIs over dyes. The most important perhaps is that expression of GECI can be performed in a cell specific manner, which reduces unwanted background contamination from cells not of interest. Another advantage of GECIs over traditional Ca2+ indicators is that photobleaching is reduced (as fluorescence and consequentially photobleaching only occurs when cells are active), as compared to dye-loaded specimens, particularly at high magnification and high rates of image capture34. Thus, imaging with GECIs, such as the GCaMP series of optogenetic sensors, affords investigators the ability to record brief, localized sub-cellular Ca2+ signals in situ and investigate Ca2+ signaling in cells within their native environments that have not been possible previously. To maximize recovery of information from such high-resolution recordings, appropriate spatial and temporal analysis of the Ca2+ signals is required. It should be noted that while GECIs can offer some clear advantages, recent studies have revealed that Ca2+ imaging can be successfully performed from large populations of different neurochemical classes of neurons simultaneously using conventional Ca2+ indicators that are not genetically encoded into the animal35. This approach used post hoc immunohistochemistry to reveal multiple different classes of neurons firing at high frequency in synchronized bursts, and avoided the potential that genetic modifications to the animal may have interfered with the physiological behavior the investigator seeks to understand35,36.
The protocols outlined in this paper facilitate more complete analysis and quantification of Ca2+ signals recorded from cells in situ using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signalling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC). ICC are specialized cells in the gastrointestinal (GI) tract that exhibit dynamic intracellular Ca2+ signaling, as visualized using mice expressing GCaMPs37,38,39,40,41,42. Ca2+ transients in ICC are linked to activation of Ca2+-activated Cl- channels (encoded by Ano1) that are important in regulating the excitability of intestinal smooth muscle cells (SMCs)43,44,45. Thus, the study of Ca2+ signaling in ICC is fundamental to understanding intestinal motility. The murine small intestine offers an excellent example for this demonstration, as there are two classes of ICC that are anatomically separated and can be visualized independently: i) ICC are located in the area between the circular and longitudinal smooth muscle layers, surrounding the myenteric plexus (ICC-MY). These cells serve as pacemaker cells and generate the electrical activity known as slow waves46,47,48,49; ii) ICC are also located amongst a plexus rich in the terminals of motor neurons (deep muscular plexus, thus ICC-DMP). These cells serve as mediators of responses to enteric motor neurotransmission37,39,40,50. ICC-MY and ICC-DMP are morphologically distinct, and their Ca2+ signaling behaviors differ radically to accomplish their specific tasks. ICC-MY are stellate in shape and form a network of interconnected cells via gap junctions51,52. Ca2+ signals in ICC-MY manifest as brief and spatially localized Ca2+ release events occurring at multiple sites asynchronously through the ICC-MY network as visualized within a FOV (imaged with a 60X objective)38. These asynchronous signals are organized temporally into 1 second clusters that, when tabulated together, amount to a net 1 s cellular rise in Ca2+. These signals propagate cell-to-cell within the ICC network and therefore organize Ca2+ signaling, generated from sub-cellular sites, into a tissue wide Ca2+ wave. Temporal clustering and summation of Ca2+ signals in ICC-MY has been termed Ca2+ transient clusters (CTCs)38. CTCs occur rhythmically (e.g. quite similar durations and similar periods between CTCs) 30 times per minute in the mouse. Conversely, ICC-DMP are spindle shaped cells, some with secondary processes, that distribute between SMCs and varicose nerve processes and do not independently form a network51,52. ICC-DMP form gap junctions with SMCs, however, and function within this greater syncytium, known as the SIP syncytium53. Ca2+ signals occur at multiple sites along the lengths of cells, but these transients are not entrained or temporally clustered, as observed in ICC-MY37. Ca2+ signals in ICC-DMP occur in a stochastic manner, with variable intensities, durations and spatial characteristics. The protocols below, using the example of Ca2+ signaling in ICC-MY and ICC-DMP, describe techniques to analyze complex signaling in specific types of cells in situ. We utilized the inducible Cre-Lox p&#160;system to express GCaMP6f exclusively in ICC, after inducing activation of Cre-Recombinase (Cre) driven by an ICC specific promoter (Kit).

<table>
	<tbody>
		<tr>
			<td>Image J software</td>
			<td>NIH</td>
			<td>1.5a</td>
			<td>Image J software</td>
		</tr>
		<tr>
			<td>Volumetry</td>
			<td>GWH</td>
			<td>Volumetry 8Gd</td>
			<td>Custom made analysis software</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, Deerfield, IL, USA</td>
			<td>NDC 10019-360-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>GCaMP6F mice</td>
			<td>Jackson Laboratory</td>
			<td>Ai95 (RCL-GCaMP6f)-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Kit-Cre mice</td>
			<td>Gifted From Dr. Dieter Saur</td>
			<td>c-Kit+/Cre-ERT2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-Aaper</td>
			<td>SDA 2B-6</td>
			<td></td>
		</tr>
	</tbody>
</table>

applications of spatio-temporal mapping and particle analysis techniques to quantify intracellular ca2+ signaling in situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Using Kit-Cre-GCaMP6F mice (Figure 1), dynamic Ca2+ signaling behaviors of ICC in the gastrointestinal tract can be imaged in situ. With confocal microscopy, high-resolution images of specific populations of ICC can be acquired without contaminating signals from other populations of ICC within the same tissue but in anatomically distinct planes of focus (Figure 2A)37,<sup class="xref"...

Watch this Scientific Journal Video about Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ at JoVE.com

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

The capacity to acquire large amounts of data from calcium imaging experiments has drastically improved, especially with tissue imaging experiments. Our protocols describe appropriate analysis techniques to quantify and describe these data sets. Our protocols generate a range of spatial, temporal, and intensity values that are intrinsic to calcium signaling in entire tissues and this can be lost in more rudimentary-based analysis.One hour before the imaging transfer a small intestine harvested from a mouse with interstitial cells of Cajal or ICC that specifically express a genetically-encoded calcium indicator to the stage of a spinning disk confocal microscope. And continuously perfuse the tissue with 37 degrees Celsius Krebs-Ringer bicarbonate or KRB solution. Using a 60 to 100 X magnification locate the stellate-shaped myenteric plexus ICC or ICC-MY between the circular and longitudinal smooth muscle layers of the small intestine.Then locate the spindle-shaped deep muscular plexus ICC or ICC-DMP in a single plane between the circular smooth muscle layer and the submucosal plexus and save the movies as a stack of TIFF images. To create spatiotemporal maps or STMs of the ICC-DMP calcium activity open the image stack in ImageJ, and click Image, Type and 32-bit to modify the image quality. To create a line scan right-click on the line selection tool and select segmented line.Using single left clicks draw a line along the mid access of an individual ICC-DMP, double-clicking when the entire cell has been outlined. Select Image, Stacks and Reslice. A pop-up window will appear.Select Rotate 90 degrees to orient the line scan from left to right so that it can be calibrated and read against time on the x-axis with space on the y-axis and click OK.The spatiotemporal map will appear. To accurately measure the amplitude of the calcium signals from the map, select rectangular selection and draw a region of interest around the area within the STM that displays the most uniform and least intense area of fluorescence. Click Analyze and Histogram to obtain a mean value of the intensity within the selected region of interest and click Edit, Selection, Select All to select the entire STM.Next, click Process, Math and Divide and enter the mean value of the intensity with the selected STM region of interest in the pop-up box. The STM will turn black. Correct this by clicking Image, Adjust, Brightness/Contrast, and Auto in the pop-up window.The line scan will now be calibrated for the amplitude with the intensity of the fluorescence expressed as the fluorescence divided by the zero fluorescence. The STM will display the number of frames in the TIFF stack on the x-axis and the number of pixels representing the length of the cell on the y-axis. To quantify the temporal and spatial data from the calcium signals click Image and Properties and enter the appropriate values to fully calibrate the STM for space and time in the pop-up window.To analyze the individual calcium events click Straight Line and click and drag on the STM to draw a straight horizontal line through the center of a calcium event parallel to the x-axis. Then click Analyze and Plot Profile. A box will appear showing the plot profile of the calcium event.For particle-based analysis of the ICC-MY calcium signaling data, open the movie file in Volumetry and right-click to select STK Filter, Differentiate. Right-click again to input a value to differentiate. Press Enter and left-click to initiate the differentiation.To create particles, use the middle mouse button to scroll through the movie to select a 20 to 40 frame section that includes a quiescent period, followed by the occurrence of a calcium transient cluster and 20 to 40 frames that follow right after the cluster. When all of the frames have been selected right-click in the movie window to access STK OPS Ramp DS particle info and click to run a particle analysis routine that progressively ramps the threshold from the maximum to the minimum intensity. To ensure a successful particle analysis, ensure that you incorporate as much pre-calcium transient cluster non-activity, and as much post-calcium transient cluster non-activity as you can.This will help to boost the signal-to-noise ratio. The analysis will be displayed graphically in the plot window as plot of noise and in the traces window as three color traces with the green trace representing the number of particles, the red trace representing the average particle size, and the blue trace representing the absolute intensity threshold. To histogram balance the plot of particle noise press H on the keyboard.Then press the right bracket key to cycle through color schemes until the background is white with the color traces on top. Press F on the keyboard one time to bring up a measuring tool. Then press F a second time to mark a single vertical line in the scroll bar of the traces window on the plot at the intersection where the colored plots begin to shift to the right-hand side.Within the traces window use the middle mouse button to scroll from the inflection point of the red trace to the marked white vertical line. After noting the displayed Y average, click the middle mouse button again inside the traces window to deselect the blue trace. In the movie window click C to bring up a color wheel, and D to adjust the threshold.Valid particles will now be shown in red, and those that saturate will be white. Use the left mouse button to scroll until the white areas are removed and use the middle mouse button to adjust the yellow numerical value in the color wheel to the Y average value to assign everything in red as an active calcium transient particle at the determined threshold point. To save the data as a coordinate-based particle file right-click to access STK 3D, save particle zero, and left-click to save the file.To create particle activity heat maps open the saved particle file and right-click in the movie window to access particle STKops, StatMap Flag and enter flag assignments to analyze. Click to apply the analysis and a heat map showing the total particles for the entire length of recording with different colors representing the percent occurrence throughout the recording. Right-click to save the heat map as a TIFF file.Then right-click in the movie window to access particle measure, particle stats STK equals, and enter a flag assignment for the analysis. A series of traces will be generated in the trace window. STM analysis provides the ability to monitor and record the spatial characteristics of calcium signaling, such as the spatial spread and propagation velocity.This information can be amassed to provide a rather complete view of calcium signaling behaviors within cells in their native environments. Using particle analysis, quantitative information about calcium signaling can be calculated by measuring the particle area and the particle count, which together can be used to determine the spatial ranges of calcium signal activation within a specified field of view. Particle analysis also allows the in-depth quantification of subcellular calcium signaling through examination of the location and firing probabilities of calcium firing sites.By allocating the particles into different flags based on their temporal characteristics, the initiating particles can be accurately mapped. And a wealth of hard data acquired on the number of initiation sites, the size of the site in pixels and micrometers, probability of each initiation site firing either once or multiple times during each calcium transient cluster, and the percentage of firing sites that fire during each calcium transient cluster cycle can be obtained. Consistency is the key to success in this protocol.All data sets and all movies must be treated exactly the same to ensure reliable, repeatable results. This is especially true when dealing with particle files. Our protocols allow a deep characterization of calcium signaling in situ, and a range of statistical methods, when used appropriately, can assess differences between a range of spatial and temporal parameters.These analysis techniques in our protocol have allowed researchers to ask and answer previously unaddressable questions relating to intestinal pacemaking, and intestinal innervation, as well as the neuronal control of the lower urinary tract.

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Biology

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...