Funding was provided by the NIDDK, via P01 DK41315.

The authors have nothing to disclose.

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 morphology of ICC-DMP make them well suited to STM analysis derived from the STMs outlined above. However, this analysis is not well suited to ICC-MY that are stellate shaped and connected in a network (Figure 3A). Furthermore, the Ca2+ signaling patterns in ICC-MY are more complex, manifesting as propagating CTCs from multiple sites of origin throughout the ICC-MY network. Thus, in order to quantify the activity occurring in the entire ICC-MY network within a FOV, particle (PTCL) analysis was implemented using custom made software (Volumetry, version G8d, GWH, operable on Mac OS, contact Grant Hennig grant.hennig@med.uvm.edu regarding inquires for Volumetry access and use).
STM analysis allows all Ca2+ events within single cells and within all of the cells in a FOV to be analyzed critically across a range of spatial and temporal parameters. The protocol described illustrates how these techniques can be applied to ICC-DMP of the mouse small intestine. By fully quantifying Ca2+ signaling in ICC-DMP, as shown in Figure 2B-G, Ca2+ signaling patterns have been characterized in detail37. These analyses have been applied to recordings where ICC-DMP undergo interventions to finely quantify the effects of blocking or stimulating Ca2+ release / Ca2+ influx / neurotransmission pathways37,39,40,41. These techniques can be easily applied to other intact tissue preparations. For example, STM analysis as described here has been utilized to identify new mechanistic pathways involved in the generation of intracellular Ca2+ waves recorded in urethral smooth muscle in situ57.
The preparation of STMs in Volumetry requires some caution, as the function in Volumetry that creates the STM from the drawn ROI (Figure 2A) is an average value of intensity. Thus, the amplitude of Ca2+ signals could potentially be diluted if the ROI is drawn wider or longer than the Ca2+ event or cell of interest. Thus, users should be careful to draw ROIs that are as tightly fitting as possible to the Ca2+ signals or particular cell that they are analyzing in order to alleviate this issue. Similarly, creating STMS using single pixel linescans in ImageJ means that accurate mapping of Ca2+ events is subject to the proximity of the Ca2+ signal to the drawn line. Such concerns are minor in thin spindle shaped cells such as ICC-DMP, however other cell types with a more stellate or round morphology may make this type of analysis inappropriate to map all Ca2+ signals accurately. When preparing STMs for analysis, regardless of whether they were made in Volumetry or with ImageJ linescans, there are a few areas to be highlighted for troubleshooting purposes. It is important to change the image quality to 32-bit before performing any calibration on the STMs. Failure to do so, or doing so after calibrating for F/F0 can lead to inconsistent measurements across experiments. Always check the image quality of the STM, which is stated in the top area of the white border of the STM itself when opened with ImageJ. Another potential area of inconsistency is selecting the F0 value when calibrating for amplitude. It is vital that for selecting the region for F0, that it covers an area of the cell that is uniform and in focus. For this reason, areas of the cell that have an unstable basal fluorescence or that change due to movement or other artefacts are not ideal and rigorous motion stabilization protocols should be employed in these cases.
Within in situ or cultured preparations containing interconnected cellular networks, such as ICC-MY in the small intestine, PTCL analysis provides a streamlined technique to quantify complex, subcellular Ca2+ events occurring in the network. Moreover, it also allows all Ca2+ events in the network within a given FOV to be analyzed, rather than using arbitrary ROIs, which only provide information on frequency and intensity within the ROI. An advantage of the PTCL analysis described here is that by applying differential and Gaussian smoothing filters to recordings, a large amount of noise can be removed from movies that may contain contaminating light from cells not of interest or due to non-dynamic bright spots or inclusions. It is important to note that the amount of differentiation applied to recordings will depend largely on the acquisition rate used by the experimenter. Differentiating movies as described in the protocol provides a means of applying a filter to the movie to remove high frequency noise from recordings. Applying a differentiation value of &#39;2&#39; when acquiring at 33FPS works well to remove background noise while maintaining good signal to noise (if the value is too low, noise will be picked up but signal to noise will be compromised if the value is too high). The differentiation value applied should be increased with faster acquisition rates, for example at 100 FPS, a differentiation value of &#39;7&#39; gives approximately the same signal to noise ratio as a value of &#39;2&#39; to a 33FPS recording. Experimenters will need to optimize these settings accordingly for their preparations and recording conditions.
The thresholding protocol described in Figure 3D allows a consistent thresholding procedure to be applied to different recordings made on different systems with different acquisition software. This flexibility allows data from multiple investigators working on different systems to compile their recordings into the same datasets. By using the FLAG system in Volumetry, PTCL analysis allows the visualization and quantification of individual Ca2+ firing sites within a network in detail. Information can be gathered on the number of initiation sites, the size of the site in pixels and &#181;m2, and the average duration of PTCLs occurring at that site. This PTCL analysis allowed the first characterization of CTC activity in the small intestine at a sub-cellular level, and, using the different FLAGs in Volumetry software, PTCLs at both the network and individual firing site level were quantified in intact tissue preparations from Kit-Cre-GCaMP3 mice38. From these initial observations, this analysis has been further utilized to study novel Ca2+ influx pathways in GI ICC-MY such as store-operated-Ca2+-entry42 and the role of mitochondrial Ca2+ signalling on GI pacemaking41. Much like STM analysis described above, PTCL analysis can be easily adapted to different intact preparations other than that described in this protocol. For example, a recent study used PTCL analysis to study novel rhythmic Ca2+ events occurring in the intact cellular networks of the lamina propria of the rat urinary bladder58,59 and thus could be easily applied to other complex, intact cellular systems such as neuronal systems. While this paper focused on Ca2+ imaging in intact tissues with GECIs, these analysis techniques can also be run on isolated cells and tissues loaded with traditional Ca2+ indicator dyes. The STM based analysis has been used to successfully quantify localized Ca2+ signals and Ca2+ waves from spindle shaped interstitial cells and smooth muscle cells from a variety of preparations11,60,61,62,63. Furthermore, the PTCL analysis routines described here have also been applied to in situ network preparations visualized with Cal 52058,59. However, these studies also retain the disadvantages of such dye loading protocols such as ambiguous cell identification and problems with signal to noise.
The examples illustrated above demonstrate that both STM and PTCL analysis are highly malleable techniques that can be used to quantify complex Ca2+ signaling in a diverse range of intact tissue preparations. The approaches offer many benefits over traditional ROI based intensity plots that have been routinely used previously and should provide investigators with more valuable quantitative information on Ca2+ signaling than could be previously achieved.

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)37,39,40,41. It is possible to record brief (&#60;100 ms), localized Ca2+ events that were not possible with membrane permeable Ca2+ indicators. Spatio-temporal mapping with Volumetry or ImageJ software can be used to generate STMs of all Ca2+ events within cells in situ. Using this approach, Ca2+ events in an entire FOV can be visualized and mapped (Figure 2B,C), rather than just recording the limited activity of a single ROI. These methods can be extended to each cell within a given FOV, ensuring representative data collection from all cells and providing quantitative information about relative amplitudes, transient durations, rate of rise and fall of transients, etc. (Figure 2E,F). STM analysis, as opposed to ROI-based intensity plots, also provide the ability to monitor and record spatial characteristics of Ca2+ signaling, such as spatial spread and propagation velocity, as shown in Figure 2G. This information can be amassed to provide a rather complete view of Ca2+ signaling behaviors in cells in their native environments (Figure 2H).
PTCL analysis can be used to quantify more complex Ca2+ signaling behaviors, such as those occurring within interconnected cellular networks. An example of this application is provided by the analysis performed on ICC-MY (Figure 3A). Usually in such complex preparations, background noise and signal to noise can be an issue. However, using Volumetry software to apply differential and smoothing filters on movies of Ca2+ activity and then applying noise threshold protocols to filter out noise (Figure 3B-D) background noise can be removed from complex recordings of dynamic activity. Using PTCL analysis such as those shown in Figure 3E-G, quantitative information about Ca2+ signaling can be calculated by measuring the PTCL area, PTCL count and PTCL size which indicate spatial ranges of activation of Ca2+ signals in a FOV. These data can be compiled as shown in Figure 3H and analyzed statistically, as appropriate. Figure 4 illustrates how PTCL analysis allows in depth quantification of sub-cellular Ca2+ signaling by examining the location and firing probabilities of Ca2+ firing sites. By allocating PTCLs into different FLAGs based on their temporal characteristics, initiating PTCLs can be accurately mapped as shown in Figure 4C-E and a wealth of hard data acquired on the number of initiation sites (referred to as &#39;domains&#39;), the size of the site in pixels and &#181;m2, probability of each initiation site firing either once or multiple times during each CTC (given as a%), the average duration and size of PTCLs occurring at the initiation site, the number of CTC cycles (as defined in step 4.23) and the% of firing sites that fired during each CTC cycle. These techniques allow a high level of data mining and quantification of in situ Ca2+ signals occurring within an intact cellular network that are not possible with ROI-based analyses.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58989/58989fig1.jpg" /> 
Figure 1: Generation of KitGCaMP6f mice. Schematic diagram of how Ai95 (RCL-GCaMP6f)-D (GCaMP6f mice) were crossed with c-Kit+/Cre-ERT2 (Kit-Cre mice) to generate Kit-Cre-GCaMP6f mice. These mice are injected with tamoxifen at ages of 6-8 weeks to induce Cre Recombinase and subsequent GCaMP6f expression exclusively in ICC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58989/58989fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58989/58989fig2.jpg" /> 
Figure 2: Analysis of stochastic Ca2+ signals in ICC-DMP using spatio-temporal mapping (STM). (A) Representative image of several ICC-DMP from the small intestine of a Kit-Cre-GCaMP6F mouse in situ. A green ROI indicates the size and orientation of ROI to draw around a single ICC-DMP within the FOV to create an STM in Volumetry. (B) STM of Ca2+ activity in the ICC-DMP highlighted in panel A after it has been properly calibrated for amplitude, space and time. (C) The same STM shown in panel B after it has been color coded with a lookup table (QUBPallete). (D) Expanded image of ICC-DMP Ca2+ transients displayed on a color coded STM, indicating where to draw a line through a Ca2+ event across its time (x) axis to create a plot profile of its activity in ImageJ. (E) Plot profile of the Ca2+ event highlighted in panel D, indicating where lines shown be drawn to accurately measure the amplitude and duration of the event. (F) Plot profile of the Ca2+ event highlighted in panel D, indicating where lines shown be drawn to accurately measure the rate of rise and rate of fall of the event. (G) Expanded image of ICC-DMP Ca2+ transients displayed on a color coded STM, indicating where to draw a line through a Ca2+ event across its space (y) axis to accurately measure its spatial spread. (H) Representative histograms of pooled data from ICC-DMP illustrating how to graphically display the amplitude, duration, and spatial spread values acquired from following the above steps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58989/58989fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58989/58989fig3.jpg" /> 
Figure 3: Quantification of CTCs in ICC-MY using particle based analysis. (A) Representative image of an ICC-MY network from the small intestine of a Kit-Cre-GCaMP6F mouse in situ. (B) Image taken from the recording shown in panel A after it has undergone a differential filter of &#916;t = &#177;66&#8211;70 ms and a Gaussian filter of 1.5 x 1.5 &#181;m, StdDev 1.0. (C) Image taken from the video in B after thresholding was completed with PTCLs above threshold shown in red. (D) Traces of PTCL count and mean PTCL size in a thresholding protocol to eliminate noise in the movie shown in panel B. PTCLs were created using a flood-fill algorithm that marked the structure of all adjoining pixels that had intensities above threshold, Ca2+ transient PTCLs were larger than noise PTCLs. The threshold at which large numbers of small sized noise PTCLs emerged and began to reduce the mean size of PTCLs can be used as a common threshold for all recordings. (E) Representative image from the coordinate-based Ca2+ PTCL file created from the thresholded recording in C. (F) Representative image taken from the PTCL file of E after a screening criteria of &#62;6 &#181;m2 (diameter ~2 &#181;m or smaller) was applied; PTCLs above this limit are flagged (FLAG 1) as light purple particles and considered valid PTCLs. (G) Heat map showing the total PTCLs (FLAG 1) for the entire recording of the video shown in panel F, with total PTCLs summated with colors representing occurrence throughout the recording (warm colors indicate increased occurrence at that location). (H) Representative traces of PTCL area (blue) and PTCL count (red) derived from the PTCL file created in panel A-G. Representative histograms of pooled data from several experiments are shown below the traces. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58989/58989fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58989/58989fig4.jpg" /> 
Figure 4: Analysis of Ca2+ firing sites in ICC-MY using particle based analysis. (A) Representative image taken from the PTCL file of Figure 3E after FLAG 1 PTCLS are further refined into FLAG 3, the flag status for Ca2+ firing sites. FLAG 3 PTCLs are displayed as lime green (only those PTCLs that did not overlap with any particles in the previous frame but overlap with particles in the next 70 ms were considered firing sites). (B) Heat map showing the total PTCLs (FLAG 3) for the entire recording of the video shown in panel A, with total PTCLs summed with colors representing occurrence throughout the recording (warm colors indicate increased occurrence at that location). (C) Representative map of Ca2+ firing sites shown in panel B, with each different firing site allocated a different identifying color. (D) Representative traces of PTCL area (blue) and PTCL count (red) derived from the PTCL file created in Figure 3A-G. (E) An occurrence map of the activity of individual firing sites. Each firing site within the FOV is displayed as a colored block in its own &#39;lane&#39; against time. (F) Representative histograms of pooled data from several experiments are shown illustrating values accumulating for Ca2+ firing site firing probability / CTC and the number of Ca2+ firing sites in a FOV. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58989/58989fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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9.2K Views.  University of Nevada Reno School of Medicine. 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.

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

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 ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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 ...

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

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Chapters in this video

Techniques for Imaging Ca²⁺ Signaling in Human Sperm

Visualization of Vascular Ca²⁺ Signaling Triggered by Paracrine Derived ROS

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

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

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

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

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Automated Analysis of Dynamic Ca²⁺ Signals in Image Sequences

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

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