Name: applications of spatio-temporal mapping and particle analysis techniques to quantify intracellular ca2+ signaling in situ
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 9 min 34 s
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.

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>ImageJ software</td><td>NIH</td><td>1.5a</td><td>ImageJ 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&lt;sup&gt;+/Cre-ERT2&lt;/sup&gt;</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 ...

applications-spatio-temporal-mapping-particle-analysis-techniques-to

Nanyang Technological University

Research

JoVE Journal

Biology

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

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Neuroscience

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

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+ ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-Resolution Imaging Technique for Detecting Ca2+ Microdomains in T Cells

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

Author Spotlight: Unraveling the Initial Activation of the Adaptive Immune Response for Therapeutic Intervention

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

Immunology and Infection

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Measuring Fast Calcium Fluxes in Cardiomyocytes

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Dissection of Local Ca²⁺ Signals in Cultured Cells by Membrane-targeted Ca²⁺ Indicators

Automated Analysis of Dynamic Ca²⁺ Signals in Image Sequences

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Imaging Local Ca²⁺ Signals in Cultured Mammalian Cells

Imaging Initial Ca²⁺ Microdomains in Primary T Cells

Spatiotemporal Analysis of Microglial Ca²⁺ Activity at Single-Cell Resolution

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells