Acknowledgment is given to Bailong Xiao from Tsinghua University for sharing the single-cell Ca2+ imaging system and the temperature control operating system, as well as for the support and assistance in this project. This research was funded by the National Natural Science Foundation of China (32000705), the Young Elite Scientists Sponsorship Program by the China Association of Chinese Medicine (CACM-(2021&#8211;QNRC2&#8211;B11)), Fundamental Research Funds for the Central Universities (2020&#8211;JYB&#8211;XJSJJ&#8211;026), (2024-JYB-KYPT-06).

The experimental methods were approved by and followed the IACUC guidelines of Tsinghua University and Beijing University of Chinese Medicine. This protocol introduces single-cell Ca2+ imaging methods for various cell types, including primary keratinocytes isolated from the skin of several newborn mice (within three days of birth, with sex-randomized littermates, C57BL/6 mice). Details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Cell preparation
NOTE: Primary cells, cell lines with endogenous target genes, or those transfected with overexpressed plasmids are all suitable for single-cell Ca2+ imaging. The plasmids used in this study were obtained from Professor Xiao Bailong&#39;s laboratory at Tsinghua University. These plasmids were constructed by incorporating sequences of GFP fluorescent protein with human STIM1, DsRed fluorescent protein with human Orai1, mRuby fluorescent protein with rabbit TRPV1, as well as the red fluorescent protein mCherry into phage plasmid vectors10.
<ol>
	<li>Prepare sterile glass slides with an 8 mm diameter in a 24-well plate. Add 500 &#956;L of poly-D-lysine (PDL) buffer (50 &#956;g/mL in DPBS) to each well.</li>
	<li>Incubate the slides at 37 &#176;C for 1 h to allow coating, then discard the coating solution using a pipette.</li>
	<li>Wash the slides once with DPBS and set them aside for later use.</li>
	<li>Culture primary cells and cell lines separately according to their specific cultivation methods10.</li>
	<li>Seed cells onto the prepared 24-well plate at a density of approximately 1.5 x 105 cells per well. Use the cells for Ca2+ imaging once they have adhered to the coverslip.</li>
	<li>For cells overexpressing plasmids, transfect10,15 the target plasmid (1 &#956;g/well) using Lipofectamine 2000 (or Lipofectamine 3000) transfection reagent at a 1:1 ratio, and incubate in a cell culture incubator for about 24 h. 
	NOTE: A longer incubation time may be necessary for larger expressed proteins.</li>
</ol>
2. Preparation of Fura-2/AM working solution
<ol>
	<li>Add 50 &#956;L of dimethyl sulfoxide (DMSO) to a tube containing 50 &#956;g of Fura-2/AM powder and mix well to prepare a 1 &#956;g/&#956;L stock solution of Fura-2/AM.</li>
	<li>Mix the Fura-2/AM stock solution and Pluronic F-127 into Hank&#39;s buffer containing 1.3 mM Ca2+. 
	NOTE: The final concentration of Fura-2/AM and Pluronic F-127 in the working solution is 2.5 &#956;g/mL. The Hank&#39;s buffer is prepared by adding 10 mM HEPES to 1x HBSS buffer.</li>
	<li>Use aluminum foil to protect the Fura-2/AM working solution from light.</li>
</ol>
3. Cell pretreatment for single-cell Ca2+ imaging
<ol>
	<li>Transfer the glass slides with cells to a new 24-well plate containing Hank&#39;s buffer for washing.</li>
	<li>Discard the buffer using a pipette and add 500 &#956;L of Fura-2/AM working buffer to each well.</li>
	<li>Incubate at room temperature in the dark for 30 min to allow probe loading.</li>
	<li>Remove the Fura-2/AM working buffer and wash the cells three times with Hank&#39;s buffer to eliminate excess Fura-2/AM. The cells are now ready for use.</li>
</ol>
4. Starting the Ca2+ imaging system
NOTE: In this study, a fluorescence microscope is used for Ca2+ imaging.
<ol>
	<li>Start the following components in sequence: DG4 light (xenon lamp), camera, white-light source, microscope stage controller, microscope, computer, and fluorescence imaging software. 
	NOTE: If detecting the activation of ion channels by temperature, also turn on the following components: perfusion system, heating system, temperature controller, and liquid circulation heating/cooling device.</li>
</ol>
5. Cellular Ca2+ response procedure
<ol>
	<li>Open the fluorescence imaging software (see Table of Materials).
	<ol>
		<li>Choose Protocol, then select File, followed by Load Protocol. Select the protocol and click on OK.</li>
		<li>Configure the experiment (on the menu list).</li>
		<li>Select New Experiment.</li>
	</ol>
	</li>
	<li>Mount the perfusion chamber on the microscope. 
	NOTE: Always lower the objective fully when mounting or removing chambers using the rough focus knob to prevent damage to the objective.</li>
	<li>Remove the Fura-2/AM treated cell slides and place them in the chamber containing Hank&#39;s buffer.</li>
	<li>Start the perfusion system.</li>
	<li>Select the 20x DIC objective and adjust the focus under white light.</li>
	<li>Click on Cfg Exp on the taskbar.
	<ol>
		<li>Select the desired fluors for imaging.</li>
		<li>Determine the acquisition frequency and display settings on the screen (Acquire: check for 340, 380, GFP; Acquire Interval: 1 s; Save Interval: 1 s).</li>
	</ol>
	</li>
	<li>Focus
	<ol>
		<li>On the experiment control panel, click on the Focus button.</li>
		<li>Adjust the acquisition time (usually 100 ms) and gain as needed, then &#34;save for this wave.&#34; 
		NOTE: For UV light, use gain instead of exposure time.</li>
		<li>Choose the desired wavelength for focusing (e.g., 380) and click on Start Focusing.</li>
		<li>Switch the view from the binoculars to the computer.</li>
		<li>Ensure that a faint greenish color is visible through the binoculars.</li>
		<li>Focus on the cells, using rough focus first to get the objective close, then fine focus. 
		NOTE: The microscope will beep if it gets too close to the stage. If this happens, lower the objective, realign the plate on the stage, and focus again. Minimize time spent focusing to reduce laser-induced cell damage.</li>
		<li>Check the fluorescence intensity of the cells during the focusing process.</li>
		<li>Adjust the fluorescence intensity of the cells by modifying the exposure time and gain. 
		NOTE: The exposure time and gain for 340 and 380 must be consistent.</li>
		<li>Once a good focus is achieved, press the button on the microscope to switch the view to the computer.</li>
		<li>Refocus as needed to obtain the sharpest image on the computer, then click on Stop Focusing.</li>
	</ol>
	</li>
	<li>Alternate procedure for finding GFP positive cells
	<ol>
		<li>Use the 340/380 procedure first to achieve a good focus on the cells.</li>
		<li>Select FITC and then click on Start Focusing.</li>
		<li>Use the stage controller to find GFP-positive cells.</li>
		<li>Switch the view to the computer and then click on Stop Focusing.</li>
	</ol>
	</li>
	<li>Region selection
	<ol>
		<li>Click on the Region button in the menu bar.</li>
		<li>Select the illumination type of choice (340/380/FITC/TRITC). FITC and TRITC filters are selected for GFP and mCherry/DsRed/mRuby, respectively, for cells overexpressing the targeted plasmids; otherwise, select Fura-2. 
		NOTE: Avoid selecting cells that are in poor condition or dead, such as those that are obviously rounded or have overexposed fluorescent proteins.</li>
		<li>Click on Acquire Images, then OK.</li>
		<li>A new window will appear; select cells by clicking on the oval tool and then clicking over the cell.</li>
		<li>Select the desired cells under the fluorescence carried by the target protein (FITC or TRITC). Select the control cells that do not express the target protein under the 380 nm condition.</li>
		<li>Undo a region by right-clicking on the circle and then selecting Delete Region.</li>
		<li>Select a background sample as the last region and record its ID number.</li>
		<li>Click on Save and then Done.</li>
	</ol>
	</li>
	<li>Background subtraction
	<ol>
		<li>Click on the menu button References.</li>
		<li>Indicate the number of the background region. 
		NOTE: If the background was the last region selected, entering a very high number will automatically switch to the last region picked.</li>
		<li>Check the box Subtract References and then click on OK.</li>
	</ol>
	</li>
	<li>Log data
	<ol>
		<li>Click on the Log Data button on the experiment control panel. 
		NOTE: Images are saved only if there is a desire to replay the experiment later; normally, just checking the data box is sufficient.</li>
		<li>Ensure that a prompt appears asking for the preferred data file type; selecting .xlsx format is recommended.</li>
		<li>An &#34;.xlsx&#34;&#160;type worksheet will open. Minimizing the worksheet will prevent it from taking up screen space.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>In the control panel&#39;s Time Lapse section, set the data acquisition interval to 1 s. 
		NOTE: This can be adjusted according to actual needs.</li>
		<li>Click on Zero Clock and Acquire on the experiment control panel to start the experiment. Baselines will be visible on a graph indicating each of the selected cell regions.</li>
		<li>Perform a series of treatments on the cells according to the experimental requirements.</li>
		<li>To observe the temperature response of the cells, heat the buffer in the perfusion system to an appropriate temperature using a temperature controller.</li>
		<li>To observe the effects of drugs on cells, perfuse or manually add drug-containing buffer and record the cell changes.</li>
		<li>After data acquisition is complete, click on Pause. 
		NOTE: Data acquisition can be paused, and the clock can be reset at any time during the experiment.</li>
		<li>Save and analyze the data.</li>
	</ol>
	</li>
	<li>Click on File and Close Experiment to end the experiment, and select No on the dialog box to save the protocol.</li>
	<li>Open a new experiment by clicking on New on the menu, and repeat the process.</li>
	<li>Shut down
	<ol>
		<li>Close the software and transfer the data from the computer.</li>
		<li>Reverse the start-up procedure.</li>
		<li>Log the hours on the sign-up sheet and clean up any mess.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Represent the intracellular Ca2+ concentration by the Fura-2 340/380 ratio or convert to the corresponding Ca2+ concentration.</li>
	</ol>
	</li>
</ol>

Single Cell Calcium Imaging for Channel Activation Studies

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+airway+mucus+secretion+and+pathophysiology+of+hypersecretion.">Physiology of airway mucus secretion and pathophysiology of hypersecretion.</a> Respir Care. 52 (9), 1134-1146 (2007).</li><li>Mitra, R., Hasan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Store-operated+Ca(2+)+entry+regulates+neuronal+gene+expression+and+function.">Store-operated Ca(2+) entry regulates neuronal gene expression and function.</a> Curr Opin Neurobiol. 73, 2022 (2022).</li><li>Landstrom, A. P., Dobrev, D., Wehrens, X. H. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+capsaicin+receptor:+A+heat-activated+ion+channel+in+the+pain+pathway.">The capsaicin receptor: A heat-activated ion channel in the pain pathway.</a> Nature. 389 (6653), 816-824 (1997).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stim1+thermosensitivity+defines+the+optimal+preference+temperature+for+warm+sensation+in+mice.">Stim1 thermosensitivity defines the optimal preference temperature for warm sensation in mice.</a> Cell Res. 29 (2), 95-109 (2019).</li><li>Bharate, S. S., Bharate, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+thermoreceptor+trpm8+by+cooling+compounds.">Modulation of thermoreceptor trpm8 by cooling compounds.</a> ACS Chem Neurosci. 3 (4), 248-267 (2012).</li><li>Coste, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezo+proteins+are+pore-forming+subunits+of+mechanically+activated+channels.">Piezo proteins are pore-forming subunits of mechanically activated channels.</a> Nature. 483 (7388), 176-181 (2012).</li><li>Behera, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+Ca2++dynamics+using+a+ubiquitin-10+promoter-driven+yellow+cameleon+3.6+indicator+reveal+reliable+transgene+expression+and+differences+in+cytoplasmic+Ca2++responses+in+arabidopsis+and+rice+(oryza+sativa)+roots.">Analyses of Ca2+ dynamics using a ubiquitin-10 promoter-driven yellow cameleon 3.6 indicator reveal reliable transgene expression and differences in cytoplasmic Ca2+ responses in arabidopsis and rice (oryza sativa) roots.</a> New Phytol. 206 (2), 751-760 (2015).</li><li>Nakai, J., Ohkura, M., Imoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+signal-to-noise+ca(2+)+probe+composed+of+a+single+green+fluorescent+protein.">A high signal-to-noise ca(2+) probe composed of a single green fluorescent protein.</a> Nat Biotechnol. 19 (2), 137-141 (2001).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+analysis+of+stim1+thermal+sensation.">Molecular mechanism analysis of stim1 thermal sensation.</a> Cells. 12 (22), 2613 (2023).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATF6+safeguards+organelle+homeostasis+and+cellular+aging+in+human+mesenchymal+stem+cells.">ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells.</a> Cell Discov. 4, 2 (2018).</li><li>Jiang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanosensitive+piezo1+channel+mediates+heart+mechano-chemo+transduction.">The mechanosensitive piezo1 channel mediates heart mechano-chemo transduction.</a> Nat Commun. 12 (1), 869 (2021).</li><li>Tao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Orai1-mediated+store-operated+Ca(2+)+channel/calpain+signaling+contributes+to+high+glucose-induced+podocyte+injury.">Enhanced Orai1-mediated store-operated Ca(2+) channel/calpain signaling contributes to high glucose-induced podocyte injury.</a> J Biol Chem. 298 (6), 101990 (2022).</li><li>Guo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+er+ion+homeostasis+maintained+by+an+er+anion+channel+clcc1+contributes+to+als-like+pathologies.">Disruption of er ion homeostasis maintained by an er anion channel clcc1 contributes to als-like pathologies.</a> Cell Res. 33 (7), 497-515 (2023).</li><li>Gouin, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trpv1+and+trpa1+in+cutaneous+neurogenic+and+chronic+inflammation:+Pro-inflammatory+response+induced+by+their+activation+and+their+sensitization.">Trpv1 and trpa1 in cutaneous neurogenic and chronic inflammation: Pro-inflammatory response induced by their activation and their sensitization.</a> Protein Cell. 8 (9), 644-661 (2017).</li><li>Barreto-Chang, O. 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The authors declare that they have nothing to disclose.

The application of single-cell Ca2+ imaging systems is extensive, enabling the study of Ca2+ signals in various cell types, including keratinocytes, stem cells16, liver cells, heart cells17, podocytes18, immune cells, and cell lines overexpressing target proteins10,19. This technique measures changes and absolute values of cellular Ca2+ concentrations and play...

Ca2+ plays a crucial role in cellular signal transduction, regulating various cellular functions such as muscle contraction1, nerve conduction2, secretion3, and gene expression4, thereby influencing multiple physiological processes. Abnormal Ca2+ concentrations can lead to diseases such as arrhythmias5, coagulation disorders6, and hormonal imbalances7. Therefore, studying the mechanisms of intracellular Ca2+ concentration changes is of paramount importance.
Various ion channels are involved in the regulation of Ca2+ concentration in cells, including highly Ca2+-selective calcium release-activated calcium (CRAC) channels8 and non-selective cation channels of the TRP family9. These ion channels can be activated by stimuli such as temperature10, compounds, and active ingredients found in traditional Chinese medicine11, playing a crucial role in various Ca2+-related physiological processes.
Effective monitoring of intracellular Ca2+ concentration changes is essential for studying Ca2+-related ion channels, particularly in the field of traditional Chinese medicine, where calcium signaling regulation plays a central role in many therapeutic approaches. Currently, the primary methods for measuring intracellular Ca2+ can be categorized into two types: electrical and optical measurements. The electrical measurement approach uses the patch-clamp technique to assess changes in cell membrane potential due to Ca2+ influx12.
In optical measurement, fluorescent probes specifically bind to Ca2+, allowing researchers to track changes in cellular fluorescence intensity. Common optical methods include fluorescent protein-based and fluorescent dye-based techniques. In fluorescent protein-based methods, researchers can overexpress Ca2+-sensitive fluorescent proteins like Cameleon13 and GCaMP14 in cells and monitor fluorescence signal changes using fluorescence microscopy or flow cytometry to observe shifts in cytoplasmic Ca2+ concentrations. Additionally, researchers can overexpress these proteins in mice and use two-photon fluorescence microscopy for real-time in vivo or tissue-level monitoring of intracellular Ca2+ concentrations, providing high resolution and deep tissue penetration10.
For fluorescent dye-based methods, commonly used Ca2+ probes include Fluo-3/AM, Fluo-4/AM, and Fura-2/AM10. Researchers incubate cells in a solution containing these fluorescent probes, which cross the cell membrane and are cleaved by intracellular esterases to form active compounds (e.g., Fluo-3, Fluo-4, and Fura-2) that remain within the cell. These probes exhibit minimal fluorescence in their free ligand form but emit strong fluorescence when bound to intracellular Ca2+, thereby indicating changes in cytoplasmic Ca2+ concentrations. Compared to other fluorescent proteins and dyes, Fura-2 is typically excited at 340 nm and 380 nm wavelengths. When bound to intracellular free Ca2+, Fura-2 undergoes an absorption shift, moving the excitation wavelength peak from 380 nm to 340 nm, while the emission peak near 510 nm remains unchanged. There is a quantitative relationship between fluorescence intensity and bound Ca2+ concentration, allowing calculation of intracellular Ca2+ concentration by measuring the fluorescence intensity ratio at these two excitation wavelengths. Ratio measurements reduce the effects of photobleaching, fluorescent probe leakage, uneven loading, and differences in cell thickness, yielding more reliable and reproducible results (Figure 1).
Single-cell Ca2+ imaging systems primarily utilize microscopy techniques and the Ca2+ indicator Fura-2/AM to detect intracellular Ca2+ concentrations. These systems comprise a fluorescence microscope, a Ca2+ imaging light source, and fluorescence imaging software, enabling real-time, quantitative monitoring of Ca2+ changes in the cytoplasm of multiple cells simultaneously (up to 50 cells per field of view). Results are saved in &#34;.xlsx&#34;&#160;format for subsequent analysis. The system offers a rapid analysis speed (approximately 1 min for analyzing a group of cells within one field of view) and generates intuitive change curves, significantly enhancing detection efficiency. Single-cell Ca2+ imaging is an essential technical approach for studying Ca2+-related channels and has considerable value in ion channel-related biomedical research. Its application in single-cell calcium imaging technology is expected to greatly advance research on the mechanisms underlying traditional Chinese medicine.

<table><tbody><tr><td>Camera</td><td>Nikon</td><td></td><td></td></tr><tr><td>Capsaicine</td><td>Sigma</td><td>211275</td><td></td></tr><tr><td>CL-100 temperature controller</td><td>Warner Instruments</td><td></td><td></td></tr><tr><td>Cyclopiazonic Acid (CPA)</td><td>Sigma</td><td>C1530</td><td></td></tr><tr><td>DG-4 light</td><td>Sutter Instrument Company</td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)&amp;nbsp;</td><td>Amresco</td><td>231</td><td></td></tr><tr><td>DPBS</td><td>Thermofisher</td><td>14190144</td><td></td></tr><tr><td>Fluorescence imaging software (MetaFluor, Paid software)&amp;nbsp;</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Fura-2/AM</td><td>Invitrogen</td><td>F1201</td><td></td></tr><tr><td>HBSS buffer</td><td>Gibco</td><td>14175103</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Invitrogen</td><td>L3000008</td><td></td></tr><tr><td>Pluronic F-127</td><td>&amp;nbsp;Beyotime</td><td>ST501</td><td></td></tr><tr><td>poly-D-lysine</td><td>&amp;nbsp;Beyotime</td><td>ST508</td><td></td></tr><tr><td>SC-20 liquid circulation heating/cooling device&amp;nbsp;</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>White-light source</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

single-cell calcium imaging for studying the activation of calcium ion channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca2+ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca2+) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. By measuring the 340/380 ratio, the Ca2+ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca2+ changes in multiple cells. The results are stored in &#8220;.XLSX&#8221; format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca2+ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca2+ imaging.

Temperature response detection 
Primary keratinocyte 
Primary keratinocytes were isolated from newborn mice and prepared according to established protocols10. These cells were seeded into 24-well plates containing glass slides. Following the loading of the Fura-2 probe, the focus was adjusted under the microscope at a wavelength of 380 nm to achieve a clear visualization of cell morphology, as illustrated in Figure 2A. If the probe ...

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...

single-cell-calcium-imaging-for-studying-activation-calcium-ion

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

473 Views.  Beijing University of Chinese Medicine. This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Video: Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

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, ...

Biology

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

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 ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Summary

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Chapters in this video

Functional Calcium Imaging in Developing Cortical Networks

In vivo Neuronal Calcium Imaging in C. elegans

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Imaging Local Ca²⁺ Signals in Cultured Mammalian Cells

Imaging Initial Ca²⁺ Microdomains in Primary T Cells

Subcellular Imaging of Neuronal Calcium Handling In Vivo

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Calcium Imaging in Neurons