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

Summary

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.

Abstract

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 “.XLSX” 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.

Introduction

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 ".xlsx" 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.

Protocol

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'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.

1. Prepare sterile glass slides with an 8 mm diameter in a 24-well plate. Add 500 μL of poly-D-lysine (PDL) buffer (50 μg/mL in DPBS) to each well.
2. Incubate the slides at 37 °C for 1 h to allow coating, then discard the coating solution using a pipette.
3. Wash the slides once with DPBS and set them aside for later use.
4. Culture primary cells and cell lines separately according to their specific cultivation methods¹⁰.
5. 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.
6. For cells overexpressing plasmids, transfect^10,15 the target plasmid (1 μg/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.

2. Preparation of Fura-2/AM working solution

1. Add 50 μL of dimethyl sulfoxide (DMSO) to a tube containing 50 μg of Fura-2/AM powder and mix well to prepare a 1 μg/μL stock solution of Fura-2/AM.
2. Mix the Fura-2/AM stock solution and Pluronic F-127 into Hank'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 μg/mL. The Hank's buffer is prepared by adding 10 mM HEPES to 1x HBSS buffer.
3. Use aluminum foil to protect the Fura-2/AM working solution from light.

3. Cell pretreatment for single-cell Ca²⁺ imaging

1. Transfer the glass slides with cells to a new 24-well plate containing Hank's buffer for washing.
2. Discard the buffer using a pipette and add 500 μL of Fura-2/AM working buffer to each well.
3. Incubate at room temperature in the dark for 30 min to allow probe loading.
4. Remove the Fura-2/AM working buffer and wash the cells three times with Hank's buffer to eliminate excess Fura-2/AM. The cells are now ready for use.

4. Starting the Ca²⁺ imaging system

NOTE: In this study, a fluorescence microscope is used for Ca²⁺ imaging.

1. 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.

5. Cellular Ca²⁺ response procedure

1. Open the fluorescence imaging software (see Table of Materials).
   1. Choose Protocol, then select File, followed by Load Protocol. Select the protocol and click on OK.
   2. Configure the experiment (on the menu list).
   3. Select New Experiment.
2. 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.
3. Remove the Fura-2/AM treated cell slides and place them in the chamber containing Hank's buffer.
4. Start the perfusion system.
5. Select the 20x DIC objective and adjust the focus under white light.
6. Click on Cfg Exp on the taskbar.
   1. Select the desired fluors for imaging.
   2. Determine the acquisition frequency and display settings on the screen (Acquire: check for 340, 380, GFP; Acquire Interval: 1 s; Save Interval: 1 s).
7. Focus
   1. On the experiment control panel, click on the Focus button.
   2. Adjust the acquisition time (usually 100 ms) and gain as needed, then "save for this wave."
      NOTE: For UV light, use gain instead of exposure time.
   3. Choose the desired wavelength for focusing (e.g., 380) and click on Start Focusing.
   4. Switch the view from the binoculars to the computer.
   5. Ensure that a faint greenish color is visible through the binoculars.
   6. 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.
   7. Check the fluorescence intensity of the cells during the focusing process.
   8. 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.
   9. Once a good focus is achieved, press the button on the microscope to switch the view to the computer.
   10. Refocus as needed to obtain the sharpest image on the computer, then click on Stop Focusing.
8. Alternate procedure for finding GFP positive cells
   1. Use the 340/380 procedure first to achieve a good focus on the cells.
   2. Select FITC and then click on Start Focusing.
   3. Use the stage controller to find GFP-positive cells.
   4. Switch the view to the computer and then click on Stop Focusing.
9. Region selection
   1. Click on the Region button in the menu bar.
   2. 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.
   3. Click on Acquire Images, then OK.
   4. A new window will appear; select cells by clicking on the oval tool and then clicking over the cell.
   5. 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.
   6. Undo a region by right-clicking on the circle and then selecting Delete Region.
   7. Select a background sample as the last region and record its ID number.
   8. Click on Save and then Done.
10. Background subtraction
    1. Click on the menu button References.
    2. 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.
    3. Check the box Subtract References and then click on OK.
11. Log data
    1. 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.
    2. Ensure that a prompt appears asking for the preferred data file type; selecting .xlsx format is recommended.
    3. An ".xlsx" type worksheet will open. Minimizing the worksheet will prevent it from taking up screen space.
12. Data acquisition
    1. In the control panel's Time Lapse section, set the data acquisition interval to 1 s.
       NOTE: This can be adjusted according to actual needs.
    2. 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.
    3. Perform a series of treatments on the cells according to the experimental requirements.
    4. To observe the temperature response of the cells, heat the buffer in the perfusion system to an appropriate temperature using a temperature controller.
    5. To observe the effects of drugs on cells, perfuse or manually add drug-containing buffer and record the cell changes.
    6. 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.
    7. Save and analyze the data.
13. Click on File and Close Experiment to end the experiment, and select No on the dialog box to save the protocol.
14. Open a new experiment by clicking on New on the menu, and repeat the process.
15. Shut down
    1. Close the software and transfer the data from the computer.
    2. Reverse the start-up procedure.
    3. Log the hours on the sign-up sheet and clean up any mess.
16. Data analysis
    1. Represent the intracellular Ca²⁺ concentration by the Fura-2 340/380 ratio or convert to the corresponding Ca²⁺ concentration.

Results

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 ...

Discussion

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 proteins^10,19. This technique measures changes and absolute values of cellular Ca²⁺ concentrations and play...

Materials

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