Advanced calcium imaging techniques

Advanced calcium imaging techniques provide a powerful means to study intracellular calcium concentrations, crucial for various cellular processes. By visualizing calcium dynamics, Dr. Sandra H. Vaz investigates their role in neuronal signaling, synaptic plasticity, and essential brain functions. Her work enhances our understanding of these mechanisms, offering insights into both normal physiology and neurological disorders.

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1.   Why use this method?

Calcium Imaging is integral part of studying intracellular calcium concentrations,
a crucial mediator in diverse cellular processes occurring everywhere in the body starting from the cell cycle, to heartbeat. This approach provides insights into both spatial and temporal dynamics of calcium transients, enabling a deeper understanding for their regulatory mechanisms. It employs specialized indicators such as bioluminescent proteins, chemical calcium indicators, and genetically encoded calcium indicators (GECIs) to facilitate the visualization of calcium dynamics with high specificity and sensitivity.

In neuroscience, this method enables real-time visualisation of neuronal and glial calcium siganling activity, where calcium serves as second messenger in neuronal communication and synaptic plasticity, processes that underlie essential brain functions such as learning, memory and behaviour. Calcium Imaging provide non-invasive, targeting monitoring in living tissues, or even awake living animals, and is applicable across multiple scales. Moreover, it can be integrated with other techniques to establish links between observed calcium signals and functional outcomes.

2.   What you’ll need

Equipment

• Inverted Epifluorescence Microscope
  • Example: Axiovert 135 TV (Zeiss).
  • Equipped with a Xenon lamp for excitation.
• Filter System
  • Band-pass filters with wavelengths of 340 nm and 380 nm (e.g., Lambda DG-4plus/DG-5plus).
• CCD Camera
  • Example: Photometrics CoolSnap fx.
  • Used for capturing images of cells.

Consumables

• Ratiometric Dye
  • Fura-2 AM (5 µM).
  • Dye operates with two excitation wavelengths, and recordings are typically made at one wavelength.
• Cell Culture Plates
  • Multi-chamber plates or Petri dishes with glass coverslips at the bottom.

Note: The glass bottom is essential for proper imaging.

Solutions

• HEPES Solution (pH 7.4, adjusted with NaOH) (Gonçalves-Ribeiro et al., Glia, 2024):
  • 125 mM NaCl.1.25 mM NaH2PO4.3 mM KCl.10 mM D(+) glucose.2 mM CaCl2.2 mM MgCl2.
  • 10 mM HEPES.

Software

• MetaFluor® Software
  • Used for image acquisition and analysis.
• Microsoft Excel
  • For data processing and visualization.

3.   Step-by-step instructions

1. Cell Incubation with Fura-2AM

1.1. Prepare a 5 µM solution of Fura-2AM (Invitrogen, Fura-2, AM, cell permeant (1 mM Solution in Anhydrous DMSO) in HEPES buffer.

1.2. Incubate the cells in the prepared solution for 45 minutes at room temperature.

Note: The exact incubation time may vary depending on the cell type.

2. Mounting the Glass-Bottom Dishes

2.1. After incubation, carefully mount the glass-bottom dishes containing the cells onto the stage of an inverted epifluorescence microscope.
2.2. Ensure proper alignment and focus of the cells in the field of view.

3. Setting Up the Apparatus

3.1. Adjust the microscope settings to enable excitation with band-pass filters at 340 nm and 380 nm.
3.2. Confirm the operation of the CCD camera and configure the acquisition software.
3.3. Set the exposure times and calibration parameters for accurate fluorescence ratio measurements.

4. Image Acquisition

4.1. Capture images corresponding to:

• Excitation at 340 nm (F340).
• Excitation at 380 nm (F380).
• Calculate and record the F340/F380 ratio for ratiometric analysis.

4.2. Select the cells of interest for detailed analysis.

5. Data Analysis and Graph Construction

5.1. Use the software to construct graphs representing the cellular response over time to specific stimuli.
5.2. Export the recorded data to an Excel file for further analysis.

Protocol Variations

A. Bath Incubation

1. Incubate cells with a specific drug (e.g., GABA) in a bath solution.
2. Measure:
   • Calcium activity.
   • Frequency and amplitude of calcium transients.

B. Puff Application

1. Apply the specific drug (e.g., ACEA) using a puff pipette to target cells locally.
2. Treat cells with different agonists and antagonists (e.g., CGS).

C. Bath Incubation – Evaluation of Neural Stem Cell Differentiation

1. Establish a 5-minute baseline recording.
2. Incubate cells with 50 mM KCl for 2 minutes to induce depolarization in neuronal cells.
3. Wash out the KCl from the cells.
4. Incubate cells with 100 µM histamine for 2 minutes.

Record calcium activity and analyze transient responses

4.   Practical tips

• An inverted epifluorescence microscope allows observation of calcium signalling  in the cells that are only in the flat monolayer on a specific plate.
• The Lambda DG-4plus/DG-5plus filter is important because its fast shutter
  (500 microseconds) allows quick switching between wavelengths
  (200 ms each for 340 nm and 380 nm). This speed is necessary to capture fast calcium changes, which can be harder to detect with slower microscopes like the confocal one.
• MetaFluor is recommended software for this experiment. It is used for real-time ratio calculations between wavelengths, streamlining data analysis by removing the need for additional calculations. This enables direct assessment of key parameters, such as transient frequency and amplitude, during the analysis process.

5.   Critical appraisal & implications for future research

Calcium imaging is a powerful tool, providing insights into cellular and network-level neural activity. Unfortunately, it has several limitations and areas for improvement. While this method offers high spatial resolution, its temporal resolution may not match direct electrophysiological recordings, making it less suitable for capturing action potential events. Additionally, variability of indicators available for the method can cause hardship in choosing the correct one for given experiment, and when imaging session is prolonged there is a risk of phototoxicity and photobleaching.

Moreover, the practical constraints like access to advanced tools (high-quality imaging systems that are costly and not always accessible) and need for complex data analysis should be addressed.

Future research, to overcome these limitations and maximize the efficacy of calcium imaging, should focus on improving both the tools and methodologies associated with this technique. Development of new calcium indicators, that are more sensitive and photostable, could enable the creation of indicators optimized for specific experimental goals, allowing more accurate measurements of intracellular calcium dynamics. Refinements in imaging technologies, could minimize phototoxicity and improve temporal resolution. Additionally, making these tools more accessible through cost-effective imaging systems and open-source data analysis platforms will make calcium imaging more broadly available to researchers. By addressing these aspects, this method can continue to evolve as a vital tool in neuroscience, supporting both fundamental and translational approaches.

References

Gonçalves-Ribeiro J, Savchak OK, Costa-Pinto S, Gomes JI, Rivas-Santisteban R, Lillo A, Sánchez Romero J, Sebastião AM, Navarrete M, Navarro G, Franco R, Vaz SH. Adenosine receptors are the on-and-off switch of astrocytic cannabinoid type 1 (CB1) receptor effect upon synaptic plasticity in the medial prefrontal cortex. Glia. 2024 Jun;72(6):1096-1116. doi: 10.1002/glia.24518. Epub 2024 Mar 14. PMID: 38482984.

Rodrigues RS, Ribeiro FF, Ferreira F, Vaz SH, Sebastião AM, Xapelli S. Interaction between Cannabinoid Type 1 and Type 2 Receptors in the Modulation of Subventricular Zone and Dentate Gyrus Neurogenesis. Front Pharmacol. 2017 Aug 10;8:516. doi: 10.3389/fphar.2017.00516. PMID: 28848435; PMCID: PMC5554396.

Silva TP, Bekman EP, Fernandes TG, Vaz SH, Rodrigues CAV, Diogo MM, Cabral JMS, Carmo-Fonseca M. Maturation of Human Pluripotent Stem Cell-Derived Cerebellar Neurons in the Absence of Co-culture. Front Bioeng Biotechnol. 2020 Feb 14;8:70. doi: 10.3389/fbioe.2020.00070. PMID: 32117945; PMCID: PMC7033648.

This protocol is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license, allowing sharing and adaptation for non-commercial purposes with proper attribution.