Calcium is an important second messenger of a plethora of signaling pathways. Spatial and temporal variations in intracellular calcium levels can affect gene transcription, mRNA translation, and even post-translational protein modification. Normally, cytoplasmic calcium concentration is maintained at a much lower concentration (100-300 nM) compared to extracellular spaces (1-3 mM) and endoplasmic reticulum (10-100 µM). This is to ensure that calcium is a specific activator or inhibitor of certain signaling pathways.
The ability to measure changes in intracellular calcium levels is of great interest to researchers in several fields. One of the earliest fields to investigate calcium dynamics is neuroscience. Neuroscientists are interested in knowing how calcium dynamics within neural networks can affect synaptic plasticity that is implicated in memory and learning. Scientists studying cardiac and muscle cells are also highly interested in monitoring calcium levels, as these cells are excited by calcium. Calcium is also an essential second messenger and acts as a signal to produce insulin by beta cells of the pancreas. Recently, immunologists also found that calcium dynamics are implicated in antigen recognition by T-lymphocyte cells.
Below, I will introduce some tools scientists can consider for measuring changes in intracellular calcium levels.
This tool uses a setup known as patch clamp, which consists of a bunch of sensitive electronics and glass pipettes. A hollow glass pipette with a wire at its core is inserted into the cell of interest. When a Giga-ohm seal is formed between the pipette and the cell membrane, it means there is no ion leakage and the electrical wire is used to measure ion exchange between the cell and fluid in the pipette. To specifically measure only calcium dynamics, scientists typically add other blockers of ion channels. For instance, tetrodotoxin is added to prevent influx of sodium ions.
- Highly (and most) sensitive technique, as it can measure calcium dynamics in time scale of µs.
- Patch clamp is an expensive system and not available in many laboratories.
- There is a steep learning curve. One of the key skills is troubleshooting, as there are many electronics and mechanical components involved.
- Low throughput, as only one cell is typically monitored at a time. This also means significant time must be spent for statistically relevant results.
Chemical calcium indicators
Due to the high cost and technical complexity of operating a patch clamp system, there have been developments in creating chemicals such as Rhod-4 and Fluo-4 that can detect changes at the intracellular level. These chemicals are basically molecules that exhibit increases in fluorescence when bound to calcium. They also come in different fluorescence colors, allowing scientists to choose suitable ones.
- Cheap and extremely easy to use.
- Relatively useful if one is only interested in changes in calcium level and not time resolution.
- The best time resolution is ~5-10 ms, which makes these chemicals unsuitable for probing µs calcium dynamics in activities like neuronal communications.
- Signals can be affected by high autofluorescence or background fluorescence that reduces the signal-to-noise ratio. In such cases, the chemical, probenecid, is usually added. Probenecid extends the time in which calcium is retained in the cells to reduce the loss of fluorescence too quickly. However, this also reduces the time resolution/sensitivity to a few seconds time-scale.
Genetically encoded fluorescence calcium indicators
With the rise of optogenetics, there is also increased interest in using genetically encoded fluorescence calcium indicators. The most popular version is GCaMP, which is basically a fusion protein of green fluorescent protein and calmodulin molecules that binds to calcium. Cells can be transfected with GCaMP, and the changes in green fluorescence signals can be used to determine changes in intracellular calcium levels. Research over the last decade has improved the sensitivity of GCaMP down to sub-ms time-scale and also expanded the range of fluorescence wavelengths to include red signals.
- Plasmids for transfection are available on Addgene.org with various calcium sensitivity.
- Can be used in conjunction with optogenetics tools.
- Requires growth of bacterial host, plasmid extraction, and then plasmid transfection, which can be time-consuming. Furthermore, not all labs have the necessary equipment for bacterial culture.
- The efficiency of plasmid transfection can vary based on the technique. Additionally, some cells are resistant to transfection, such as T-cells, while the age of cells can also affect the transfection efficiency.
Quartzy is the world’s No. 1 lab management platform. We help scientists easily organize orders, manage inventory, and save money. We’re free and always will be. Visit Quartzy.com or reach out at firstname.lastname@example.org.
Interested in writing for The Q? Send us an email!
Andy Tay is a graduate student in the University of California, Los Angeles and an instructor in the National University of Singapore. His research focuses on magnetic neural stimulation and magnetotactic bacteria. He enjoys science communication and using the gym in his free time.