Types and Applications of Calcium Indicators for Calcium Imaging

Overview

Calcium ions play a crucial role within cells, and accurately measuring their dynamics is extremely important in cellular biology and neuroscience research.

However, many researchers may be unsure about which calcium indicator to use.

There are many different types of calcium indicators, including Fura-2, Fluo-4, and Rhod-2. Each indicator has different wavelength characteristics and dissociation constants, so it is necessary to use them appropriately depending on the specific research purpose and experimental conditions.

This article explains the types of reagents used in calcium imaging and how to choose them.

What are Calcium Indicators?

Calcium indicators are chemical substances used to measure the concentration of calcium ions (Ca²⁺) within cells. They are also known as fluorescent calcium dyes or calcium-sensitive dyes.

Calcium ions are essential for physiological processes such as muscle contraction and neural transmission. Calcium indicators emit fluorescence when they bind to calcium ions, allowing the visualization of calcium concentration changes.

Types of calcium indicators

There are several types of calcium indicators.

(1) Calcium-sensitive fluorescent dye using calcium chelating agent

These include Ca²⁺-selective chelators like EGTA and BAPTA. Examples of such dyes are Quin-2/AM, developed by R.Y. Tsien in 1980, and others like Fura-2, Fluo-3, Indo-1, and Rhod-2.

(2) Calcium-sensitive luminescent proteins such as Aequorin

These proteins emit weak light when they bind to Ca²⁺.

(3) Calcium-sensitive fluorescent proteins encoded by genes such as Cameleon and GCaMP (Genetically Encoded Calcium Indicator: GECI)

These use Ca²⁺-binding calmodulin to create fluorescent proteins.

This article focuses on calcium-sensitive fluorescent dyes and explains their principles and types.

Mechanism of Action of Calcium Indicators

Calcium indicators are primarily composed of chelators and fluorescent substance. When Ca²⁺ binds to the chelator part of the indicator, the fluorescent properties change, resulting in an increase in fluorescence intensity or a shift in the fluorescence spectrum.

BAPTA, for example, is a derivative of EGTA with a benzene ring replacing the methylene group. BAPTA has high Ca²⁺ selectivity similar to EGTA but is less affected by pH changes and has faster Ca²⁺ binding and release kinetics.

Mechanism of Fluorescence Generation

Fluorescent substances absorb light at specific wavelengths. This light energy lifts the electrons within the fluorescent material from the ground state to the excited state.

The absorbed energy moves the electron to a higher energy excited state. This state is unstable, and the electrons quickly lose energy and try to return to the ground state.

When the electrons return to their ground state, they emit part of the absorbed light energy as light. This emitted light typically has a longer wavelength and therefore lower energy than the absorbed light. This phenomenon is known as Stokes shift.

The emitted light is observed as fluorescence. The wavelength and intensity of fluorescence depend on the properties of the fluorophore and the surrounding environmental conditions.

The most efficient wavelengths for these transitions are the maximum excitation and emission wavelengths.

Types of calcium indicators

There are different types of calcium indicators. There are two main types: fluorescent indicators and absorption indicators, but the absorption type is currently not widely used.

Fluorescent types can be further divided into (1) those whose fluorescence intensity changes according to changes in ion concentration, and (2) those whose excitation or fluorescence spectrum changes according to changes in ion concentration.

(1) Fluorescence intensity changes according to changes in ion concentration

(1-1) 1-wavelength excitation / 1-wavelength fluorescence (e.g. Fluo-3, Fluo-4, Rhod-2, etc.)

(2) Excitation or fluorescence spectra change according to changes in ion concentration

(2-1) 2-wavelength excitation / 1-wavelength fluorescence (e.g. Fura-2)

(2-2) 1-wavelength excitation / 2-wavelength fluorescence (e.g. Indo-1)

List of Major Calcium Indicators: High Affinity

List of Major Calcium Indicators: Low Affinity

Which Calcium-Sensitive Fluorescent Dye Should I Choose?

1. Non-cytotoxicity.
2. Sufficiently fast response rate compared to the changes in Ca²⁺ concentration to be measured
3. Affinity for Ca²⁺ (Kd value) close to physiological levels
4. Excitation light does not damage cells
5. Excitation and fluorescence wavelengths of the reagent match the optical path characteristics of the microscope
6. Minimal interference from other ions

Which calcium-sensitive fluorescent dye should I choose?

The fluorescent probe reagent must be introduced into the cells to be measured, but because it is water-soluble, it cannot penetrate the cell membrane as is.

Therefore, methods such as using modified reagents that increase membrane permeability or directly injecting the reagent into the cell are employed.

1. Microinjection
- Used in cells with low esterase activity
-- A microelectrode is inserted into the cell
-- Perfusion from a patch pipette

2. Introduction via Lipophilic Esters
- AM derivatives, which are commonly used, are created by esterifying the probe reagent with an Acetoxymethyl (AM) group to make it lipophilic. The AM derivative itself is non-fluorescent and has no binding ability with free ions.
- Since it easily permeates the cell membrane, when cells are immersed in a solution containing the AM derivative, it passes through the membrane and enters the cell without binding to extracellular ions. Once inside, the AM group is cleaved by esterases, allowing the probe to bind to ions and become water-soluble, thus remaining in the cytoplasm. In tissue samples where probe introduction is difficult, using surfactants like Cremophore EL can sometimes be effective.

Sample data

Calcium imaging data using MiCAM02

Data acquired with MiCAM02-HR after injecting calcium dye into a single cell