Calcium imaging

What is calcium imaging?
Two-wavelength imaging method
Filter wheel method
Simultaneous two-wavelength measurement method
Advantages of the simultaneous two-wavelength measurement method
Differences between single-wavelength imaging and two-wavelength ratio imaging
Example images
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Calcium imaging related products
Other experimental methods

What is calcium imaging?

Calcium imaging refers to a method of observing the concentration and flow of calcium ions within cells using a Ca²⁺ probe. A characteristic of this probe is that its fluorescence brightness changes in accordance with the concentration of calcium ions in the cell. Since calcium ions are involved in processes such as neurotransmitter release and muscle contraction, imaging the fluorescence intensity changes of calcium ions allows us to observe their flow and activity within cells.

There are two major types of probes for calcium imaging: Fluo-3, Fluo-4, and GCaMP, whose fluorescence intensity changes in response to changes in calcium ion concentration, and Fura-2 and Indo-1, whose excitation light and fluorescence spectrum change in response to changes in calcium ion concentration. The former measures the fluorescence intensity at one wavelength, while the latter measures the ratio of two wavelengths.

Fura-2

Figure 1: Imaging example of calcium imaging

Two-wavelength imaging method

Filter wheel method

Schematic diagram of an epifluorescence microscope using a filter wheel

Figure 2: Schematic diagram of an epifluorescence microscope using a filter wheel

In the filter wheel method, the excitation filter and fluorescence (absorption) filter holders are wheel-shaped holders that can accommodate various wavelengths by rotating a wheel containing multiple filters. Multi-wavelength imaging is possible by changing the position of the filter wheel during imaging.

Simultaneous two-wavelength measurement method

Schematic diagram of an epifluorescence microscope using W-VIEW GEMINI image splitting optics system

Figure 3: Schematic diagram of an epifluorescence microscope using W-VIEW GEMINI image splitting optics system

The simultaneous two-wavelength measurement method uses an image-splitting optical system called W-VIEW GEMINI. By separating and focusing images for each wavelength in the camera’s vertical (or horizontal) direction, it enables simultaneous capture of two wavelengths. Various combinations of wavelengths can be accommodated by adjusting dichroic mirrors and bandpass filters.

Advantages of the simultaneous two-wavelength measurement method

As shown in Figure 4, switching filters typically takes about 200 ms. For example, rapid biological phenomena such as membrane potential changes continue even during filter switching, making it impossible to accurately measure life processes that may occur during observation using the filter wheel method. To capture such high-speed biological phenomena, simultaneous measurement of two wavelengths is necessary.

Comparison of imaging timing between the filter wheel method and the simultaneous two-wavelength measurement method

Figure 4: Comparison of imaging timing between the filter wheel method and the simultaneous two-wavelength measurement method

Comparison of time resolution between the simultaneous two-wavelength measurement method (left) and the filter wheel method (right)

Differences between single-wavelength imaging and two-wavelength ratio imaging

In calcium imaging, directly interpreting changes in the fluorescence intensity of simple Ca²⁺ probes as the dynamics of the desired phenomenon or target molecule can lead to inaccurate measurements. The fluorescence intensity detected from Ca²⁺ probes varies due to factors such as fading of the fluorescent probe, variations in localization concentration, and sample movement.

For an explanation of the differences between one-wavelength imaging and two-wavelength ratio imaging, as well as the advantages of two-wavelength ratio imaging, you can refer to this page.

Differences between single and two-wavelength ratio imaging

Example images

High-speed Ca²⁺ imaging of iPS cardiomyocyte

High-speed intracellular Ca²⁺ gradient driven by UTP stimulation

Simultaneous intracellular Ca²⁺ and phase contrast imaging of spontaneously beating hiPS-CMs

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The image splitting optical system splits the incident light into two wavelengths and forms an image on the camera. It is an optical system for microscopes. By incorporating commercially available dichroic mirrors or half mirrors internally, it allows branching of light.

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The FDSS-GX Kinetic Plate Imager is a system designed for high-throughput screening (HTS) in drug discovery, leveraging our technology developed in the field. It features an FDSS-specific qCMOS^Ⓡ sensor known for its high quantification accuracy in fluorescence/luminescence measurements. The system also incorporates a dispensing head with independent metal piston cylinders (available in 1536-well, 384-well, and 96-well formats) that enables precise and reproducible micro-volume dispensing. This setup allows for simultaneous drug plate dispensing, measurement, and analysis. It is compatible with various Ca²⁺ assays.