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Calcium imaging
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.
Figure 1: Imaging example of calcium imaging
Two-wavelength imaging method
Filter wheel method
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
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.
Figure 4: Comparison of imaging timing between the filter wheel method and the simultaneous two-wavelength measurement method
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.
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.
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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With 5x faster photon number resolving capabilities, the ORCA-Quest 2 remains the apex camera for ultra low-light, quantitative imaging.
Highlights:
- Ideal for ultra low light and replacing EM-CCDS
- Exclusive quantitative CMOS for pioneering imaging
- Ultra-low noise enables single photon resolution
- High sensitivity does not require speed compromise
This ORCA is ideal for:
- TIRF
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Small pixels, big benefit
The ORCA-Fire is a unique back-thinned sCMOS that is optimized for fast, low-mag, large field of view, low-light quantitative imaging.
Highlights:
- Ideal for low light with high pixel volume and speed
- Nyquist sampling at 40x and below
- High sensitivity even at fast speeds
- Advanced bidirectional lightsheet readout modes
This ORCA is ideal for:
- Lightsheet
- Simultaneous multi-wavelength imaging
- High-throughput widefield fluorescence
- Genetically encoded voltage imaging
- Tissue mapping
Uncompromising performance
The ORCA-FusionBT sCMOS camera is the perfect synthesis of sensitivity, speed, resoution and overall quantiative, low-light performance.
Highlights:
- Ideal for very low light applications at 60x and 100x
- Superior SNR from maximized QE, minimized noise
- Three speed/noise modes for use-specific imaging
- Exclusive, high QE, back-thinned sCMOS sensor
This ORCA is ideal for:
- Spinning disk confocal
- Lightsheet
- Optogenetics
- Structured illumination microscopy
- Single-molecule localization microscopy
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.
We offer two models: W-VIEW GEMINI, which uses a single camera, and W-VIEW GEMINI-2C, which uses two cameras. W-VIEW GEMINI-2C enables wider field-of-view imaging due to its use of two cameras.
In summary, Hamamatsu's W-VIEW GEMINI image splitting optics enhance calcium imaging by facilitating simultaneous dual-wavelength acquisition, enabling accurate and rapid assessment of intracellular calcium fluctuations essential for understanding various cellular processes.
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.
Our photomultiplier tube (PMT) modules are ideal for calcium imaging with their high sensitivity, fast response, and low noise characteristics. The high quantum efficiency of Hamamatsu PMTs enables precise detection of small changes in fluorescence leading to more accurate calcium level detection. Their fast temporal response captures rapid dynamics in cellular signaling enabling real time data collection.
Additionally, Hamamatsu PMTs have low noise performance leading to clearer signal detection. The wide spectral response of Hamamatsu PMTs can be adapted to various imaging setups with a range of spectral responses, to match different calcium indicators. These features make Hamamatsu PMTs highly suitable for calcium imaging, supporting accurate and reliable observation of cellular and neuronal activity.
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