Considering Automated Calcium Imaging?
Find out what you need from a light source
The significance of calcium needs no introduction in the world of cell biology and pharmaceutical research.
As fluctuations in intracellular calcium levels can indicate physiological activity, this makes calcium imaging using fluorescence microscopy an invaluable technique for life science researchers, or pharmaceutical scientists profiling drug responses.
What are the requirements for a calcium imaging light source?
Calcium imaging isn’t just limited to standard fluorescence microscopy setups, and can be integrated into automated imaging systems for faster and more efficient investigations. This can either be based on microplate readers for higher throughput, or automated imaging systems to provide extra context for deeper insights. And the light source is a crucial component for this application, whatever the platform.
While each component must be carefully optimised to maximise the performance in calcium imaging, our speciality at CoolLED is the LED light source. And the two most crucial features to consider are spectral coverage and triggering speed.
Spectral coverage – Many different dyes and genetically encoded calcium indicators (GECIs) are now available for calcium imaging, each requiring precise excitation at a specific wavelength. To ensure compatibility with as many of these as possible (both now and in the future), the light source must have complementary wavelengths across the spectrum.
CoolLED’s LED technology offers consistent and stable excitation across a variety of different wavelengths, allowing assay flexibility to suit the needs of the end user – and this even includes 340 nm and 380 nm LEDs for Fura-2 ratiometric experiments.
Another aspect to consider is which of these LEDs are available on individually controllable channels, and in the case of ratiometric imaging for example, this is crucial to allow fast sequential excitation.
Triggering speed – Ratiometric calcium imaging requires fast LED triggering speeds in order to capture real-time changes in calcium levels.
CoolLED light sources achieve industry leading channel switching speeds below 7 µs, maximising data accuracy.
For all imaging applications, higher speed is of course beneficial for getting through experiments faster, allowing a higher sample throughput in the lab.
Calcium Imaging in Action
In a recent study of Mitochondrial flickers by Dr Martin Bootman, Reader in Biomedicine, Faculty of Science, Technology, Engineering & Mathematics (The Open University) – the CoolLED pE-340fura was used extensively.
Analysing cellular signalling and sub-cellular processes are important aspects of cell biology research. For this, they relied heavily on rapid, high resolution fluorescence measurement techniques – including Fura-2 ratiometric calcium imaging and the ability to stably image fluorophores in small cellular compartments.
They performed ratiometric Fura-2 calcium measurements using the pE-340fura Illumination System, which includes 340 and 380 nm LEDs (in addition to a white light LED channel).
Fura-2 calcium imaging with the pE-340fura
Cardiac myocytes loaded with Fura-2 using standard conditions (i.e. incubation with 2 micromolar Fura-2 acetoxymethyl ester for 30 minutes,
followed by an additional 30 minutes for de-esterification – Videos acquired by Martin Bootman and Katja Rietdorf
The left-hand and right-hand videos show the Fura-2-loaded cells with 340 nm and 380 nm excitation, respectively.
Conclusion
For manufacturers of automated imaging systems, integrating calcium imaging capabilities is a strategic move that can significantly enhance product functionality and appeal. To achieve the best results, choosing the right light source is paramount – and CoolLED’s Amora Series of custom microscopy illumination is well-suited to meet the demanding requirements of calcium imaging assays.
By partnering with CoolLED, you can ensure that your high-content imaging systems are equipped with the most advanced and reliable technology available, enabling your customers to push the boundaries of scientific and pharmaceutical research.
