Calcium Imaging Light Source

Choosing a calcium imaging light source

Calcium imaging is a powerful technique for understanding the many biological processes controlled and mediated by Ca2+ oscillations. Ratiometric calcium imaging with dyes such as Fura-2 is a popular technique and selecting the best calcium imaging light source is crucial. Where traditional arc lamps with a monochromator were the go-to approach, LEDs have evolved significantly over the last decade. LEDs which can quickly switch between 340 nm and 380 nm now provide the ideal solution as a powerful, flexible, cost-effective and controllable calcium imaging light source.

CoolLED and calcium imaging light sources

CoolLED is a UK-based company specialising in calcium imaging light source technology for the life sciences. Since launching the first commercially available LED illumination system in 2006, we have led the way with our technical know-how and unique blend of optical engineering and life science expertise.

We draw on the latest technology to design and manufacture a vast range of cutting-edge LED microscope lighting solutions, from fluorescence microscopy to ratiometric calcium imaging light sources.

Why use LEDs as a Fura-2 calcium imaging light source?

• High-speed triggering (20 µs) for high temporal resolution
• Narrow bandwidths for optimum excitation at 340 nm and 380 nm
• Improved signal-to-noise ratio
• Irradiance modulation
• Stable and homogenous illumination
• Variety of wavelengths for everyday fluorescence
• Easy to install to microscope
• No laser safety requirements
• Cost-effective

Calcium imaging Light Source optimised for Fura-2: CoolLED pE-340^fura

The CoolLED pE-340^fura is a bespoke LED Illuminator for Fura-2 ratiometric calcium imaging. The Light Source allows high-precision, stable, high-throughput imaging with video-rate time resolution. Resulting from our collaboration with the University of Strathclyde, the pE-340^fura utilises the successful pE-300 Series platform, and also supports everyday fluorescence microscopy in a compact and affordable package.¹

The CoolLED pE-340^fura includes 340 nm and 380 nm LEDs for ratiometric calcium imaging with Fura-2.  Adding flexibility to this calcium imaging Light Source, this also includes white light for everyday fluorescence microscopy.

Ultimate control of your calcium imaging light source

Control is where LEDs come into their own as a calcium imaging light source. Precise temporal control is crucial, as slower recordings reduce experimental accuracy. The solid-state nature of LED calcium imaging light sources means LEDs have fast and precise on/off switching. As with any peripheral device, latencies can be introduced by USB serial communications and computer operating system overheads. This can reduce switching speeds by as much as 100 ms and may also impact system synchronisation, e.g. between the camera and calcium imaging light source.

For users wishing to capitalise on the maximum speed of LEDs, electronic control via BNC TTL offers switching speeds of around 20 μs. The trade-off here is cost, and TTL control often requires specialist computer cards and components which can be expensive.

To overcome this and make TTL a cost-effective means of achieving high-speed imaging, the CoolLED pE-340^fura LED Illumination System includes the Sequence Runner program. Sequence Runner uses the TTL output signal available on most scientific cameras and cycles though LEDs in a user-selected sequence for each TTL signal. This is a simple approach and provides high-speed microsecond switching speeds that are precisely synchronised to the camera exposure.

Improved signal-to-noise

Work by Sandrine Prost et al., from the University of Edinburgh, has shown that with independent wavelength controllable LED sources, signal-to-noise is dramatically improved over bulb systems and even over some available white broad-spectrum LED sources.

High levels of autofluorescence and fast photobleaching of specific fluorescence when illuminating Qdots with Metal Halide.² 

Preventing phototoxicity and photobleaching with your calcium imaging Light Source

When combining the narrow spectral spread of each wavelength with TTL control, this limits unnecessary sample irradiance, minimising phototoxicity and photobleaching. Helping to reduce these effects, it is also possible to modulate the brightness of the LED calcium imaging Light Source to optimally excite the Fura-2 dye. The irradiance of LED Illumination Systems has been increasing year-on-year, and modern systems feature simple and precise irradiance modulation in 1% steps (0-100%), which can be controlled from software, analogue connection or the control pod.

With the resulting improvement in cell viability, this achieves not only insightful but accurate data. It also reduces costs since less Fura-2 is required per experiment.

Stable, powerful and homogenous illumination

Modern LED calcium imaging light sources provide stable illumination over time and they also achieve high levels of homogeneity.

In 2006 when CoolLED introduced the first LED Illumination System for the life sciences, the technology was still in its infancy. Modern calcium imaging light sources such as the pE-340^fura have come along way, however the topic of optimal illumination levels has been surrounded by complex, confusing and sometimes inaccurate measurements and terminology. Even the term ‘intensity’ is inaccurate, and a key issue has so far been an industry-wide lack of a standardised approach to measure illumination.

We have created a white paper which sets out to change this by introducing a protocol whereby scientists can quantify the amount of light at the sample plane and therefore directly compare systems to find their ideal solution.

Not sure? Try it out!

References

1. TINNING, P. W., FRANSSEN, A. J. P.M., HRIDI, S. U., BUSHELL, T. J. and MCCONNELL, G. (2017), A 340/380 nm light-emitting diode illuminator for Fura-2 AM ratiometric Ca²⁺imaging of live cells with better than 5 nM precision. Journal of Microscopy. doi:10.1111/jmi.12616

2. Prost S et al (2016) Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections. PLoS ONE 11(9): e0162419. doi:10.1371/journal.pone.0162419