Resulting from our collaboration with the University of Strathclyde, the pE-340^fura is a bespoke LED Illuminator for Fura-2 ratiometric calcium imaging. The system utilises the successful pE-300 Series platform, and also supports everyday fluorescence microscopy in a compact and affordable package.

 

Description

The 340 nm and 380 nm LED illumination system provides the optimum excitation wavelengths for Fura-2-based calcium imaging, allowing high-precision, stable, high-throughput imaging with video-rate time resolution.

High-speed acquisition

Until recently, the response time of illumination systems for Fura-2 imaging has been limited to milliseconds due to mechanical switching of the wavelengths in arc lamp and monochromator systems.  However, the new pE-340^fura can be controlled via convenient BNC TTL connections for precise illumination control in as little as 20 microseconds.

Improved cell viability and cost

Using the new pE-340^fura LED Illumination System, less Fura-2 dye can be loaded into the cells whilst still maintaining the same measured calcium concentration and good signal-to-noise ratio.  The reduction in required dye not only improves cell-viability due to reduced dye toxicity, but also results in a cost reduction per experiment.

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  

• Direct or light guide delivery options – flexibility of UV optimised attachment options

340 nm Excitation

380 nm Excitation

Figure 1: cardiac myocytes.  The cells were loaded with Fura-2 using standard conditions (incubation with 2 mM Fura-2 acetoxymethyl ester for 30 minutes, followed by an additional 30 minutes for de-esterification). Images were obtained by Martin Bootman and Katja Rietdorf, School of Life, Health and Chemical Sciences, The Open University, UK.

Spontaneous Ca²⁺ events are induced in Mg²⁺-free HBS. (A) Representative trace from a single hippocampal neuron of Mg²⁺-free induced Ca²⁺ events imaged at 0.5 Hz and (B) representative trace from two hippocampal neurons of Mg²⁺-free induced Ca²⁺ events imaged at 24.39 Hz.¹

Comparison of Ca²⁺ increases obtained from the application of trypsin (100 nM) to tsA-201 cells loaded with different concentrations of Fura-2 AM.¹

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

Performance

• Dedicated 340 nm and 380 nm outputs for Fura-2 ratiometric calcium imaging
• Broad white output (435 nm – 645 nm) for imaging standard fluorophores
• Microsecond switching (340/380) for capturing high-speed events
• Stable LED illumination minimises noise below that of experiment, reducing false positives and increasing precision.
• Higher signal-to-noise gives cleaner images and data whilst requiring less Fura-2 dye, reducing toxicity and costs
• UV optimised optics delivers maximum irradiance on standard microscope configurations
• Individual channel triggering via TTL in microseconds
• Removable inline excitation filter holders: no moving parts for fast acquisition
• Sequence Runner – sequenced excitation from a single TTL-out
• Individual channel selection controls the level of excitation of each dye independently on a multi-stained sample
• Compatible with most imaging software for integrated control with imaging set up
• Precise irradiance control in 1% steps (0-100%): no ND filters required

Convenience

• Specify for existing single and multi-band filter sets: no need to buy new filters
• Simple to fit, simple to use: no alignment, a once only adjustment
• Wide range of microscope adaptors: fits most microscopes

Specifications

Power requirements
100-240 V a.c. 50/60 Hz, 1.4 A

Power consumption
Standby Max 2 W
1 band (white) at 100% irradiance Max 24 W
2 bands (340 + white) at 100% irradiance Max 30 W
3 bands (All) at 100% irradiance 36 W

Dimensions
pE-340^fura Light Source: 77 mm(w) x 186 mm(d) x 162 mm(h) – Weight 1.40 kg
pE-340^fura Control Pod: 88 mm(w) x 125 mm(d) x 37 mm(h) – Weight 0.32 kg
pE-340^fura Power Supply: 167 mm(w) x 67 m(d) x 35 mm(h) – Weight 0.62 kg

pE-Universal Collimator: 44 mm(w) x 86 mm(d) x 44 mm(h) – Weight 0.17 kg

Environment and Safety

• Mercury-free and Laser-free
• Energy Efficient: 80% less power
• Long lifetime
• No bulb replacements
• Reduced risk of eye damage
• Quiet operation
• No special disposal regulations or issues

Warranty

Industry-leading 36 months (please note, the 340 nm LED is warranted for 3000 hours accumulated use).