Overview

Resulting from our collaboration with the University of Strathclyde, the pE-340fura 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.

pe-340fura-spectrum
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-340fura 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-340fura 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 power 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 intensity 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
    Plus all the benefits of LED technology

    The system comprises a pE-340fura Light Source, Control Pod, set of three Excitation Filter Holders and Power Supply.

    Control

    Control pod for operation and access to settings and usage history.

    Manual:
    Manual control for instant on/off and intensity control in 1% steps from 0 – 100%
    Individual bands can be selected/deselected and controlled independently as desired

    Remote:
    Via global and individual channel TTL for on/off control of selected channels using a BNC connection on the light source.
    Triggering speed <20 µs at full power

    Connectivity:
    Remote via USB (B type) for imaging software control (see Imaging Software)

    Sequence Runner:
    Single TTL input to step through user defined sequence.
    Triggering speed <20 µs at full power

    Choose direct fit or liquid light guide

    There are two pE-340fura‑configuration options:

    Direct-fit: Higher intensity is possible when directly connecting to a microscope. Select from a range of microscope adaptors which covers all current and most older models.  A simple once-only adjustment will allow optimisation to the optical path of the microscope.

    Liquid Light Guide: If there is a need to keep the source of illumination remote from the microscope, the Liquid Light Guide variant is available for use with a 3 mm diameter Liquid Light Guide. An optional pE-340fura Universal Collimator can be specified in conjunction with a microscope adaptor if required, containing optics optimised to transmit the 340 nm.  We now include a light source stand for the pE-340fura Illumination System ordered for light guide delivery.

    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% intensity Max 24 W
    2 bands (340 + white) at 100% intensity Max 30 W
    3 bands (All) at 100% intensity 36 W

    Dimensions
    pE-340fura Light Source: 77 mm(w) x 186 mm(d) x 162 mm(h) – Weight 1.40 kg
    pE-340fura Control Pod: 88 mm(w) x 125 mm(d) x 37 mm(h) – Weight 0.32 kg
    pE-340fura 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
    System: 36 months (NB 340 nm LED warranted for 3000 hours accumulated use)

    • I can absolutely recommend this light source for any kind of Fura-2 measurements.

      Lars Kaestner and Quinghai Tian, Molecular Cell Biology, University of Saarland, Germany

    Improving laboratory sustainability

    Improving laboratory sustainability

    Configuring Olympus cellSens

    Configuring Olympus cellSens

    A 340/380nm LED Illuminator for High Precision Fura-2 Ca2+ Imaging

    A 340/380nm LED Illuminator for High Precision Fura-2 Ca2+ Imaging

    An Introduction to Calcium Imaging

    An Introduction to Calcium Imaging

    Light SourceEmission ColourTypeCoolLED LEDChromaSemrock
    pE-340furaBlueSingle38039000 AT Dapi49028 ET Dapi 395LED-DAPI-B
    pE-340furaCyanSingleBlu/Grn (410-750)49001 ET eCFP49013 ET TealLED-CFP-ALED-mTFP-A
    pE-340furaGreenSingleBlu/Grn (410-750)49054 CoolLED GFP49002 ET eGFPGFP-4050BGFP-A-Basic
    pE-340furaYellow-GreenSingleBlu/Grn (410-750)49303 ET Green#149003 ET eYFP
    pE-340furaOrangeSingleBlu/Grn (410-750)49004 ET Cy349005 ET dsRedTxRed-4040CLED-TRITC-A
    pE-340furaRedSingleBlu/Grn (410-750)49055 CoolLED mCherry #149055 CoolLED mCherry #2
    pE-340furaRedSingleBlu/Grn (410-750)49008 ET mCherry39010 AT Tex. RedYFP-2427BLED-mCherry-A
    pE-340furaFar RedSingleBlu/Grn (410-750)49006 ET CY539007 AT Cy5Cy5-4040C
    pE-340furaFuraPinkel340, 380T400lp & ET510/80mFF409-Di03FF01-510/84
    pE-340furaFura-GreenPinkel340, 380, White (BLU/GRN)T495lpxru, ET470/40x, ET535/70m
    pE-340furaFura-RedPinkel340, 380, White (BLU/GRN)79003bs, 79003m, ET555/25x
    pE-340-FR-D-YYY-ZZ: pE-340fura Illumination System.  Direct Fit.  Includes Light Source, exchangeable microscope adaptor (YYY) to customer-specified microscope, remote manual control pod, set of three excitation filter holders (25mm diameter), Excitation Filters for 340nm & 380 nm, and power supply (ZZ).
    pE-340-FR-L-ZZ: pE-340fura Illumination System.  For use with 3 mm Liquid Light Guide.  Includes Light Source, stand, remote manual control pod, set of three excitation filter holders (25 mm diameter), Excitation Filters for 340 nm and 380 nm, and power supply (ZZ).
    pE-1910 3 m long, 3 mm diameter UV liquid light guide for use with pE-340fura
    pE-340-FR-COLL-YYY pE-340fura Universal Collimator for use with a single liquid light guide. Includes Microscope Adaptor (YYY) to customer-specified microscope

    To specify microscope code (YYY) see Adaptors
    To specify power cable (ZZ):  10 = Australia, 20 = Europe, 30 = UK, 40 = USA

    More information is available on the Accessories tab.

    Liquid Light Guide

    Liquid Light Guide

    pE-340<sup>fura</sup> Universal Collimator

    pE-340fura Universal Collimator

    pE-6501

    pE-6501

    The CoolLED pE Driver is first required for software integration, and can be downloaded here.

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