LEDs – More Than Just a Light Source Presented by Danielle Love

CoolLED Overview Based one hour west of London, UK Design, develop and manufacture specialist illumination technology Key market is the microscopy industry Using market knowledge to drive product development - Based in Andover, which is about one hour west of London. Supply worldwide and have a number of distributors across the globe Our aim is to produce high quality illumination systems. Everything happens in Andover, from design to production and shipping. Our main market is Micrscopy and we are always looking to provide the best for the customer. We use customer feedback and research to drive our product development, along with our technical knowledge and ambition to contrast improve and be at the forefront of technology. In 2016, we were purchased by Judges scientific. They invest in scientific manufacturing companies. You may have heard of Scientifica.

Overview CoolLED Timeline Arc Lamps LEDs CoolLED Products Summary

Illumination Timeline 2006 Launch of pE-1 2012 Launch of pE-300 2018 Launch of enhanced pE-4000 1900’s First fluorescence microscope using transmitted light 2003 Development of first commercial LED system 2010 Launch of pE-100 2014 pE-100wht & pE-300white launched 1595 First compound microscope 1665 Robert Hook microscope with illuminator and condenser 2006 Introduced the first commercially available LED illumination system for microscopy 2011 Research into white light 2016 Launch of pE-300lite 1595 – first compound microscope – 9x mag 1665 – Robert hook, added focussing system, illuminator and condenser 1900s – First fluorescence microscope using transmitted light, Arc Lamps, 1930s – Mercury arc lamps and around this time EpiFluorescence 1930’s Mercury lamps used for Epi-Fluorescence 2007 Launch of 4 colour pE-2 2013 Launch of pE-4000

Arc Lamps Traditionally used for fluorescence microscopy. Mercury arc lamp are most common. Many fluorophores designed around mercury peaks. High intensity, broad spectrum light source. Michael W. Davidson - National High Magnetic Field Laboratory, The Florida State University

Arc Lamp – Disadvantages Short life time – bulbs last approx. 200 hours. Bulb alignment and focusing required with each new bulb. Bulb intensity decreases with use – can lead to misleading data. Uneven intensity across the spectrum – ND required due to intensity difference in between fluorophores. High pressure bulb can explode – care must be taken when changing. Strong in UV, can be harmful to samples and user – UV filter required. Mercury is harmful and requires careful disposal. Inherent amplitude instability. Beacher J, Biophotonics International 2008 Broad spectrum illuminates at all wavelengths - a filter wheel is required to excite at specific wavelengths. Warm up period required before lamp can be used, on/off switching can shorten lamp life - shutter required.

LEDs Past Performance LEDs not powerful enough to illuminate some fluorophores Narrow bandwidth – don’t cover full spectrum LEDs not the correct wavelengths for fluorophores As LED technology became increasingly powerful, manufacturers began to realise that they could provide a much needed alternative for fluorescence imaging illumination. LEDs provide narrow bandwidth illumination and previously there were not enough available LEDs to cover the full visible spectrum. In addition to this, the LEDs that were available were not bright enough for fluorescence imaging. This meant that LEDs were not able to provide a suitable replacement for mercury or metal halide bulbs. However, with improvements in technology, there is now a large number of powerful LEDs that cover the spectrum from the UV across the visible range and into the IR. This means that regions of the spectrum in which mercury and metal halide lamps do illuminate strongly can now be illuminated by LEDs. This has also made way for the development of new fluorescent molecules that are no longer dependent on the emission spectrum of a mercury bulb. The introduction of LEDs into the world of fluorescence imaging has also brought about many additional advantages. Firstly, they can be much more easily controlled than traditional mercury or metal halide lamps. They can be switched on and off with ms timing precision, which removes the need for a mechanical shutter and improves the temporal resolution of experiments. Secondly, where a mercury/metal halide lamp would need to be left on for a days’ worth of experiments, LEDs can be switched off easily when not in use. This reduces energy consumption. In addition to this LEDs are more energy efficient and emit much less heat. Thirdly, their intensity can be electronically controlled, which removes the need for neutral density filters. Fourthly, mercury bulbs last approximately 300 hours, with a decline in intensity over their lifetime. They also require specialist disposal due to the high pressure blub and mercury content. In contrast, LEDs last upwards of 10,000 hours without any drop in intensity (Figure 2). Finally, the tricky alignment required after frequently replacing bulbs is no longer needed. LEDs can be factory aligned and are ready to be fitted to the microscopy without complicated alignment procedures. Figure 2 Graphical representation of intensity of various light sources across their life span. The fact that LEDs illuminate in narrow bandwidths allows for ultimate flexibility when choosing a light source. Many manufacturers provide multiple solutions, from single wavelength light sources to sources combing multiple LEDS to cover the full visible spectrum. This is where optimal filtering becomes essential and where LED technology really comes into its own. LEDs produce white light or wavelengths of defined bandwidth: Reduces photobleaching from unwanted wavelengths Multiple LEDS can be combined in one light source for multi colour imaging Filters can be placed in front of LEDs – no filter wheel required Intensity is controllable: Different intensities for different wavelengths. Allows for colour balancing of different fluorophores. ND filters not required.

LEDs Today LEDs are now powerful enough Larger number of LEDs – UV, Visible, IR Design of new fluorophores As LED technology became increasingly powerful, manufacturers began to realise that they could provide a much needed alternative for fluorescence imaging illumination. LEDs provide narrow bandwidth illumination and previously there were not enough available LEDs to cover the full visible spectrum. In addition to this, the LEDs that were available were not bright enough for fluorescence imaging. This meant that LEDs were not able to provide a suitable replacement for mercury or metal halide bulbs. However, with improvements in technology, there is now a large number of powerful LEDs that cover the spectrum from the UV across the visible range and into the IR. This means that regions of the spectrum in which mercury and metal halide lamps do illuminate strongly can now be illuminated by LEDs. This has also made way for the development of new fluorescent molecules that are no longer dependent on the emission spectrum of a mercury bulb. The introduction of LEDs into the world of fluorescence imaging has also brought about many additional advantages. Firstly, they can be much more easily controlled than traditional mercury or metal halide lamps. They can be switched on and off with ms timing precision, which removes the need for a mechanical shutter and improves the temporal resolution of experiments. Secondly, where a mercury/metal halide lamp would need to be left on for a days’ worth of experiments, LEDs can be switched off easily when not in use. This reduces energy consumption. In addition to this LEDs are more energy efficient and emit much less heat. Thirdly, their intensity can be electronically controlled, which removes the need for neutral density filters. Fourthly, mercury bulbs last approximately 300 hours, with a decline in intensity over their lifetime. They also require specialist disposal due to the high pressure blub and mercury content. In contrast, LEDs last upwards of 10,000 hours without any drop in intensity (Figure 2). Finally, the tricky alignment required after frequently replacing bulbs is no longer needed. LEDs can be factory aligned and are ready to be fitted to the microscopy without complicated alignment procedures. Figure 2 Graphical representation of intensity of various light sources across their life span. The fact that LEDs illuminate in narrow bandwidths allows for ultimate flexibility when choosing a light source. Many manufacturers provide multiple solutions, from single wavelength light sources to sources combing multiple LEDS to cover the full visible spectrum. This is where optimal filtering becomes essential and where LED technology really comes into its own. LEDs produce white light or wavelengths of defined bandwidth: Reduces photobleaching from unwanted wavelengths Multiple LEDS can be combined in one light source for multi colour imaging Filters can be placed in front of LEDs – no filter wheel required Intensity is controllable: Different intensities for different wavelengths. Allows for colour balancing of different fluorophores. ND filters not required.

Benefit 1: Improved Temporal Resolution Fast switching No mechanical shutter Improved temporal resolution Pulsing for decreased photobleaching They can be switched on and off with ms timing precision, which removes the need for a mechanical shutter and improves the temporal resolution of experiments. Our light sources have a range of triggering options and can be triggered by global or individual TTL. <20us Camera sync

Benefit 2: Environmental Reduced energy bills – cooler rooms – no hazardous chemicals Instant on/off means that LEDs can be switched off when not in use Lower power consumption Energy efficient, emit less heat Mercury-free

Evans, A, Green Light Laboratories LTD, 2017

Benefit 3: No ND filters required Intensity electronically controlled Get the exact intensity you require Allows for colour balancing in multi colour experiments

Benefit 4: No changing bulbs and misleading data Very long lifetime - No need to change bulbs Factory aligned and focussed No decline in intensity Very long lifetime – no need to change bulbs meaning no refocussing or realigning the light source. Intensity of LED maintained for the life of the LED, as such measurements are not affected by the age of light source. Avoid misinterpreting data.

Benefit 5: Improved signal to noise LED illumination offers much higher amplitude stability than arc lamps. LED illumination offers much higher amplitude stability than arc lamps. This means the user can get higher precision from their experiments when using LEDs. D. A. Wagenaar, PLoS One, 2012

Benefit 6: Filter friendly Single wavelength imaging: No need to change your existing filter set up Can use multiband filter sets

Benefit 6: Filter friendly Slow speed multi fluorophore imaging: Multiple LEDs in one light source No need to change existing filters. Can place exciters in front of LED Can use multiband filter sets – avoid pixel shift

Benefit 6: Filter friendly High speed multi fluorophore imaging: Single band sets – too slow Filter wheels – too slow/vibrations Solutions: Multi-band set or Exciters in light source with multiband dichroic: No filter wheel time delay No filter wheel vibrations Only limited by camera exposure No pixel shift

Benefit 6: No filter wheel

Where can CoolLED LEDs be used for? Transmitted illumination Standard white light for 100W halogen replacement Phase contrast techniques Single wavelength for IR-DIC or specialist applications Fluorescence illumination: Replacement for fluorescence arc lamps Single wavelength Multi-wavelength imaging High speed multi-wavelength imaging

pT-100 – Transmitted Applications pT-100 has 4 variants for transmitted imaging techniques including brightfield, darkfield, DIC, Dodt gradient contrast & phase contrast: Broad white output – pT-100-WHT (formerly known as pE-100wht ) a powerful white LED illumination system designed to replace a 100W halogen lamp.  Unlike a Halogen lamp, the colour balance of the pT-100-WHT Illumination System does not vary with intensity, removing the need to make any adjustment.

pT-100 – Transmitted Applications 2. Narrow bandwidth at 525nm – pT-100-525 is optimised for the spectral response of most scientific cameras and reduces background noise from a phosphored LED. 3. Narrow bandwidth at 635nm – pT-100-635 provides deeper penetration, excellent for revealing detail in thicker samples 4. Narrow bandwidth at 770nm – pT-100-770 provides deeper sample penetration techniques that require the use of infrared light

pE-100 One LED = one channel Select wavelength to match fluorophore Perfect for clinical applications, optogenetics & electrophysiolgy Manual control pod, Instant on/off, 0-100% intensity control Fast switching by TTL trigger (<150us) Direct-fit, LLG and Fiber options Combine 2 pE-100s eg CFP/YFP fret Electrophysiology e.g. remote light delivery by LLG or Fiber

pE-300 Series Three models – same spectral output, different control options White light, broad spectrum output for fluorescence Fits all new and most older microscopes Replaces mercury and metal-halide lamps

The New pE-340fura Bespoke LED Illuminator for Fura-2 ratiometric calcium imaging Dedicated 340nm and 380nm outputs Broad white output 435nm-645nm Until recently, the response time of illumination systems for Fura-2 imaging have 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.

pE-4000 Flexible Microscopy Illumination System pE-4000 is a powerful 4-channel high-specification LED Illumination system. It is flexible, controllable and environmentally friendly. 16 selectable available LED wavelengths, it offers the broadest spectrum of illumination available.

Summary Long lasting Stable Controllable Provide equivalent of: filter, attenuator, shutter and sequence generator Safer for user and environment Wide range of available wavelengths.

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