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

2. 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.

3. Overview
CoolLED Timeline
Arc Lamps
LEDs
CoolLED Products
Summary

4. 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

5. 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

6. Arc Lamp – Disadvantages
Short life time – bulbs last approx 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.

7. 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.

8. 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.

9. 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

10. 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

11. Evans, A, Green Light Laboratories LTD, 2017

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

13. 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.

14. 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

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

16. 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

17. 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

18. Benefit 6: No filter wheel

19. 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

20. 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.

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

22. 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

23. 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

24. 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.

25. 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.

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

27. Thank You! CoolLED Ltd. CoolLED USA t +44 (0) 1264 323040e w
CoolLED USA t e w
Thank You!