Any microscopy lab is familiar with the angst that comes with mercury lamps for widefield fluorescence. Long boot up times, high energy consumption, short lifespan, wait times between power cycles, the patience required to train new users. Metal halide lamps offer a marginal improvement but carry the same challenges.
It’s perhaps unsurprising that LED lighting alternatives quickly gained a foothold in microscopy despite having relatively few applications due to early technical limitations. Rapid advances over the last decade have had an enormous impact on the technological landscape, resulting in a steadily increasing userbase across many applications. The factors underlying its success and rapid adoption include impressive performance capabilities, low running cost, improved uniformity across space and time, improved control, and far greater environmental sustainability.
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Why LEDs Are Changing the Face of Fluorescence Microscopy
Labs are increasingly turning to advanced LED lighting systems as an alternative to traditional mercury and metal halide lamps for their lower costs, greater uniformity, finer control, and environmental sustainability
Any microscopy lab is familiar with the angst that comes with mercury lamps for widefield fluorescence. Long boot up times, high energy consumption, short lifespan, wait times between power cycles, the patience required to train new users. Metal halide lamps offer a marginal improvement but carry the same challenges.
It’s perhaps unsurprising that LED lighting alternatives quickly gained a foothold in microscopy despite having relatively few applications due to early technical limitations. Rapid advances over the last decade have had an enormous impact on the technological landscape, resulting in a steadily increasing userbase across many applications.
Readers who recall the limitations in wavelength selection and irradiance of early LED lighting systems may not be aware of the extent of recent technological advancements. Modern LEDs include systems from one to eight or more discrete LED channels with a range of wavelengths to choose from. This expansion has played a large role in extending their relevance beyond simple fluorescence screening. Considerable increases in irradiance and control have led to LED systems becoming cost-effective solutions for applications like high-speed live cell imaging, Förster resonance energy transfer for capturing protein-protein interactions, optogenetics, and precise 340/380 nm Fura-2 calcium imaging.
The factors underlying its success and rapid adoption include impressive performance capabilities, low running cost, improved uniformity across space and time, improved control, and far greater environmental sustainability.
Low-cost performance
For labs considering new fluorescence scope purchases, LED systems typically have a higher purchase price than traditional mercury or metal halide lamps. Cost of ownership soon makes up the difference, however, as illustrated by a cost analysis informed by Green Light Laboratories comparing mercury lamps and the CoolLED pE-300ultra, an LED lighting system that offers comparable widefield illumination.
Bulb replacements form a large portion of the operating costs for traditional lamps. Mercury bulbs require frequent replacement with a lifespan of just 200 hours per bulb. Metal halide lamps, introduced as an alternative to the older mercury bulbs, have a comparatively longer lifespan of 2,000 hours, but LEDs far exceed both with an expected lifespan of around 25,000 hours. Assuming costs of $150 for mercury bulbs and $175 for metal halides, that equals $18,750 and over $2,100 respectively in replacement bulbs alone over the LED’s lifespan. As both types of bulbs contain mercury, there are additional disposal costs for spent bulbs (though if you are at a university, your institution may be footing the bill). Add to this replacement costs for the liquid light guide, which gets damaged over time by UV output from the mercury and metal halide light sources.
Electricity costs form another major line item. Green Light Laboratories found that mercury lamps consume over 10 times the energy of their LED counterparts, in part due to the 20-minute warm-up and 30-minute cool- down periods required for operation. Apart from the added power draw on warm-up, the inconvenience of a minimum intersession downtime of 50 minutes leads many labs to simply leave their lamps on for eight hours at a time, despite the hit to the bulb life. LEDs, which can be turned on and off as needed, are typically on for considerably less time through the day.
Cost of ownership comparison between mercury lamps and LEDs across a 25,000-hour timespan
Equations | Mercury | LED | |
Bulbs used | timespan bulb lifespan | 125 | 0 |
Cost of bulbs | bulbs used × bulb cost | $18,750 | 0 |
Energy consumed (kWh) | energy consumption × hours | 2,850 | 250 |
Energy costs | energy consumed × electricity rate | $345.99 | $30.35 |
Daily use (hrs) | runtime+warmup | 8.33 | 8 |
Daily cost | (electricity rate × energy consumption × daily use) + ( bulb cost × daily cost) lifespan | $6.36 | $0.01 |
Total cost for 25,000 hrs | timespan × daily cost daily use | $19,095.99 | $30.35 |
Calculations assume a 200-hour lifespan and $150 replacement cost for mercury bulbs, a 25,000-hour lifespan for LEDs, and energy consumption rates of 0.114 kWh/h and 0.01 kWh/h for mercury and LED lighting systems respectively. Electricity costs were calculated using $0.1214 USD/kWh/h, the average US commercial rate in May 2022 as reported on eia.gov. All prices are in USD.
Staff-hours are a frequently overlooked added cost. Each replacement bulb must be installed, focused, and aligned, adding cumulative hours of highly trained lab staff’s time. LEDs are often factory-aligned and require one simple adjustment when installed on a microscope.
Replacing existing lighting systems with LEDs will carry a larger upfront cost, however, the cost of replacement may still be recouped through the savings accumulated in cost in ownership.
Improving consistency and repeatability
When discussing the power or intensity of lighting systems, lingering confusion remains over comparative measurements between systems. “Power” in Watts is frequently reported but is not a useful metric without defining the focal area or type of power measured. “Intensity” is power per unit area, W/m2, and often used to describe illumination, yet inconsistent use has generated confusion over which output, such as electrical, light, or heat, it is describing. “Irradiance” is radiant power per unit area (mW/mm2) and is recommended as a less ambiguous measurement allowing direct comparisons between light sources. To be most relevant, irradiance should be measured at the sample plane rather than the source. It is important to note that some manufacturers measure this at the source, so ask about the point of measurement when purchasing or enquire about equipment demonstrations or loans to experience performance first-hand.
Both mercury and metal halide bulbs suffer from temporal instability—their irradiance drops steadily across their lifespan. Both also fluctuate during use, by as much as 10 percent even in the more stable metal halide bulbs, and vary spatially in irradiance. This instability in performance negatively impacts both spatial and temporal studies as well as general repeatability.
LEDs offer exceptional temporal stability during use and across their lifespan with no fading or fluctuations in irradiance. They also offer greater spatial uniformity across the field of view.
Comparative lifetime graph showing relative irradiance over time of LEDs, metal halide lamps, mercury lamps.
Improving specimen longevity and data quality
Compromised sample longevity—and cell viability for live-cell imaging—have traditionally constrained studies using fluorescence microscopy and have negatively impacted data quality. Lighting is a major contributor through the effects of phototoxicity (damage to live cells and tissues impacting behaviour and reducing viability) and photobleaching (damage to fluorophores causing signal fading).
Limiting unnecessary irradiance through fine control is key to obtaining a high-quality image, reducing light exposure, and extending cell viability. The light source, filters, and objective lens can all contribute to blocking unwanted wavelengths, minimizing photodamage and background noise.
Shutters and filters are the traditional solutions to minimize unwanted light exposure. Even computer- operated shutters and filters introduce a lag time that limits potential imaging speeds while still generating unnecessary exposure. Filters are used to narrow the illumination to specific wavelengths, but some bleed- through can occur, diluting the signal of interest through background noise. Motorized filter turrets enable multiwavelength imaging, though their use for imaging fast cellular processes is limited by the time it takes to physically switch filters and the mechanical vibrations induced by the action.
Mercury-based bulbs emit high-intensity light in the UV range that is especially damaging to live specimens. Metal halides offer better control over intensity compared to mercury bulbs, which require neutral density filters, but still cause high autofluorescence and rapid, significant photobleaching in comparison studies of lighting.
LEDs provide dimmable illumination restricted to discrete wavelengths, limiting light exposure even without filters, which can be applied directly at the diode. Programmable LED light systems offer additional control over light intensity and timing to decrease phototoxicity and photobleaching effects. This lengthens sample lifetime and increases reliability of experimental data involving live-cell fluorescence microscopy. Image acquisition speed and illumination efficiency can be increased further by bypassing computer controls through TTL triggering synced to the camera, giving microsecond precision to illumination without the warm-up and cool-down periods associated with arc lamps. Pulsing or strobing also limits exposure of samples to photons and allows relaxation of cells and fluorophores between exposures. Introducing a relaxation measure decreases photobleaching by up to nine times by reducing the likelihood of a second photon hitting a fluorophore in triplet mode. It also increases the fluorescent signal, with reports of five to 25 times higher yield for fluorescent dyes.
LEDs can offer greater temperature control for sensitive applications. Mercury and halogen bulbs generate enough heat to raise the ambient temperature, with potentially detrimental effects on live samples. In one example of brightfield imaging, LEDs enabled greater temperature stability in IVF imaging than halogen bulbs, which raised the temperature within the isolator.
Through improved viability achieved with rapid image capture, temperature stability, and reduced light exposure, LEDs allow growth tracking and continuous observations across entire lifespans of live organisms.
Improving sustainability
The toxicity and environmental impact of mercury-based lights along with their short lifespan and other health and safety risks, not the least of which is that they form an explosive hazard, make them a popular target for sustainability efforts. A wide ban on the use of mercury seems to be forthcoming, as the United Nations pushes reducing or eliminating the use of mercury and mercury compounds globally through the Minimata Convention on Mercury, established in 2017.
Labs interested in reducing their footprint can look for the ACT label—a labeling initiative by My Green Lab to measure and display the environmental impact factor of lab supplies from manufacture to disposal. CoolLED has ACT-label certified LED lighting systems and operates a “take-back” program for recycling old systems.
Ultimately, decisions on the best microscopy light source should take into consideration the applications required, the scale, and the sensitivity of the samples to effects like photobleaching. Purchasers will also want to consider the irradiance of the light source, spectrum coupling, speed, software controls, stability, lifespan, costs, energy consumption, and environmental impact. While decisions should be made on a case-by-case basis, the list of reasons to adopt LED is lengthy and growing by the year.