Challenges and Need for Developing Green Wavelength Technology in Life Science Fluorescence Applications
Kavita Aswani* Life Sciences, Excelitas Technologies Corp., 2260 Argentia Road, Mississauga, Ontario L5N 6H7 Canada
*
kavita.aswani@
excelitas.com
Abstract: In microscopy and analytical instrumentation, a researcher or clinician may need to excite several fluorophores in a sample in order to generate a useful fluorescence map of the cell or tissue of interest. This typically would include a nuclear marker such as DAPI, a green-emitting fluorophore such as FITC or GFP, and a red-emitting fluorophore such as mCherry, TRITC, or Texas Red. These red-emitting dyes can be efficiently excited using an arc lamp with a strong peak in the 550 nm region and another at 580 nm. When technology in micro- scope light sources originally moved to light-emitting diodes (LEDs) in the early 2000s, fluorescence work in the green gap (540 to 590 nm) excitation range was a challenge with no solution. This article explains how technology has adapted to provide LEDs that match the excita- tion of fluorophores typically used in multiplex fluorescence imaging.
Keywords: green gap, light-emitting diodes, mercury lamps, xenon lamps, metal halide lamps
Introduction Imaging in microscopy and life sciences applications has
traditionally relied on mercury, metal halide, and xenon arc lamp sources for illumination as they can excite a wide range of fluorescent compounds. With advancements in solid-state technology, microscopists are requesting a switch from these lamp sources to light-emitting diodes (LEDs) for life science fluorescence studies. Benefits of LED technology include a long lifetime, increased stability, and elimination of con- sumables, toxic waste, and the costs associated with mercury disposal [1,2]. LED technology is used not only in fluorescence microscopy systems to image the cells or tissues under inves- tigation, but also in several other fluorescence-based tech- nologies such as fluorescence activated cell sorting (FACS) and polymerase chain reaction (PCR) instruments used for diagnostic purposes. Many clinical applications requiring a high-power light source to cover various wavelength ranges are converting to LED systems that can now meet require- ments previously achievable only with mercury, metal halide, or xenon lamp technology. While LED technology is acceptable for most wavelengths
used in the majority of microscopy and medical applications, developing high-power LEDs in the green excitation range has been challenging. Tis article explains the need for green excitation light in life sciences and why this wavelength range is an issue with LEDs. It will further explain how established technology can overcome challenges in developing high- power light sources that include the wavelength gap between 540–590 nm required for life sciences applications.
The Need for Green in Life Sciences Fluorescence excitation in microscopy has traditionally
relied on the spectral properties of the mercury arc lamp, which emits from 350 nm to 750 nm (Figure 1). Tis has determined the chemistry of most common fluorophores used in biological
38 doi:10.1017/S1551929520001121
Figure 2: Bovine pulmonary artery endothelial cells labeled with Mito Tracker™ Red CMXRos, Alexa Fluor™ 488 Phalloidin, and DAPI. Magnification is 40×.
www.microscopy-today.com • 2020 July
Figure 1: Spectrum of a mercury arc lamp showing distinct peaks at wave- lengths common for many dyes used in biomedical research.
and medical research, and thus the excitation and emission filters used in fluorescence imaging (Figure 2). Te mercury arc lamp has distinct wavelength peaks around which fluo- rophores were developed and used for decades. Tese include common fluorophores such as DAPI, FITC, and TRITC. More recently, genetically expressed fluorescent proteins such as BFP (blue fluorescent protein), GFP (green fluorescent protein),
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