Award-Winning LED Illumination for Fluorescence from Fura-2 to Cy7.5 and Beyond
Kavita Aswani Excelitas Technologies Corporation, Mississauga, ON L5N 6H7, Canada
kavita.aswani@
excelitas.com Abstract: Light-emitting diode (LED) illumination for fluorescence
microscopy systems has come a long way since the turn of the century, when the LED sources available had low optical power and a limited range of wavelengths. While the benefits of LED technology were attractive, their limitations prevented large-scale adoption. Twenty years later, advances in the semi-conductor industry have eliminated many of those fundamental hurdles. Today, LED technology is rapidly replacing the lamps used in microscopy and fluorescence imaging, even for applications on the extreme ends of the light microscopy spectrum. This article describes the latest advances in illumination for fluorescence imaging, from near-UV to near-IR fluorophores. Case studies demonstrate the new X-Cite®
NOVEM™ as successful
replacements for traditional arc lamps in calcium imaging applications, producing equivalent results with the convenience of LEDs. For its usefulness to the microscopy community, the X-Cite NOVEM system was awarded the prestigious 2021 Microscopy Today Innovation Award.
Keywords: LED, Fura, ICG, fluorescence imaging, microscopy illumination
Introduction Microscopy and imaging in the life sciences are advancing
from traditional lamps to solid-state light-emitting diode (LED) technology. Moving to LEDs allows users to benefit from long lifetimes with increased stability while negating the need to replace or dispose of toxic bulb waste. Microscopy and fluores- cence excitation have traditionally relied on the spectral proper- ties of the mercury arc lamps, which has defined the chemistry of fluorophores, as well as the excitation and emission filters used in fluorescence imaging. Mercury arc lamps have discrete peaks around which the most common fluorophores, namely DAPI, FITC, and TRITC, have been developed and used for decades. With technology moving to LEDs, users must be aware of the differences in the peak optical power between lamps and LEDs and ensure optimization of their filters to achieve maxi- mum excitation efficiency of their fluorophores. Te X-Cite NOVEM (Figure 1) was awarded the 2021
Microscopy Today Innovation Award, which recognizes inno- vations to make microscopy more powerful, productive, and easier to accomplish [1]. Te X-Cite NOVEM is a nine-channel LED illumination system for fluorescence imaging, with config- urations spanning from near-UV wavelengths of the spectrum to the near-IR. Te unit features a patented LaserLED Hybrid Drive® technology, which provides users with high-power exci- tation in the previously challenging 500–600 nm Green Gap. Each of the nine channels can be controlled individually or in combination with other channels. Rapid channel switching allows for high-throughput imaging. Te unit has five independent LEDs, outfitted with excita-
tion filters, allowing for imaging of popular fluorophores such as DAPI, CFP, GFP/FITC, and Cy5. Four additional channels (500–600 nm) are powered by the LaserLED Hybrid Drive® technology with a 4-position motorized filter changer to select
24 doi:10.1017/S1551929522000608
specific wavelengths within this range for popular green excited fluorophores such as YFP, TRITC, and mCherry. Te prein- stalled filters and motorized filter changer make it easy to image with multiband filter cubes without bleed-through (Table 1). Te X-Cite NOVEM couples to most standard research micro- scopes with a liquid light guide and an X-Cite microscope adap- tor (Figure 1). Each of the nine channels can be individually controlled or combined with others. Intensity control of 1% per channel can be achieved using the
intuitive speedDIAL hand controller, or via USB, TTL, or third- party soſtware control. Even operating at full power, the acoustic noise generated by the X-Cite NOVEM is exceptionally low when compared to the X-Cite Xylis and 120Q systems ( Figure 2).
Imaging with Fura-2 to IR800/Indocyanine Green (ICG) Intracellular calcium signals drive many cellular processes
including muscle contraction, cell proliferation, synaptic trans- mission, and gene transcription. Detecting changes in intracellu- lar calcium is of interest to researchers because it provides insight to these dynamic processes in living cells. Techniques for detect- ing intracellular calcium signals using Fura-2 imaging are well established [2,3]. Te excitation maxima of Fura-2 shiſts from 380 nm to 340 nm upon binding of calcium. Tis shiſt can be used to quantify intracellular calcium fluxes. Xenon lamps have been the gold standard for Fura-2 imaging because they provide a uniform intensity of light over a wide range of wavelengths. How- ever, these arc lamps have their own drawbacks. Since arc lamps are broad-spectrum, they must be used in combination with filter wheels to isolate and constantly change between 340 nm and 380 nm excitation wavelengths. Tis results in a mechani- cal limit on imaging speed. In addition, xenon lamps require frequent replacement, making them a nuisance and expensive to maintain. In addition, since arc lamps are broad-spectrum, they must be used in combination with filter wheels to isolate and constantly flip between the 340 nm and 380 nm excitation wavelengths, putting a mechanical limit on imaging speed. Hence, there has been a general move toward dyes like Fluo-
4, which are single-excitation. Single-wavelength dyes can detect changes in calcium but cannot measure calcium concentrations in the cytoplasm. Fura-2 imaging requires alternately exciting the fluorophore at 340 nm and 380 nm. Image pairs are recorded, and the signal excited by 340 nm is divided by the signal excited by 380 nm to produce a ratiometric image. A change in the ratio over time corresponds to a change in intracellular calcium [2]. With the development of LEDs at lower wavelengths, it is now possible to use solid-state technology for this application. In an LED system, each wavelength can be controlled sepa-
rately, allowing simple adjustment of the 340 nm versus 380 nm LEDs to capture the images at the two wavelengths at similar
www.microscopy-today.com • 2022 May
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