pE-800 Microscope Illuminator in CFP-YFP FRET application
Researchers discover how cell membrane dynamics affects activity of HCN volage-gated ion channels involved in health and disease.
In the latest of our Science Snapshot articles exploring the different applications of the popular 8-channel pE-800 Illumination System (including spatial biology and optogenetics), we cover how scientists have utilised CFP-YFP FRET as part of their research.
The research paper, “Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization”, published in Nature Communications, explores the role of the cell membrane in the activity of a particular type of voltage-gated membrane receptor, HCN channels, which are important for regulating heartbeat and have a role in pain and epilepsy.
Live cell analysis is vital in order to provide an accurate picture of HCN activity, and combining molecular biology techniques with microscopy tools presents an ideal approach for understanding receptor protein behaviour. As part of this groundbreaking research, authors Lucas J. Handlin and Gucan Dai utilised the 8-channel pE-800 LED Illumination System for Förster resonance energy transfer (FRET), a fluorescence microscopy technique which allows the researchers to observe the proximity of labelled molecules.
Lipidated peptides which localise to either ordered or disordered membrane domains, were labelled with CFP to act as FRET donors, and these probes were excited using the 435 nm LED of the CoolLED pE-800. The FRET acceptor, YFP, was tagged to human HCN molecules. In order to help calculate the FRET efficiency, the YFP acceptor was also excited individually using the 500 nm LED of the pE-800.
The FRET results were complemented by Fluorescence Lifetime Imaging Microscopy (FLIM) using a confocal microscope setup, to provide greater detail on the FRET results – and determine any difference in the FRET results between peptides localising in either the ordered or disordered areas of the membrane.
In summary, this research indicated that disordered membrane lipid domains have a direct effect on the HCN receptors and leads to increased activity, which is relevant in disease.
Benefits of the pE-800 for FRET
The pE-800 is ideal for FRET, owing to several features including:
- Eight individual LEDs – the wide spectral coverage from eight individually controllable LEDs includes the most popular fluorophores used in FRET, such as CFP and YFP, with the ability to tailor the excitation precisely to the fluorophore using optical filters.
- Fine intensity control – providing the flexibility to modulate individual channel intensities with 0-100% control enables a balance between the brightest visualisation and minimising photobleaching.
- Industry-leading <7 µs TTL triggering – Fast imaging maximises temporal resolution when studying molecular interactions.
About the pE-800
The CoolLED pE-800 includes eight LED channels covering the major fluorophores and opsins.
The ability to have any combination of these switched on and control the intensity allows multiple fluorophores to be excited in parallel, while high-speed TTL triggering and synchronisation enables accurate and precise control of complex pulsing protocols.
This is particularly useful for neuroscience applications.
CoolLED specialises in LED microscope lighting and, since our team of four introduced the first commercially available LED microscope illuminator in 2006, we have led the way in designing and manufacturing cutting edge LED Illuminators for Microscopes using the latest technology.