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Shining a light on solutions for multiplexed fluorescence detection
Keely Portway discovers some of the latest techniques to increase the capabilities of multiplexed fluorescence detection, and why the right illumination solutions are essential
M
ultiplex microscopy is widely used by researchers, particularly in the
biological sciences, to obtain information that will help them gain a greater understanding of biological processes. The multiplexed fluorescence detection technique can simultaneously detect and quantify multiple targets, such as proteins or nucleic acids in a single sample. This method uses fluorescent dyes or labels that emit distinct signals when excited by different wavelengths of light.
In multiplexed fluorescence
detection, multiple fluorescently labelled probes or antibodies are used, each of which targets a specific analyte. Probes are labelled with unique fluorescent dyes, which allows them to be distinguished and detected separately. The sample is then analysed using a specialised instrument, such as a microarray scanner or a flow
26 Electro Optics May 2023
cytometer, which can detect and measure the fluorescent signals emitted by each dye. The technique has been
used throughout the world for years in applications such as gene expression analysis, protein analysis, pathogen detection, environmental monitoring and drug discovery and development, to name just a few. This is because of the advantages it offers as a cost- effective, accurate, flexible and efficient form of detection.
Multiplexed fluorescence detection: the challenges However, as demand from biologists and researchers grows to be able to detect more biological targets than ever before, multiplexed fluorescence detection has also encountered its share of challenges – largely due to the risk of spectral crosstalk if more than four or five targets are attempted. Iain Johnson, Ph.D., Director of Technical
Support at Lumencor explains: “In my experience, if you ask a biologist how many samples they need to target, the answer they always provide is ‘more’. If they can identify four, then next they want to identify 40, and then 400 and 4,000. There are billions of different protein molecules in a single cell that produce its activity, and you can only get so far looking at four of them at any one time. So, what happens when four or five is not enough? How do you get to 400, and then 4,000?” A number of techniques
have been developed to help increase the number of targets that can be detected, each with their own advantages and challenges, and each of which requires its own specific chain of component parts that must fit together to guarantee the right results, and each of which relies on the illumination solution as the first step to success. These techniques include the introduction of
quantum dot nanocrystals with narrow emission spectra, molecular barcode scanning for gene expression analysis, and multiplexed error- robust fluorescence in situ hybridisation (also known as MERFISH).
Looking specifically at MERFISH, the technique works as a single-cell ribonucleic acid (RNA) sequencing method that uses fluorescent probes to detect and quantify multiple RNA targets in individual cells. It can detect hundreds to thousands of genes simultaneously, so it can be a powerful tool for biologists looking to identify more targets. Continues Johnson: “MERFISH is a state-of-the- art technique where you can actually look at 10,000 targets to some extent. That’s how far things have come. Admittedly, it is a much more complex and expensive technique to implement, but the biological imperative for doing it is so
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