31 Aug 2023
Boston University S-RACE platform uses Raman rather than fluorescence in flow cytometry.
The principle involves illuminating cells while they pass along a channel narrow enough to force the cells into roughly single file, usually after tagging them with fluorescent markers.
Recent improvements to this principle have included research at NIST into using bespoke waveguides allowing multiple measurements to be made of each individual cell, reducing the measurement-dependent variations that can arise using the flow cytometry approach.
A project at Boston University has now investigated a different route to improving the process, by using Raman spectroscopy rather than the more usual fluorescence methods. The work will be presented at the Frontiers in Optics+Laser Science conference in October 2023.
"Although most current high-throughput cell sorting methods rely on fluorescence signals for sorting, fluorescence labels can disturb cell function and can't be used with small molecules," said the Boston team.
"Raman spectroscopy is a promising alternative because it offers label-free and non-destructive single-cell measurement by obtaining a chemical fingerprint of the cell. However, it has been difficult to achieve both a strong Raman signal and a practical microfluidic setup for imaging cells."
The project tackled these hurdles by using stimulated Raman spectroscopy, the variant technique in which non-linear effects are exploited to transform more of the incident light into useful Raman scattering and improve the signal to noise ratio, potentially by several orders of magnitude.
Pathogens directly captured from their natural habitat
In the Boston platform, stimulated Raman images are acquired and used to identify the objects or cells of interest. 2D galvo mirrors and an acousto-optic modulator then direct single pulses of a 532 nanometer laser to the desired cells and push them into a collector, with each ejection taking about 8 milliseconds.
In trials, this stimulated Raman-activated cell ejection (or S-RACE) method was applied to a mixture of 1 micron polymer beads, achieving around 95 percent purity and 98 percent throughput with about 14 ejections performed each second, according to the Boston project. The trials also showed that the method could also be used with fixed bacteria.
For tests on live yeast cells, the researchers added a thin layer of agar to the ejection module and used an agar dish as a collector to provide cushioning and moisture during cell landing. The system ejected approximately 340 yeast cells, which were healthily growing in the receiving dish around 40 hours afterwards. Other genomic analysis approaches, such as quantitative polymerase chain reaction, could be integrated with this sorting approach.
"Our S-RACE approach offers an innovative way to sort cells based on their intracellular chemical composition in a high-throughput manner," commented Boston University's Jing Zhang.
"Various downstream phenotypic or genomic analysis could be applied to the separated cell populations, and its compatibility with small cells is advantageous for sorting bacteria and other microorganisms. By employing S-RACE, pathogens or cells exhibiting specific metabolic profiles could be directly captured from their natural habitat, for example water bodies, soil, or the gastrointestinal tract."