Researchers at the Laboratory for Lightwave Metrology and at Attoworld have achieved the first successful field-resolved spectroscopy (FRS) flow cytometry measurements, marking a significant stride in the realm of cellular and particle analysis. Flow cytometry has long been recognized as a powerful tool for studying individual cells and particles, providing crucial insights into biological and medical phenomena. However, its reliance on fluorescent labels has been a limitation, prompting the quest for label-free alternatives.
The team’s pioneering work, to be presented at the International Congress on Biophotonics (ICOB) in Jena in March 2024 [1], focuses on overcoming the challenges posed by fluorescent labels through the application of vibrational spectroscopy. This promising technique combines minimal sample preparation, non-destructive acquisition of chemically-specific signals, and very short acquisition times.
Utilizing FRS with brilliant, waveform-controlled femtosecond-laser-based infrared (IR) sources, the researchers successfully carried out high-speed IR-FRS measurements of particles in flow. An FRS instrument with rapid fingerprint acquisition at 38 kHz enabled the investigation of individual particles and cells in flow, achieving measurement times as low as 1 ms per cell .
The team collected a dataset comprising more than 25,000 spectra, from various particle types including PMMA and PS beads of different diameters, as well as yeast and THP-1 cells. By employing data analysis techniques such as density-based spatial clustering and t-distributed stochastic neighbour embedding, the researchers demonstrated the ability to distinguish and separate all measured particle types.
These results showcase the potential of FRS in realizing label-free, IR-based flow cytometry and fast, label-free sorting of large numbers of human cells for the first time. The ability to identify different particle types at the individual level without the need of labelling and with acquisition times on the order of 1 ms per particle heralds a new era in high-speed cellular analysis. The team anticipates future applications to benefit from next-generation spectrometers, expanding the scope of vibrational fingerprinting in biological systems and enabling high-throughput phenotyping of immune-cell activation states as well as screening for low-abundance circulating tumour cells.
[1] Marinus Huber, Daniel Gerz, Holger Mirkes, Florian Lindinger, Mark Kielpinski, Thomas Henkel, Mihaela Zigman, Ferenc Krausz, Jürgen Popp, Ioachim Pupeza, “Label-free infrared-vibrational flow cytometry,” International Congress on Biophotonics, Jena, 3 – 7 March 2024.