Comprehensive single-shot biophysical cytometry using simultaneous quantitative phase imaging and Brillouin spectroscopy.

Single-cell analysis, or cytometry, is a ubiquitous tool in the biomedical sciences. Whereas most cytometers use fluorescent probes to ascertain the presence or absence of targeted molecules, biophysical parameters such as the cell density, refractive index, and viscosity are difficult to obtain. In this work, we combine two complementary techniques-quantitative phase imaging and Brillouin spectroscopy-into a label-free image cytometry platform capable of measuring more than a dozen biophysical properties of individual cells simultaneously. Using a geometric simplification linked to freshly plated cells, we can acquire the cellular diameter, volume, refractive index, mass density, non-aqueous mass, fluid volume, dry volume, the fractional water content of cells, both by mass and by volume, the Brillouin shift, Brillouin linewidth, longitudinal modulus, longitudinal viscosity, the loss modulus, and the loss tangent, all from a single acquisition, and with no assumptions of underlying parameters. Our methods are validated across three cell populations, including a control population of CHO-K1 cells, cells exposed to tubulin-disrupting nocodazole, and cells under hypoosmotic shock. Our system will unlock new avenues of research in biophysics, cell biology, and medicine.

Multi-wavelength excitation Brillouin spectroscopy.

We propose and demonstrate, first on simulated spectra and then experimentally, a novel approach to correct the undesired background distortions in the Brillouin spectra caused by molecular filter's absorption, fluorescent emission, ambient room light or any other constant contaminant. The developed multi-wavelength excitation Brillouin spectroscopy method computationally reconstructs the pure Brillouin component of the signal from multiple Brillouin spectra acquired using different excitation wavelengths. By removing the baseline distortions, the approach improves the goodness of fit of the Brillouin peaks, enabling accurate Brillouin shift and linewidth measurements from a wide range of challenging samples. In the present report, we explain the principle behind the method on a set of simulated spectra and present experimental application on an intentionally strongly-distorted spectrum. Utilizing the multi-excitation Brillouin spectroscopy approach, we successfully reconstruct Brillouin spectra of a highly-scattering sample, initially rendered not analyzable by excessive iodine absorption and contamination by out-of-focus light.