Brillouin microscopy monitors rapid responses in subcellular compartments.

Measurements and imaging of the mechanical response of biological cells are critical for understanding the mechanisms of many diseases, and for fundamental studies of energy, signal and force transduction. The recent emergence of Brillouin microscopy as a powerful non-contact, label-free way to non-invasively and non-destructively assess local viscoelastic properties provides an opportunity to expand the scope of biomechanical research to the sub-cellular level. Brillouin spectroscopy has recently been validated through static measurements of cell viscoelastic properties, however, fast (sub-second) measurements of sub-cellular cytomechanical changes have yet to be reported. In this report, we utilize a custom multimodal spectroscopy system to monitor for the very first time the rapid viscoelastic response of cells and subcellular structures to a short-duration electrical impulse. The cytomechanical response of three subcellular structures - cytoplasm, nucleoplasm, and nucleoli - were monitored, showing distinct mechanical changes despite an identical stimulus. Through this pioneering transformative study, we demonstrate the capability of Brillouin spectroscopy to measure rapid, real-time biomechanical changes within distinct subcellular compartments. Our results support the promising future of Brillouin spectroscopy within the broad scope of cellular biomechanics.

Rapid and precise tracking of water influx and efflux across cell membranes induced by a pulsed electric field.

Quantitative measurements of water content within a single cell are notoriously difficult. In this work, we introduce a single-shot optical method for tracking the intracellular water content, by mass and volume, of a single cell at video rate. We utilize quantitative phase imaging and a priori knowledge of a spherical cellular geometry, leveraging a two-component mixture model to compute the intracellular water content. We apply this technique to study CHO-K1 cells responding to a pulsed electric field, which induces membrane permeabilization and rapid water influx or efflux depending upon the osmotic environment. The effects of mercury and gadolinium on water uptake in Jurkat cells following electropermeabilization are also examined.

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.

Dynamic nitrogen vacancy magnetometry by single-shot optical streaking microscopy.

Nitrogen vacancy diamonds have emerged as sensitive solid-state magnetic field sensors capable of producing diffraction limited and sub-diffraction field images. Here, for the first time, to our knowledge, we extend those measurements to high-speed imaging, which can be readily applied to analyze currents and magnetic field dynamics in circuits on a microscopic scale. To overcome detector acquisition rate limitations, we designed an optical streaking nitrogen vacancy microscope to acquire two-dimensional spatiotemporal kymograms. We demonstrate magnetic field wave imaging with micro-scale spatial extent and ~400 µs temporal resolution. In validating this system, we detected magnetic fields down to 10 µT for 40 Hz magnetic fields using single-shot imaging and captured the spatial transit of an electromagnetic needle at streak rates as high as 110 µm/ms. This design has the capability to be readily extended to full 3D video acquisition by utilizing compressed sensing techniques and a potential for further improvement of spatial resolution, acquisition speed, and sensitivity. The device opens opportunities to many potential applications where transient magnetic events can be isolated to a single spatial axis, such as acquiring spatially propagating action potentials for brain imaging and remotely interrogating integrated circuits.

Visualizing bleb mass dynamics in single cells using quantitative phase microscopy.

Understanding biological responses to directed energy (DE) is critical to ensure the safety of personnel within the Department of Defense. At the Air Force Research Laboratory, we have developed or adapted advanced optical imaging systems that quantify biophysical responses to DE. One notable cellular response to DE exposure is the formation of blebs, or semi-spherical protrusions of the plasma membrane in living cells. In this work, we demonstrate the capacity of quantitative phase imaging (QPI) to both visualize and quantify the formation of membrane blebs following DE exposure. QPI is an interferometric imaging tool that uses optical path length as a label-free contrast mechanism and is sensitive to the non-aqueous mass density, or dry mass, of living cells. Blebs from both CHO-K1 and U937 cells were generated after exposure to a series of 600 ns, 21.2 kV/cm electric pulses. These blebs were visualized in real time, and their dry mass relative to the rest of the cell body was quantified as a function of time. It is our hope that this system will lead to an improved understanding of both DE-induced and apoptotic blebbing.

Strobe photography mapping of cell membrane potential with nanosecond resolution.

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious electrostimulation). The imagery from this system allows for direct comparison of membrane voltage change to both computationally simulated external electric fields and time-dependent membrane charging models. Acquisition of a full microscope field of view enables the selection of data from multiple cell locations experiencing different electrical fields in a single image sequence for analysis. Using this system, more realistic membrane parameters can be estimated from living cells to better inform predictive models. As a proof of concept, we present evidence that within the range of membrane conductivity used in simulation literature, higher values are likely more valid.

Quantitative phase microscopy monitors subcellular dynamics in single cells exposed to nanosecond pulsed electric fields.

A substantial body of literature exists to study the dynamics of single cells exposed to short duration (<1 µs), high peak power (~1 MV/m) transient electric fields. Much of this research is limited to traditional fluorescence-based microscopy techniques, which introduce exogenous agents to the culture and are only sensitive to a single molecular target. Quantitative phase imaging (QPI) is a coherent imaging modality which uses optical path length as a label-free contrast mechanism, and has proven highly effective for the study of single-cell dynamics. In this work, we introduce QPI as a useful imaging tool for the study of cells undergoing cytoskeletal remodeling after nanosecond pulsed electric field (nsPEF) exposure. In particular, we use cell swelling, dry mass and disorder strength measurements derived from QPI phase images to monitor the cellular response to nsPEFs. We hope this demonstration of QPI's utility will lead to a further adoption of the technique for the study of directed energy bioeffects.