We apply our recently developed QPI methodologies, which quantify and track intracellular water content, to investigate the role of aquaporin (AQP) proteins in pulsed electric field (PEF) induced water uptake in cells. QPI imaging is performed on Jurkat and glial cells subjected to PEF simulation. The effects on the transmembrane water flux of the cells are investigated when solutions contain mercury, a known broad spectrum AQP blocker, or commercially available AQP blockers. Our study demonstrates the utility of QPI for rapid intracellular water quantification and shows a path forward for identifying biophysical mechanisms using label-free imaging.
Water is the most basic requirement of life, acting as a medium of exchange, transport, homeostasis and diffusion. We present a technique which leverages quantitative phase imaging (QPI) and a two-component mixture model to quantify, in absolute terms, the intracellular water fraction (by mass and volume) as well as the intracellular water volume in femtoliters. We demonstrate our technique by applying a single microsecond-duration electric pulse, and observe water influx in physiological buffer, water efflux in hyperosmotic buffer, and no movement across the membrane of synthetic vesicles. Dose response curves and channel blockers are employed to better understand underlying phenomena.
We present a novel strobe photography system capable of recording a full dynamic sequence in a single camera acquisition, with an effective frame rate of 1 million FPS. This system has been utilized to study pulsed laser ablation from a 1064 nm, 6 ns laser pulse incident on biological tissue. Image sequences enable the extraction of physical parameters, include shock wave and ejecta velocities, and accelerations. Encoding contrast with shadow photography enables the estimation of the mass ejected for a single ablation pulse.
A great deal of research has been focused on the study of the dynamics of single cells exposed to short duration, (<1µs) high peak power (~1 MV/m) transient electric fields. Currently, most of this research is limited to the use of traditional fluorescence-based microscopy techniques, which introduce exogenous agents to the culture and are only sensitive to a specific molecular target depending on the dye used. Quantitative phase imaging (QPI) is a coherent imaging modality which uses optical pathlength (OPL) as a label-free contrast mechanism, and has proven highly effective for the study of single-cell dynamics. In this work, we demonstrate how QPI can be used to monitor biophysical properties of cells undergoing pulsed electric field (PEF) exposure. We introduce new QPI image processing methods to monitor the cellular dry mass, refractive index, mass density, and water content of cells from a single snapshot. These parameters are tracked following exposure to a microsecond-duration pulse. We hope QPI will continue to be used for the study of electroporation-induced bioeffects.
Our group recently published direct observation of membrane charging in FluoVolt labeled CHO-K1 cells by nanosecond electrical pulses using a streak camera. Using this technique, called Streak Camera Microscopy (SCM), we imaged the membrane charging dynamics in giant unilamellar vesicles (GUVs) during AC exposures up to 6 MHz and compared these results to existing capacitive circuit models of membranes. This work shows further application of Streak Camera Microscopy for evaluation of high speed biological events.
Recently, Beier et al. a new imaging system called a streak camera microscope (SCM) that is able to resolve sub nanosecond membrane potential changes in cells loaded with FluoVolt dye. This technique opens the window into the response of cell membrane potential to rapidly applied electric fields. Having demonstrated this response on single bipolar and unipolar electric pulses, we have begun to investigate the rapid charging and discharging of the plasma membrane during bursts of AC frequencies. We believe that understanding the dynamics of plasma membrane charging during AC pulses will better inform those using such pulses to modify cell behavior.
Compressed ultrafast photography (CUP) is a new imaging technique which incorporates the high-speed imaging capabilities of a streak camera with principles from compressed sensing to allow for single-shot, ultrafast imaging of optical phenomena at up to 100 billion frames per second1. While this technique has primarily been utilized to observe physical phenomena, it has broad ranging application to biological imaging including fluorescence lifetime microscopy, and single-shot hyperspectral imaging2. Additionally, similar optical systems can be used to for streak camera microscopy (SCM), enabling the resolution of sub microsecond changes in cellular membrane potential in response to electrical stimulation3. Here, we present several applications for CUP based imaging and spectroscopy as a novel tool to enable more rapid biological imaging and sensing.
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