Abstract
Although cell volume is a critical cell parameter and acute changes of its regulation mechanisms can reveal pathophysiological states, its accurate measurement remains difficult. On the other hand, custom-made devices are now possible through the application of 3D printing that meet the needs of certain specific applications. Therefore, in this article we describe the development of a low-cost, open-source, and 3D-printed flow chamber optimized to perform non-invasive accurate measurements of important cell biophysical parameters. This measurement development includes the absolute cell volume, mean cell thickness, and whole-cell refractive index using quantitative-phase digital holographic microscopy. Specifically, this advancement makes it possible to develop a flow chamber that preserves cell viability while exhibiting a fast washout process for the two-liquid decoupling procedure - an experimental procedure allowing to separately measure the intracellular refractive index and cell thickness from the quantitative-phase signal - characterized by a homogeneous return of the extracellular refractive index over the entire imaging slit toward the known value of the last perfusion solution. Such a fast washout process has been extensively characterized by monitoring the time required to obtain a stabilization of the quantitative-phase signal following the perfusion of the two liquids. A more rapid quantitative-phase signal stabilization time results in fewer cell changes, including important cell position and shape modifications. These changes likely take place over the washout process time and ultimately result in important artefacts to all cell biophysical measurements. The proposed flow chamber has been used to perform measurements of the whole-cell refractive index, mean cell thickness, and absolute cell volume of three cell types in culture during resting state, and hypo-osmotic and hyper-osmotic challenges. For a typical cell body, these biophysical parameters are measured with a precision of 0.0006, 100 nm, and 50 µm3 respectively. Finally, the design files of the flow chamber will be shared with the scientific community through an open-source model.
Highlights
IntroductionMovements of water across the membranes are in large part dictated by osmotic pressure gradients
Animal cell membranes are highly permeable to water
In the context of the two-liquid decoupling approach, these dead volumes and unstirred layers are detrimental to obtaining a fast-homogeneous return of the extracellular refractive index toward either nm1 or nm2
Summary
Movements of water across the membranes are in large part dictated by osmotic pressure gradients. Even at constant extracellular osmolarity, volume constancy of any mammalian cell is permanently challenged by its own activity including, for example, movements of different ionic species across the cell membrane or transport of osmotically active substances involved in cell metabolism all of which induce osmotic water movements [1, 2]. Cells have developed numerous efficient volume regulatory mechanisms involving multiple ion transport processes including channels, cotransporters, and exchangers which operate to maintain a constant ionic balance and hydrostatic pressure gradient. Osmotically active solutes (mainly inorganic ions such as Na+, K+, Cl−, or organic osmolytes) are gained or lost to regulate the cell volume [5]
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