Dialéctica de la triplicidad, entendiendo a Lefebvre

Using digital holographic microscopy (DHM) we developed a medium-throughput flow-based quantitative technique to measure deformability. DHM determines the height of a sample and has the advantage over other techniques being non-invasive and label-free. DHM can map three-dimensional fluctuations in the shape and volume of multiple cells simultaneously at in situ conditions. In the case of erythrocytes, height and volume are reliant on the deformability of the cell and thus can be used as an indirect measure for deformability.

We initially validated the DHM imaging technique on stationary erythrocytes, which were fixed onto slides through air drying, and showed that DHM could resolve the height and topology of individual erythrocytes up-to the nanomolar range (He et al. 2016). It was then further confirmed in a microfluidic-based deformability assays to quantify deformability in fluidic conditions reflective of the microvasculature environment. The deformability model consisted of erythrocytes artificially stiffened with varying amounts of glutaraldehyde, which allowed to test the limits of quantification. We tracked the displacement of the centre of mass (COM) in the x-axis and z-axis directions, in response to external fluidic pressure, as a measure of overall cell displacement and height (membrane) displacement respectively. DHM was able to discern the variation in displacement between various concentrations of glutaraldehyde and untreated cells where we observed a non-linear inverse relationship between cell deformability and the concentration of glutaraldehyde.

The technique was then tested on wild type infected erythrocytes and showed that infected erythrocytes had a lower rate of deformation in response to fluctuating fluidic pressure in comparison to wild type erythrocytes. We observed a significant difference (p<0.0001) in overall cell deformability between infected and uninfected erythrocytes but were not able to differentiate membrane deformability as changes in height were inconsistent between infected erythrocyte cells.

This lack of resolution on membrane deformability likely comes from the unit of measurement used (COM displacement) rather than the microscopy technique itself. Centre of mass

Chapter 6

COM reduces a cell to a single mean spatial integral and does not reflect the non-uniform fluctuations in the membrane. DHM can however also resolve the height at multiple points on the membrane, depending on the magnification used. Sampling the height of the cell rather than height of COM would give a better estimation of membrane deformability. Thus, future experiments on membrane deformation should require inferring the height of the cell rather that height of COM. The experiments performed here show that DHM, in a flow-based system, is capable of resolving single cell deformability of multiple cells at a nanomolar resolution of overall cell displacement. In terms of throughput efficiency, DHM can analyse multiple cells in one field of view and in the case of uninfected erythrocytes analysed up to 14 cells in one field of view (under 400x magnification). The limiting factor in the efficiency of the current technique is the rate of cell binding to Con A and thus optimising binding would improve the efficiency.

Quantitively, comparison to current deformability techniques in terms of resolution and sensitivity would require more data on infected erythrocytes for both cell and membrane deformability. This includes more cells per technical repeat but also more technical and biological repeats in the form of different donors, as deformability profiles can also differ between donors. Although the deformability quantification method requires optimisation, the DHM microscopy technique does fill a gap in the range of available population and single cell techniques. High-throughput population-based techniques such as ektacytometry cannot differentiate between the several factors that contribute towards overall deformability and cannot recognise variations in deformability that fit outside the expected patterns of cell distortion, such as those manifested in sickle cell disease (Rabai et al. 2014). Furthermore, through analysis of population of erythrocytes, these techniques assume that deformability between cells in a population is consistent and does not account for the inherent inter-cell age heterogeneity of erythrocytes found in the blood stream, which affects deformability (Baskurt et al. 2009). DHM, on the other hand, can measure multiple cells simultaneously, and produce a value that is an average of cells in a population rather than population deformability, thus accounting for the older more rigid erythrocytes.

On the other end of the spectrum, micropipette aspiration (MPA), despite the high resolution, is low throughput and only measures one spot on each cell (Hochmuth 2000). This can be

problematic for erythrocytes as the plasma membrane contains several cholesterol and sphingomyelin-enriched lipid microdomains. Microdomains render the membrane heterogenous in its elasticity and fluidity (Chabanel et al. 1983; D. A. Brown 2006), which would affect the quantification of membrane deformability if measured at a singular point on the membrane. Additionally, host cell modifications are biased towards microdomains as several exported parasite proteins are associated with detergent-resistant membranes (Sanders et al. 2007). DHM can sample several spatial points on the surface of one cell and map the variation in membrane displacement of single cells as a proximation of heterogenous membrane deformation. Thus, to optimize measurement of membrane deformability, future experiments would simply require re-evaluation of the entire technique with multiple points per cell.

In summary, DHM was able to resolve changes in deformability in infected cells in a flow-based system that stimulates in vivo conditions. However, it still requires further optimisation to increase throughput efficiency and a change in the unit of measurement to allow to measure also membrane deformability. The use of a quantitative phase imaging technique such as DHM provides the ease of use and increased efficiency of a population-based technique but with the ability to discern individual cells and is a valuable addition to the current repertoire of techniques.