Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

Abstract

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.

High-throughput physical phenotyping of cell differentiation

“…However, both methods are time and labor intensive (tens of cells per hour), posing challenges for examining large populations of cells to either obtain statistically valid conclusions or identify rare sub-populations. Recent advances in micro-/nano-fabrication technologies have opened up a range of new mechanophenotyping technologies that can measure deformations of tens to hundreds of cells per second [11][12][13][14][15][16][17][18][19] . We recently reported a technology, called deformability cytometry, in which a cross-slot microfluidic channel is employed to generate a hydrodynamic extension zone where individual cells are exposed to uniform hydrodynamic stress and deformed 20 .…”

Imaging of a linear diode bar for an optical cell stretcher

“…Because of this, microfluidics and optical forces have been combined to create optical stretchers [5][6][7] which impart stresses on the surface of deformable microscale objects [5][6][7][8][9][10][11][12], an approach that has seen significant recent interest because of the non-invasive nature of optical traps and the desire to measure cell mechanical properties [13,14]. Currently, the simplest linear optical stretcher employs a single inexpensive, high-power diode laser bar source [15][16][17] to stretch cells along the laser long axis [18][19][20]. In this, opposing antipodal stretching forces are generated along the linear stretcher when the beam width and length are shorter and longer than the cell size, respectively [19].…”

“…The short laser dimension (fast axis) of linear diode bar sources typically diverges at ~30°, corresponding to a 0.5 NA, while the beam typically diverges at ~10° (slow axis) along the length of the extended source. In previous reports, we have used a pair of microscope objectives to collimate the laser output and relay the image of the laser bar to the specimen plane [18,20]. In such a design, the laser source is collimated by the first microscope objective, but due to the elongated geometry of the linear source, the collimated beam continues to experience extension along the long axis.…”

High‐throughput linear optical stretcher for mechanical characterization of blood cells

“…In the case of a linear optical trap, antipodal stretching forces generated parallel to the trap long axis act to elongate the cell (37), while the relative deforming influence of the hydrodynamic forces remains low (40). By applying optical forces independent of flow forces cell viscoelastic behavior has been accurately quantified via a mechanical model (41). Such techniques can maintain flow velocities capable of deforming 10 3 210 4 cells in 10-20 min while employing geometries very similar to current light-scattering and fluorescence-based imaging cytometers.…”

“…Here, we use linear optical stretchers described previously (41,42) to stretch cells continuously in flow. To avoid changes in laser intensity and beam profile due to the linear emitter geometry (43)(44)(45), custom optics (37) were used to focus the stretcher at the optimal numerical aperture (NA) with efficient 1:1 imaging of the source (46).…”