Dead Yeast Cells

The final particle type included in our library our yeast cells that are non-viable. This introduces a particle type that differs in a distinct physiological way from living yeast cells. For the purpose of carrying out experiments, one way to render living cells non-viable in a uniform way is to expose them to high temperatures for an extended duration of time [51]. Dead cells prepared in this manner have an additional benefit of being able to be selectively stained with dye, allowing them to be distinguished from living cells visually. Figure 3.16 shows a sample containing a mixture of living yeast cells and dyed cells that have been rendered non-viable via heating.

Figure 3.16 Mixture of living yeast cells and dead cells that have been stained with dye

Heat-treated yeast cells have been analyzed previously in the context of dielectrophoresis [51]. The multi-shell model has been found to be able to accurately model the electrical properties of yeast cells that have been rendered non-viable in this manner. The primary difference in the models used for dead versus living yeast is that dead yeast models have fewer layers. The model we use (figure 3.17), has 3 layers: a cell wall, membrane and inner core, 2 fewer layers than is used by our viable yeast model. Although 3-layer model has been empirically shown to be better fit for heat-treated cells, the physiological reasons as to why this is the case is not known. However, it would be reasonable to suggest that the application of extreme heat disrupts the cells internal structure to the point where the periplasmic space has been destroyed and there is no longer a significant difference between the inner and outer cell walls. In this model, the inner core has a radius of rint = 3 μm, and the membrane and cell wall have respective thicknesses of tmem = 8 nm and twall = 250 nm. The conductivities of the layers are σint = 7mS/m, σmem = 160 μS/m and σwall = 1.5 mS/m with corresponding dielectric constants

of εint = 50, εmem = 6 and εwall = 60. Figure 3.18 shows the Clausius-Mossotti factor that results when this model is placed in the reference medium.

Figure 3.17 3-layer multi-shell models used for calculating the effective permittivity of non-viable yeast cells

Figure 3.18 Clausius-Mossotti factor spectrum for 3-layer mutli-shell model of non-viable yeast cells in the

Figure 3.19 Real part of Clausius-Mossotti Factor for non-viable yeast cells

Figures 3.19 shows a close up views of Re{KCM} for dead yeast cells. The plot shows that Re{KCM} is negative for all frequencies, meaning that they will always undergo negative dielectrophoresis in this medium. There is a point of inflection in the spectrum for Re{KCM}, centered around f = 285 kHz, where Re{KCM} briefly increases towards zero, but never becomes positive. At frequencies shortly after that, the spectrum quickly declines and saturates to its maximum negative value. This is quite different from the behavior of living yeast cells that can exhibit both nDEP and pDEP depending on frequency. At lower frequencies, Re{KCM} has a constant value of -0.03 and at high frequencies a constant value of -0.13 . These peak values are such that the maximum negative dielectrophoresis force that can be exerted on dead yeast is on the same order of magnitude as living yeast cells but significantly less than the maximum for the polystyrene particles.

The imaginary spectrum for dead yeast cells is shown in figure 3.20. At low frequencies Im{KCM} is zero and then slowly increases to a small positive peak of 0.007 at approximately 195 kHz, where TWDEP forces will propel particles in the direction of the phase gradient. After that peak Im{KCM} descends to a negative value of -0.07 near 1.85 MHZ, where TWDEP forces will move the cell opposite the phase gradient. At high frequencies, f > 100MHz, Im{KCM} nears zero again and dead cells will not have TWDEP forces exerted on them

When this model is introduced into the TWDEP configuration of figure 2.5, it is predicts that dead cells will have 3 primary regions of operation. At very all frequencies, and in all 3 regions, nDEP forces will cause the particle to elevate above the array. In the first region of operation, occurring at very low and high frequencies, dead cells will not exhibit any lateral motion while remaining elevated above the electrode array due to nDEP. Near the frequency at which the small positive peak in Im{KCM} occurs (195 kHz), dead cells will be propelled laterally

in the same direction as the phase gradient, while they will move laterally in the opposite direction at frequencies near the negative peak (1.85MHz). Similar to the case for living yeast cells, the model for dead cells indicates that they will experience TWDEP in two directions. However, the response for dead yeast is distinctly different in that the maximum TWDEP forces will be an order of magnitude smaller than as for living yeast and their relative maximums happen at different frequencies.