Cellular tolerance to pulsed hyperthermia

Transient heating of tissues leading to cellular stress or death is very common in medicine and biology. In procedures involving a mild (below $70°\mathrm{C}$) and prolonged (minutes) heating, such as hyperthermal tumor therapy, the cellular response to thermal stress is relatively well studied. However, there is practically no data on cell viability at higher temperatures and shorter exposures, while the demand for this knowledge is growing. Two main reasons motivate this research: (i) a growing number of laser therapies and surgical procedures involving pulsed heating, and (ii) cellular viability data at short exposures to high temperatures provide a unique insight into the understanding of processes leading to thermally induced cellular death. We designed a technique and performed a study of cell viability under pulses of heat from $0.3\text{to}100\mathrm{ms}$ in duration with peak temperatures as high as $130°\mathrm{C}$. We found that the threshold of cellular death in this range can be accurately approximated by the Arrhenius law with the activation energy of $1\mathrm{eV}$, a significantly lower value than was reported in studies based on multisecond exposures.

Figure 1

(a) Pulsed laser heating of a single layer cell culture. (b) Laser heating pulses of variable intensity and duration were produced by transmitting the $\mathrm{C}{\mathrm{O}}_2$ laser beam through a pair of polarizers that served as an attenuator and a rotating disk chopper. Thermal radiation emitted from the central region of the irradiated sample area was used to determine sample temperature. An aperture in the collection optics selected radiation emitted from the central part of the sample and a filter rejected scattered $\mathrm{C}{\mathrm{O}}_2$ laser radiation.

Figure 2

Calculated axial temperature distribution in a sample at the end of $300\mu \mathrm{s}$ and $100\mathrm{ms}$ laser pulses.

Figure 3

Calibration curve for temperature measurements using infrared thermal emission from the sample.

Figure 4

(a) Infrared emission signal from the target. Moment of explosive vaporization is marked by an abrupt change in surface emissivity and appearance of a signal from the $\mathrm{C}{\mathrm{O}}_2$ laser radiation scattered on an oscillating bubble. (b) Threshold of explosive vaporization of water adjacent to Mylar film measured by IR detection system as a function of laser pulse duration. For short laser pulses thermal emission measurements give underrated values of temperature due to optical thinness of the hot surface layer.

Figure 5

(Color) Microscope image of the stained cells after irradiation showing live (green) and dead (red) zones within the laser-irradiated spot. Temperature corresponding to the boundary between live and dead cells was defined as a threshold temperature.

Figure 6

Temperatures at the end of a laser pulse corresponding to the live-dead transition region.

Figure 7

Temporal profiles of the sample surface temperature corresponding to laser pulses of 0.3, 1, 3, 10, 30, and $100\mathrm{ms}$. Time scale is normalized to laser pulse length. Shortest normalized temperature relaxation time corresponds to $100\mathrm{ms}$ laser pulse and longest one to $0.3\mathrm{ms}$ pulse.

Figure 8

Cell viability threshold as a function of “effective time” (solid black squares) fitted with the Arrhenius law (solid line). Open symbols-experimental data of cell viability thresholds from various sources with dashed line showing general trend.