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Bioluminescence imaging as a marker for cellular Hsp70 response to thermal laser injury

Assessment of laser tissue damage is not complete without an investigation into the cellular effects that are induced. In the past, tissue damage was quantified by such macroscopically visual results as tissue mass removal, carbonization, and melting. In this research, we used heat shock protein (hsp70) transcription, to track cellular response to laser injury. A stable cell line was generated containing the luciferase reporter gene attached to the heat shock protein (Hsp70) promoter. After thermal injury with a Holmium:YAG pulsed laser (wavelength= 2.1 μm, pulsetime = 250 μs, 30 pulses, 3 Hz), luciferase is produced upon hsp70 activation and emits bioluminescence at 563 nm. The luminescence was quantified with a liquid nitrogen cooled CCD camera. A minimum pulse energy (65 mJ/pulse, 2.0 mJ/mm2) was needed to activate the hsp70 response and a higher energy (103 mJ/pulse, 3.2 mJ/mm2) was associated with a reduction in hsp70 response. Bioluminescence levels correlated well to actual hsp70 protein concentrations as determined by ELISA assay. Photon counts were normalized to the percentage of live cells by means of a flow cytometry cell viability assay. The hsp70 response followed an Arrhenius relationship in nature when constant temperature water bath and constant area laser experiments were carried out.