Principles of laser microdissection and catapulting of histologic specimens and live cells

Abstract

Rapid contact- and contamination-free procurement of specific samples of histologic material for proteomic and genomic analysis as well as separation and transport of living cells can be achieved by laser microdissection (LMD) of the sample of interest followed by a laser-induced forward transport process [laser pressure "catapulting," (LPC)] of the dissected material. We investigated the dynamics of LMD and LPC with focused and defocused laser pulses by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation. Catapulting is driven by plasma formation, when tightly focused pulses are used, and by ablation at the bottom of the sample for moderate and strong defocusing. Driving pressures of several hundred megapascals accelerate the specimen to initial velocities of 100-300 m/s before it is rapidly slowed down by air friction. With strong defocusing, driving pressure and initial flight velocity decrease considerably. On the basis of a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the temporal evolution of the heat distribution in the sample. After laser microdissection and laser pressure catapulting (LMPC), the samples were inspected by scanning electron microscopy. Catapulting with tightly focused or strongly defocused pulses results in very little collateral damage, while slight defocusing involves significant heat and UV exposure of up to about 10% of the specimen volume, especially if samples are catapulted directly from a glass slide. Time-resolved photography of live-cell catapulting revealed that in defocused catapulting strong shear forces originate from the flow of the thin layer of culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that makes the specimen wind its way out of the culture medium, thereby undergoing much less shear stresses. Therefore, the recultivation rate of catapulted cells was much higher when focused pulses were used.