Abstract
The purpose of the method presented here is to isolate living cancer stem cells in an autophagic state using laser microdissection and pressure catapulting (LMPC). Isolating stem cells is challenging because of their small numbers, size heterogeneity, and fragility. Cell characteristics are very specific under combined laser microdissection and immunohistochemistry staining, used here for the stem cell identification. LMPC is based on a contact-free dissection using an ultraviolet, pulsed N2 (UV-A, λ = 337 nm) or, more recently, an Nd:YAG laser beam focused through the objective lens of a microscope to a diameter of 2 µm. With the laser beam, target excision and catapulting depend on plasma-mediated ablation (for the dissection process) and plasma-induced pressure (for catapulting). In this way, isolated cells are recovered by catapulting them into a capture fixture using the force of a more energetic defocused laser pulse.
LMPC is a contact-free and contamination-free method. Its precision depends on the laser characteristics (wavelength and beam quality) and the microscope (magnification and the numerical aperture of the objective). It enables the direct visualization of the studied cells without any cell-size limitation. On the other hand, it can be time-consuming and requires an experienced pathologist or adequate training in cellular morphology.
