Long-term marker-free multiphoton imaging, targeted transfection, optical cleaning of stem cell clusters, and optical transport of microRNA with extreme ultrashort laser pulses

Abstract

The novel utrashort femtosecond laser scanning microscope FemtOgene (JenLab GmbH, Germany) with 12 femtoseconds at the focal plane have been employed in marker-free imaging and optical manipulation of stem cells as well as for the non-contact introduction of microRNA in cancer cells. Human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells have been investigated by femtosecond laser multiphoton microscopy. Autofluorescence based on NAD(P)H and flavoproteins and second harmonic generation due to collagen have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. Major emission peaks at 460 nm and 530 nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured in a variety of stem cells using spectral imaging and time-correlated single photon counting. During differentiation, the ratios of bound to free NAD(P)H and NAD(P)H/flavoproteins changed. In addition, the biosynthesis of lipids and collagen was detected over a long period of time of up to 5 weeks. Nanoprocessing was performed with 12 femtosecond laser pulses and low picojoule pulse energies to realize targeted transfection and optical cleaning of human adult stem cell populations. Multiphoton sub-20fs microscopes may become novel non-invasive tools for marker-free optical stem cell characterization, for on-line monitoring of differentiation within a three-dimensional microenvironment, and for optical manipulation.