The traditional Laser Induced Forward Transfer technique - originally developed for transfer of inorganic thin films from a transparent holder to a facing substrate - has been modified to allow the controlled transfer of sensitive biomaterials and cells. In our experimental arrangement the biological substrates (biomaterials, cells and conidia) are spread onto the surface of a thin metal layer coated fused silica plate. According to the name of the developed method - Absorbing-Film Assisted Laser Induced Forward Transfer (AFA-LIFT) - the target biological layer is protected from the photonic and thermal effects of the laser irradiation by the metal film which absorbs the laser energy and converts it to kinetic energy resulting in the ejection of the biological structures. The biomaterials were transferred either from dry (conidia) or wet (living cells) environment. After the process the substrates were placed in culture medium and the state of the transferred materials was monitored with optical microscopy. In case of Trichoderma longibrachiatum conidia 20 h incubation time after the transfer the highest germination ratio (number of the germinated conidia divided by the estimated number of transferred conidia) was around 75% reached at 355 mJ/cm2 laser fluence. Freshly isolated cells (rat Schwann and astroglial and pig lens epithelial cells) were transferred from solution, spread on the holder in 140-160 μm thick layer to substrate covered with a wet gelatin layer. The trypan blue dye exclusion test showed that 80-85% of the transferred cells still remained intact after transfer. The initially round-shaped cells 24 h after the transfer started to proliferate and differentiate. After 1 week the cells formed a monolayer with no signs of cellular degeneration. The dynamics of the process was studied with a fast photographic arrangement using a 1 ns pulse-length dye laser beam as a probe pulse. In case of 'dry transfer' the velocity of the front of the ejected conidia plume was 1,150 m/s, while the lower limit of the estimated initial acceleration was 109×g. For the 'wet transfer' the velocity of the liquid jet containing the cells was 'only' 122 m/s and the acceleration was above 107×g. This means that the studied conidia and cells can tolerate extremely high acceleration at the beginning of the ejection and the deceleration during the impact to the acceptor plate without significant damage. The absorbing metal film missing from the irradiated area and numerical thermal calculations both indicate that the metal is evaporated at the fused silica-metal interface by the laser beam and the expanding gas layer initiates the ejection of the biological structures. In order to avoid the possible thermal damages of the cells caused by nanosecond laser irradiation, cell transfer using femtosecond excimer laser was also studied. However, femtosecond AFA-LIFT proved to be less advantageous for cell transfer, probably due to the extreme mechanical forces produced by the short laser pulse.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)