title: Biophysical response to pulsed laser microbeam-induced cell lysis and molecular delivery.
abstract: Cell lysis and molecular delivery in confluent monolayers of PtK(2) cells are achieved by the delivery of 6 ns, lambda = 532 nm laser pulses via a 40x, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3 kDa) FITC-conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time-resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam-induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses tau(w, max) > 190 +/- 20 kPa are immediately lysed while cells exposed to tau(w, max) > 18 +/- 2 kPa are necrotic and subsequently detach. Cells exposed to tau(w, max) in the range 8-18 kPa are viable and successfully optoporated with 3 kDa Dextran molecules. Cells exposed to tau(w, max) < 8 +/- 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam-induced shear stresses and subsequent cellular outcome.
authors.affiliation: Dept. of Bioengineering, University of California, San Diego, La Jolla, CA 92093-0412, USA.
authors.forename: Amy N
authors.initials: AN
authors.lastname: Hellman
authors.forename: Kaustubh R
authors.initials: KR
authors.lastname: Rau
authors.forename: Helen H
authors.initials: HH
authors.lastname: Yoon
authors.forename: Vasan
authors.initials: V
authors.lastname: Venugopalan