Optical and ultrasonic monitoring of laser-generated intracellular contrast agents: initial cell culture studies

Acoustically monitored laser-induced optical breakdown (LIOB) can be used its an important diagnostic and therapeutic tool in living cells. With properly controlled laser parameters, optical breakdown is a minimally invasive means to target a single, transient, acoustically detectable contrast agent bubble within a cell without affecting its viability. With different laser parameters, optical breakdown can effectively destroy a target cell. Simultaneous real-time acoustic and optical microscopy can monitor this range of effects. In this study, experiments were performed on a monolayer of cells cultured on a glass coverslip. To initiate and monitor intracellular photodisruption in real time, we have developed a system integrating an ultrafast laser with optical and acoustic microscopy. A regeneratively amplified Ti:sapphire laser produced the breakdown, and a high-frequency (50 MHz) ultrasonic transducer, confocal with the laser, monitored the resultant bubble via continuous pulse-echo recordings. The photodisruption was also observed using optical microscopy, and the viability of each cell was assessed after laser exposure using conventional live/dead staining. By varying laser pulse fluence (from 7 J/cm² to 112 J/cm²) as well as the number of applied pulses, a broad range of intracellular effects was studied. When up to 5000 laser pulses were applied at the minimum fluence, a small (1μm) transient bubble was generated. Cells targeted in this regime retained viability several hours after laser exposure. In contrast, with even a few laser pulses at high fluence, at large (>6μm) stable bubble was induced, and cells were consistently killed in this regime. Geometric targeting within cells can thus generate acoustically detectable microbubbles without introducing exogenous agents. Furthermore, by incorporating biochemical targeting agents, this system can also be used as a powerful tool for minimally invasive molecular diagnostics (e.g. detecting targeted molecular agents without affecting cell viability) and therapy (e.g. by destroying labeled cells without damaging cells in the vicinity).