For diagnostic or therapeutic technologies using femtosecond laser-induced optical breakdown (LIOB) in turbid biological tissues, pulses of sufficient fluence must be delivered to the site of interest. As light attenuates and diffuses rapidly due to wavelength-dependent absorption and scattering, it is important to develop penetration optimization schemes. In this study, we use a high frequency (50MHz) ultrasonic technique to investigate the precision and penetration depth limitations of infrared femtosecond laser-induced photodisruption in excised pig skin. Optical parameters varied include laser fluence (energy density in J/cm<sup>2</sup>) and focusing numerical aperture. Our ultrasonic method uses sensitive detection of laser-induced bubbles to measure breakdown extent. Using a geometrically focused Nd:Glass laser (1053 nm, 800 fs) source, we show that acoustically detectable bubbles can be produced as deep as 900 um into excised porcine skin. As penetration exceeds several hundred microns, however, multiple bubbles stacked at different depths can be produced with a single laser excitation. Secondary bubble creation is more likely at supra-threshold fluences or with low NA (≤ 0.4) focusing, where optical self-focusing may occur near threshold fluences. However, as the numerical aperture is increased (> 0.4) for deeper focusing, aberrations can severely distort the beam, increasing the perceived LIOB-threshold with maximal penetrations of less than 500um. Using an index matching fluid (i.e. aqueous glycerol solutions) to help reduce scattering, we are able to improve penetration. However, multiple breakdown sites and the corresponding reduction in precision is still likely in skin even with glycerol treatment.
Acoustical monitoring of laser-induced optical breakdown can be used as an important tool for diagnostics and therapeutics in living cells. Laser-induced intracellular microbubbles provide measurable contrast when detected with high-frequency ultrasound, and the bioeffects of these bubbles can be controlled to be within two distinct regimes. In the nondestructive regime, a single, transient, detectable bubble can be generated within a cell, without affecting its viability. In the destructive regime, the induced photodisruption can kill a target cell. To generate and monitor this range of effects in real time, we have developed a system integrating a femtosecond pulsed laser source with optical and acoustical microscopy. Experiments were performed on monolayers of Chinese hamster ovary cells. A Ti:Sapphire laser (793 nm wavelength, 100 fs pulse duration) was pulsed at 3.8 kHz and tightly focused to a 1 μm spot within each cell, and a high-frequency (50 MHz) ultrasonic transducer monitored the generated bubble with continuous pulse-echo recordings. The photodisruption was also observed with bright field optical microscopy, and cell viability was assessed after laser exposure using a colorimetric live/dead stain. By controlling laser pulse fluence, exposure duration, and the intracellular location of the laser focus, either nondestructive or destructive bubbles could be generated.
Femtosecond pulsed laser beams can induce precise photodisruption in tissue and tissue-like materials. Both geometrical and biochemical manipulation of laser-induced optical breakdown (LIOB) produces highly localized photodisruption without residual damage to surrounding tissue. Measurable effects associated with LIOB are shock wave emission and microbubble formation. In previous work, we presented techniques for monitoring site-targeted, LIOB microbubbles with high-frequency (>50MHz) ultrasonic imaging. In this study, we used these techniques to study the stability of LIOB-induced bubbles in water-based gelatin. Successive recordings taken before, during, and after laser exposure illustrated bubble creation and stability. Bubbles with a range of lifetimes (20 - 400 ms) and dissolution behaviors were produced by varying either laser fluence (0.7 - 2.1 J/cm<sup>2</sup>/pulse) or total number of laser pulses delivered (30 - 500 pulses at 18kHz repetition rate). While both increases in pulse fluence and pulse number lengthened bubble lifetime by an order of magnitude and decreased the rate of bubble dissolution, bubble stability was nonlinearly related to total laser exposure. A few pulses at high laser fluence created initially large bubbles with long lifetimes and slow dissolution rates. In contrast, pulses at near-threshold laser fluence created initially smaller, shorter lifetime bubbles that were stabilized with subsequent pulses. Increased stability could be maintained only above a threshold bubble size. Below that critical size, dissolution rate rapidly increased, causing bubble collapse. Ultimately, these results demonstrated an ability to control the size, lifetimes, and stability of laser-induced microbubbles with various optical parameters, increasing their utility as <i>site-activated </i>contrast agents that can be sensitively monitored with high-frequency ultrasound.