Vasan Venugopalan

Investigation of laser-induced cell lysis using time-resolved imaging

Using time-resolved imaging, we investigated the lysis of confluent PtK2 cell cultures by pulsed laser microbeam irradiation. Images obtained at time delays of 0.5 ns to 50 μs demonstrate lysis to be mediated by laser-induced plasma formation resulting in pressure wave propagation and cavitation bubble formation. Image analysis enabled quantitative characterization of the pressure wave and cavitation bubble dynamics. The zone of cell damage exceeded the plasma size and serves to implicate cavitation bubble expansion as the primary agent of cell injury.

Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at lambda = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications.

Comparison of cortical bone ablations by using infrared laser wavelengths 2.9 to 9.2 μm

The purpose of this study was to compare the ablation of cortical bone at wavelengths across the near and midinfrared region.

Thermodynamic response of soft biological tissues to pulsed infrared-laser irradiation.

The physical mechanisms that achieve tissue removal through the delivery of short pulses of high-intensity infrared laser radiation, in a process known as laser ablation, remain obscure. The thermodynamic response of biological tissue to pulsed infrared laser irradiation was investigated by measuring and analyzing the stress transients generated by Q-sw Er:YSGG (lambda = 2.79 microns) and TEA CO2 (lambda = 10.6 microns) laser irradiation of porcine dermis using thin-film piezoelectric transducers. For radiant exposures that do not produce material removal, the stress transients are consistent with thermal expansion of the tissue samples. The temporal structure of the stress transients generated at the threshold radiant exposure for ablation indicates that the onset of material removal is delayed with respect to irradiation. Once material removal is achieved, the magnitude of the peak compressive stress and its variation with radiant exposure are consistent with a model that considers this process as an explosive event occurring after the laser pulse. This mechanism is different from ArF- and KrF-excimer laser ablation where absorption of ultraviolet radiation by the collagenous tissue matrix leads to tissue decomposition during irradiation and results in material removal via rapid surface vaporization. It appears that under the conditions examined in this study, explosive boiling of tissue water is the process that mediates the ablation event. This study provides evidence that the dynamics and mechanism of tissue ablation processes can be altered by targeting tissue water rather than the tissue structural matrix.

Physical factors involved in stress-wave-induced cell injury: The effect of stress gradient

We have studied the biological effects of ablation-induced stress waves in vitro. Mouse breast sarcoma cells (EMT-6) were exposed to stress waves that differed only in rise time. Two assays were used to determine cell injury: incorporation of tritiated thymidine (viability assay), and transmission electron microscopy (morphology assay). We present evidence that the rise time of stress waves can significantly modify cell viability and that cell injury correlates better with the stress gradient than peak stress.

Kinetics of cortical bone demineralization: Controlled demineralization—a new method for modifying cortical bone allografts

We investigated the kinetics of hydrochloric acid demineralization of human cortical bone with the objective of developing a method of controlled demineralization for structural bone allografts. It is known that the demineralization of cortical bone is a diffusion rate limited process with a sharp advancing reaction front. The demineralization kinetics of human cortical bone, described as the advance of the reaction front versus immersion time, were determined by measuring extraction of bone mineral in both planar and cylindrical geometries. Mathematical models based on diffusional mass transfer were developed to predict this process. The experimental data fit well with the behavior predicted by the model. The model for planar geometry is applicable to controlled demineralization of cortical bone allografts of irregular shapes such as cortical struts. The model for cylindrical geometry is appropriate when curved surfaces are involved such as in diaphyseal bone allografts. This method of demineralization has direct application to clinical modification of cortical bone allografts to potentially enhance their osteoinductive properties.

Optoacoustic tomography using time-resolved interferometric detection of surface displacement.

We introduce a minimally invasive technique for optoacoustic imaging of turbid media using optical interferometric detection of surface displacement produced by thermoelastic stress transients. The technique exploits endogenous or exogenous optical contrast of heterogeneous tissues and the low attenuation of stress wave propagation to localize and image subsurface absorbers in optically turbid media. We present a system that utilizes a time-resolved high-resolution interferometer capable of angstrom-level displacement resolution and nanosecond temporal resolution to detect subsurface blood vessels within model tissue phantoms and a human forearm in vivo.

Stress generated in polyimide by excimer‐laser irradiation

Stress transients are generated in polyimide by irradiation with excimer‐laser pulses at radiant exposures between 3×10−3 and 102 J/cm2. The duration and peak stress of these transients are measured using piezoelectric film transducers. For all the wavelengths tested (193, 248, 308, 351 nm) we determine three ranges of radiant exposure within which different physical mechanisms govern the stress generation. The scaling of stress with radiant exposure depends on wavelength only in the low fluence regime. In this regime the stresses observed are attributed to subsurface thermal decomposition at 351 and 308 nm and to photodecomposition at 248 and 193 nm. At higher radiant exposure the stress generation is governed either by the thermal expansion of the gaseous ablation products or by the formation and expansion of a dense plasma. The boundary between these two regimes is identified from the variation of the mechanical coupling coefficient with radiant exposure. The results also indicate that heat conduction ...

Biophysical Response to Pulsed Laser Microbeam-Induced Cell Lysis and Molecular Delivery

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.

Optoacoustic imaging using interferometric measurement of surface displacement

We describe an optoacoustic imaging technique based on time-resolved measurements of laser-induced thermoelastic expansion. Tomographic images of tissue phantoms are formed using such measurements made at several locations following irradiation with a Q-switched Nd:YAG (λ=1064nm) laser pulse. Our system is based on a modified Mach–Zehnder interferometer that measures surface displacement with a temporal resolution of 4ns and a displacement sensitivity of 0.3nm. Images formed from data sets acquired from several highly scattering tissue phantoms provide better than 200μm resolution and show great promise for high-resolution noninvasive imaging of heterogeneous tissues at depths approaching 1cm.