Pedro A. Quinto-Su

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.

Birth and growth of cavitation bubbles within water under tension confined in a simple synthetic tree.

Water under tension, as can be found in several systems including tree vessels, is metastable. Cavitation can spontaneously occur, nucleating a bubble. We investigate the dynamics of spontaneous or triggered cavitation inside water filled microcavities of a hydrogel. Results show that a stable bubble is created in only a microsecond time scale, after transient oscillations. Then, a diffusion driven expansion leads to filling of the cavity. Analysis reveals that the nucleation of a bubble releases a tension of several tens of MPa, and a simple model captures the different time scales of the expansion process.

Generation of laser-induced cavitation bubbles with a digital hologram

We demonstrate a method using a spatial light modulator (SLM) to generate arbitrary 2-D spatial configurations of laser induced cavitation bubbles. The SLM acts as a phase hologram that controls the light distribution in the focal plane of a microscope objective. We generate cavitation bubbles over an area of 380 x 380 microm(2) with a 20x microscope objective through absorption of the pulsed laser light in a liquid ink solution. We demonstrate the ability to accurately position up to 34 micrometer sized bubbles using laser energies of 56 microJ.

Mechanisms of laser cellular microsurgery.

This chapter reviews the optics of pulsed laser microbeams and the use of basic instrumentation to provide pulsed laser microbeam capabilities within a microscope platform. Moreover, we review the principal mechanisms by which laser microbeams produce microsurgical effects in cellular targets. We discuss the principal photothermal, photomechanical, and photochemical damage mechanisms as well as their relationship to critical laser microbeam parameters, including wavelength, pulse duration, and numerical aperture. We relate this understanding of damage mechanisms to laser microbeam applications reported in the literature.

Characterization and use of laser-based lysis for cell analysis on-chip

We demonstrate the use of a pulsed laser microbeam for cell lysis followed by electrophoretic separation of cellular analytes in a microfluidic device. The influence of pulse energy and laser focal point within the microchannel on the threshold for plasma formation was measured. The thickness of the poly(dimethylsiloxane) (PDMS) layer through which the beam travelled was a critical determinant of the threshold energy. An effective optical path length, Leff, for the laser beam can be used to predict the threshold for optical breakdown at different microchannel locations. A key benefit of laser-based cell lysis is the very limited zone (less than 5 μm) of lysis. A second asset is the rapid cell lysis times (approx. microseconds). These features enable two analytes, fluorescein and Oregon Green, from a cell to be electrophoretically separated in the channel in which cell lysis occurred. The resolution and efficiency of the separation of the cellular analytes are similar to those of standards demonstrating the feasibility of using a pulsed laser microbeam in single-cell analysis.

Interaction between two laser-induced cavitation bubbles in a quasi-two-dimensional geometry

We report on experimental and numerical studies of pairs of cavitation bubbles growing and collapsing close to each other in a narrow gap. The bubbles are generated with a pulsed and focused laser in a liquid-filled gap of 15 μm height; during their lifetime which is shorter than 14 μs they expand to a maximum radius of up to R max = 38 μm. Their motion is recorded with high-speed photography at up to 500 000 frames s -1 . The separation at which equally sized bubbles are created, d, is varied from d = 46-140 μm which results into a non-dimensional stand-off distance, y = d/(2R max ), from 0.65 to 2. For large separation the bubbles shrink almost radially symmetric; for smaller separation the bubbles repulse each other during expansion and during collapse move towards each other. At closer distances we find a flattening of the proximal bubbles walls. Interestingly, due to the short lifetime of the bubbles (≤ 14 μs, the radial and centroidal motion can be modelled successfully with a two-dimensional potential flow ansatz, i.e. neglecting viscosity. We derive the equations for arbitrary configurations of two-dimensional bubbles. The good agreement between model and experiments supports that the fluid dynamics is essentially a potential flow for the experimental conditions of this study. The interaction force (secondary Bjerknes force) is long ranged dropping off only with 1/d as compared to previously studied three-dimensional geometries where the force is proportional to 1/d 2 .

Controlled Manipulation and in Situ Mechanical Measurement of Single Co Nanowire with a Laser-Induced Cavitation Bubble

The flow induced by a single laser-induced cavitation bubble is used to manipulate individual Co nanowires. The short-lived (<20 μs) bubble with a maximum size of 45 μm is created in an aqueous solution with a laser pulse. Translation, rotation, and radial motion of the nanowire can be selectively achieved by varying the initial distance and orientation of the bubble with respect to the nanowire. Depending on the initial distance, the nanowire can be either pushed away or pulled toward the laser focus. No translation is observed for a distance further than approximately 60 μm, while at closer distance, the nanowire can be bent as a result of the fast flow induced during the bubble collapse. Studying the dynamics of the shape recovery allows an estimation of the Youngs modulus of the nanowire. The low measured Youngs modulus (in a range from 9.6 to 13.0 GPa) of the Co nanowire is attributed to a softening effect due to structural defects and surface oxidation layer. Our study suggests that this bubble-based technique allows selectively transporting, orienting, and probing individual nanowires and may be exploited for constructing functional nanodevices.

Mechanisms of Pulsed Laser Microbeam Release of SU-8 Polymer “Micropallets” for the Collection and Separation of Adherent Cells

The release of individual polymer micropallets from glass substrates using highly focused laser pulses has been demonstrated for the efficient separation, collection, and expansion of single, adherent cells from a heterogeneous cell population. Here, we use fast-frame photography to examine the mechanism and dynamics of micropallet release produced by pulsed laser microbeam irradiation at lambda = 532 nm using pulse durations ranging between 240 ps and 6 ns. The time-resolved images show the laser microbeam irradiation to result in plasma formation at the interface between the glass coverslip and the polymer micropallet. The plasma formation results in the emission of a shock wave and the ablation of material within the focal volume. Ablation products are generated at high pressure due to the confinement offered by the polymer adhesion to the glass substrate. The ablation products expand underneath the micropallet on a time scale of several hundred nanoseconds. This expansion disrupts the polymer-glass interface and accomplishes the release of the pallet from its glass substrate on the microsecond time scale (approximately 1.5 micros). Our experimental investigation demonstrates that the threshold energy for pallet release is constant (approximately 2 microJ) over a 25-fold range of pulse duration spanning the picosecond to nanosecond domain. Taken together, these results implicate that pallet release accomplished via pulsed laser microbeam irradiation is an energy-driven plasma-mediated ablation process.

Cavitation within a droplet

The rapid dynamics of vapor bubbles, the so-called cavitation bubbles, in confined geometries may lead to surprisingly rich dynamics. The example presented here in Fig. 1 a shows a cavitation bubble expanding and collapsing inside a liquid droplet, which creates two high-speed liquid jets shooting upwards. The primary jet is formed during the initial bubble expansion and collapse, and the secondary cylindrical jet forms when the bubble rebounds, i.e., after the collapse. The top picture captures both jets taken 400 s after the cavitation bubble has been created. The central jet is surrounded by the circular one. Both jet tips become unstable and pinch off droplets. The cavitation bubble inside the droplet creates a jet flow downwards and transforms into a torus, which disintegrates; its remains are still visible. The bottom row shows the stages leading to the double jet. Figure 1 b depicts the bubble 40 s after creation at the apex of the droplet. Bubble collapse occurs around 120 s Fig. 1 c , leading to flow separation, with an upward jet and a jet inside the droplet flowing through the bubble’s center, deforming the bubble into a torus. During re-expansion of the bubble a second upward pointing, now cylindrical, jet emanates; see Fig. 1 d . Technical details: a Nd:YAG laser =532 nm with 6 ns pulse duration is focused with a 40 microscope objective from below through a microscope slide into a sessile water droplet. The laser pulse energy 3 mJ explosively vaporizes the water and creates a bubble of approximately 1 mm maximum radius. The stroboscopic pictures are taken with a commercial digital camera Nikon D200 equipped with a macrolens. The droplet is illuminated from behind with a high power light emitting diode Seoul Semiconductor, P7 with a strobe duration 2 s . The light is mildly diffused to obtain a clear view into the droplet interior. For each picture, a new droplet is placed with a precision syringe onto a partially hydrophobic microscope slide.

Fast temperature measurement following single laser-induced cavitation inside a microfluidic gap

Single transient laser-induced microbubbles have been used in microfluidic chips for fast actuation of the liquid (pumping and mixing), to interact with biological materials (selective cell destruction, membrane permeabilization and rheology) and more recenty for medical diagnosis. However, the expected heating following the collapse of a microbubble (maximum radius ~ 10–35 µm) has not been measured due to insufficient temporal resolution. Here, we extend the limits of non-invasive fluorescence thermometry using high speed video recording at up to 90,000 frames per second to measure the evolution of the spatial temperature profile imaged with a fluorescence microscope. We found that the temperature rises are moderate (< 12.8°C), localized (< 15 µm) and short lived (< 1.3 ms). However, there are significant differences between experiments done in a microfluidic gap and a container unbounded at the top, which are explained by jetting and bubble migration. The results allow to safe-guard some of the current applications involving laser pulses and photothermal bubbles interacting with biological material in different liquid environments.