Imaging in vivo neuronal transport in genetic model organisms using microfluidic devices.

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

In vivo nanosecond laser axotomy: cavitation dynamics and vesicle transport.

Nanosecond laser pulses (lambda = 355 nm) were used to cut mechanosensory neurons in Caenorhabditis elegans and motorneurons in Drosophila melanogaster larvae. A pulse energy range of 0.8-1.2 microJ and < 20 pulses in single shot mode were sufficient to generate axonal cuts. Viability post-surgery was >95% for C. elegans and 60% for Drosophila. Cavitation bubble dynamics generated due to laser-induced plasma formation were observed in vivo by time-resolved imaging in both organisms. Bubble oscillations were severely damped in vivo and cavitation dynamics were complete within 100 ns in C. elegans and 800 ns in Drosophila. We report the use of this system to study axonal transport for the first time and discuss advantages of nanosecond lasers compared to femtosecond sources for such procedures.

Pulsed-laser-induced damage in rat corneas: time-resolved imaging of physical effects and acute biological response.

Laser-induced damage is studied in the rat corneal epithelium and stroma using a combination of time-resolved imaging and biological assays. Cavitation bubble interactions with cells are visualized at a higher spatial resolution than previously reported. The shock wave is observed to propagate through the epithelium without cell displacement or deformation. Cavitation bubble expansion is damped in tissue with a reduction in maximum size in the range of 54 to 59%, as compared to 2-D and 3-D cultures. Bubble expansion on nanosecond timescales results in rupture of the epithelial sheet and severe compression of cell layers beyond the bubble rim. In the stroma, the dense collagen lamellae strongly damped bubble expansion, thus resulting in reduced damage. The acute biological response of this tissue to laser pulses is characterized by confocal fluorescence microscopy. A viability assay of the epithelium reveals that only cells around the immediate site of laser focus are killed, while cells seen to undergo large deformations remain alive. Actin morphology in cells facing this mechanical stress is unchanged. Collagen microstructure in the stroma as revealed by second-harmonic imaging around the ablation site shows minimal disruption. These cellular responses are also compared to in vitro 2-D and 3-D cell cultures.

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.

Laser-induced mixing in microfluidic channels.

We demonstrate a novel strategy for mixing solutions and initiating chemical reactions in microfluidic systems. This method utilizes highly focused nanosecond laser pulses from a Q-switched Nd:YAG laser at lambda = 532 nm to generate cavitation bubbles within 100- and 200-microm-wide microfluidic channels containing the parallel laminar flow of two fluids. The bubble expansion and subsequent collapse within the channel disrupts the laminar flow of the parallel fluid streams and produces a localized region of mixed fluid. We use time-resolved imaging and fluorescence detection methods to visualize the mixing process and to estimate both the volume of mixed fluid and the time scale for the re-establishment of laminar flow. The results show that mixing is initiated by liquid jets that form upon cavitation bubble collapse and occurs approximately 20 micros following the delivery of the laser pulse. The images also reveal that mixing occurs on the millisecond time scale and that laminar flow is re-established on a 50-ms time scale. This process results in a locally mixed fluid volume in the range of 0.5-1.5 nL that is convected downstream with the main flow in the microchannel. We demonstrate the use of this mixing technique by initiating the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and nonfluorescent N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to yield fluorescent resorufin. This approach to generate the mixing of adjacent fluids may prove advantageous in many microfluidic applications as it requires neither tailored channel geometries nor the fabrication of specialized on-chip instrumentation.

Pulsed laser microbeam-induced cell lysis: time-resolved imaging and analysis of hydrodynamic effects.

Time-resolved imaging was used to examine the use of pulsed laser microbeam irradiation to produce cell lysis. Lysis was accomplished through the delivery of 6 ns, lambda=532 nm laser pulses via a 40x, 0.8 NA objective to a location 10 microm above confluent monolayers of PtK2 cells. The process dynamics were examined at cell surface densities of 600 and 1000 cells/mm2 and pulse energies corresponding to 0.7x, 1x, 2x, and 3x the threshold for plasma formation. The cell lysis process was imaged at times of 0.5 ns to 50 micros after laser pulse delivery and revealed the processes of plasma formation, pressure wave propagation, and cavitation bubble dynamics. Cavitation bubble expansion was the primary agent of cell lysis with the zone of lysed cells fully established within 600 ns of laser pulse delivery. The spatial extent of cell lysis increased with pulse energy but decreased with cell surface density. Hydrodynamic analysis indicated that cells subject to transient shear stresses in excess of a critical value were lysed while cells exposed to lower shear stresses remained adherent and viable. This critical shear stress is independent of laser pulse energy and varied from approximately 60-85 kPa for cell monolayers cultured at a density of 600 cells/mm2 to approximately 180-220 kPa for a surface density of 1000 cells/mm2. The implications for single cell lysis and microsurgery are discussed.