Ting-Hsiang Wu

Photothermal effects of supramolecularly assembled gold nanoparticles for the targeted treatment of cancer cells.

Noble-metal nanostructures with unique photophysical properties have been considered as prime candidate agents for the photothermal treatment of cancer.[1–4] Typically, the photothermal properties of these nanostructures can be controlled by manipulating their sizes and shapes.[4,5] Over the past decade, significant endeavors have been devoted to the production of a variety of gold nanostructures, such as nanoparticles,[6,7] nanoshells,[8–10] nanorods,[11,12] and nanocages,[5,13,14] which are able to overcome limitations of organic-dye-based photothermal agents,[7] such as low light absorption and undesired photobleaching. For sufficient energy to be harvested/generated to damage tumor cells, the size of these nanostructure-based agents are required in the range of tens to hundreds nm.[15] However, the relatively “large” size of the agents often leads to poor bioclearance (i.e., accumulation in the liver, spleen, and kidneys), which is a major obstacle to their in vivo application.[16–18] Alternatively, the photophysical properties of noble-metal nanostructures can be altered systematically by the formation of aggregates through self-assembly.[19–30] The antibody-assisted aggregation of Au nanoparticles on cell membranes or in intracellular environments led to the enhancement of photothermal performance[31] as a result of the collective effects[32,33] associated with the assembled structures. Therefore, the self-assembly of small noble-metal building blocks, that is, noble-metal colloids with diameters of less than 8 nm[16–18] (compatible with renal clearance) would be a promising approach toward a new class of noble-metal photothermal agents.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade

Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SSBsa) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SSBsa activity. Although intracellular movement was essential for cell–cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell–cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SSBsa-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.

Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter

We report a high speed and high purity pulsed laser triggered fluorescence activated cell sorter (PLACS) with a sorting throughput up to 20,000 mammalian cells/s with 37% sorting purity, 90% cell viability in enrichment mode, and >;90% purity in high purity mode at 1,500 cells/s or 3,000 beads/s. Fast switching (30 μs) and a small perturbation volume (~90 pL) is realized by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel.

High-speed droplet generation on demand driven by pulse laser-induced cavitation

We report on a pulse laser-driven droplet generation (PLDG) mechanism that enables on-demand droplet generation at rates up to 10,000 droplets per second in a single-layer PDMS-based microfluidic device. Injected droplet volumes can be continuously tuned between 1 pL and 150 pL with less than 1% volume variation.

Massively parallel delivery of large cargo into mammalian cells with light pulses

We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.

Photothermal nanoblade for large cargo delivery into mammalian cells

We report a photothermal nanoblade that utilizes a metallic nanostructure to harvest laser pulse energy and convert it into a localized explosive vapor bubble, which rapidly punctures a lightly-contacting cell membrane via high-speed fluidic flows and induced transient shear stress. Integrating the metallic nanostructure with a micropipette, the nanoblade generates a micron-sized membrane access port for delivering concentrated cargo (5×108 bacteria/ml) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, 200 nm polystyrene beads to 2 µm bacteria have been delivered into multiple cell types.

Floating electrode optoelectronic tweezers: Light-driven dielectrophoretic droplet manipulation in electrically insulating oil medium

We report an optical actuation mechanism, floating electrode optoelectronic tweezers (FEOET). FEOET enables light-driven transport of aqueous droplets immersed in electrically insulating oil on a featureless photoconductive glass layer with direct optical images. We demonstrate that a 681 mum de-ionized water droplet immersed in corn oil medium is actuated by a 3.21 muW laser beam with an average intensity as low as 4.08 muWmm(2) at a maximum speed of 85.1 mums on a FEOET device. FEOET provides a promising platform for massively parallel droplet manipulation with optical images on low cost, silicon-coated glass. The FEOET device structure, fabrication, working principle, numerical simulations, and operational results are presented in this letter.

3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter

We report a 3D PDMS microfluidic pulsed laser triggered fluorescence activated cell sorter capable of sorting at 11,000 cells sec-1 with >95% purity or at 45,000 cells sec-1 with 45% purity within a single channel.

Pulsed laser activated cell sorting with three dimensional sheathless inertial focusing.

We present a Pulsed Laser Activated Cell Sorter (PLACS) integrated with 3D sheathless inertial focusing that is capable to sort at 10,000 particles sec-1 with >90% sort purity and 6,000 cells sec-1 with >80% sort purity. It is realized by exciting laser induced cavitation bubbles in a single layer PDMS microfluidic channel to create high speed liquid jets to deflect detected fluorescent samples. Fluid inertia and secondary flows induced by stepped microchannels are used to focus samples in 3 dimensions. After focusing, samples go through an expansion chamber whose function is to enlarge the separation distance between two closely positioned particles by particle-particle interaction in inertial flows and reduce the coincident events within the switching window (16 μsec in time or 32 μm in distance). This sorter uses 10 times lower initial concentration cell samples than that in sheath-based PLACS in order to avoid severe dilution effects from high volume sheath flows at the same throughput.

Nanoblade delivery and incorporation of quantum dot conjugates into tubulin networks in live cells.

Quantum dots (QDs) have not been used to label cytoskeleton structure of live cells owing to limitations in delivery strategies, and QDs conjugation methods and issues with nonspecific binding. We conjugated tubulin to QDs and applied the emerging method of photothermal nanoblade to deliver QD-tubulin conjugates into live Hela cells. This method will open new opportunities for cytosolic targeting of QDs in live cells.