Single cell analysis is the study of individual cells isolated from cell populations. A typical workflow of single cell analysis starts from a physiologically heterogeneous cell culture, and involves a high-throughput and robust isolation method based on single or multiple parameters to prepare a subpopulation of cells for further downstream processing and analysis.

New technologies are demanded to conduct single cell analysis on a finer level. Microelectromechanical systems (MEMS) provide good interface with cells because of comparable feature size, and have the advantages of device miniaturization and process integration, thus are suitable for biological studies. In this dissertation, I focus on developing heterogeneously integrated optofluidic platforms for applications in cell manipulation and sensing.

We first demonstrate a novel dielectrophoresis-integrated pulsed laser activated cell sorter (DEP-PLACS). It consists of a microfluidic channel with 3D electrodes laid out to provide a tunnel-shaped electric field profile for sheathless focusing of microparticles/cells into a single stream in high-speed microfluidic flows. DEP-PLACS has achieved a sorting purity of 91% for polystyrene beads at a throughput of 1,500 particles/sec.

To achieve enhanced DEP forces in high conductivity media, which can potentially improve manipulation throughput in a biocompatible environment, hemispherically shaped, heavily doped (N++) silicon electrode is proposed to decouple the strong electric field region from the electrode interface and provides a large interface capacitance to prevent surface charging in high conductivity media, thereby effectively suppressing electrochemical reactions. Compared to conventional metal electrode, N++ electrode can provide 3 times higher threshold voltage and a corresponding 9-fold enhancement of maximum DEP force in 1� PBS buffer with an electrical conductivity of 1 S/m.

As one kind of downstream analysis, mechanobiology has been an emerging cross-disciplinary field that studies the mechanical properties of cells and tissues and how physical forces can contribute to the changes in biological events such as cell differentiation and disease development. Over the past years several tools and approaches have been developed for quantifying mechanical properties of biological samples. However, the throughput is usually low and obtaining statistically significant data can be difficult. To address this problem, we proposed a mechanobiological measurement platform consisting of two major steps: high-throughput cell patterning and parallel pressure sensing.

For the first step we developed a novel and simple technique called lift-off cell lithography (LCL). Our approach borrows the key concept of lift-off lithography from microfabrication and utilizes a fully biocompatible process to achieve high-throughput, high-efficiency cell patterning with nearly zero background defects across a large surface area. Using LCL, we reproducibly achieved > 70% patterning efficiency for both adherent and non-adherent cells with < 1% defects in undesired areas.

For the second step we developed a pressure-sensing substrate that can monitor pressure distribution across a large area with high spatial resolution by utilizing colorimetric interferometry and image-based measurements. Vertical optical micro-cavities are constructed on a silicon substrate and covered by a thin planar layer of polymer material that deforms under pressure. Local measurement of pressure is realized by monitoring the change of reflected color spectrum from each micro-cavity. Pressure distribution across 1 cm2 area with a spatial resolution of 50 μm has been achieved. We demonstrate a measurement range of 0-5psi with < 0.2 psi error. The measurement range can be customized by tuning the design and material properties depending on the specific application such as biological cell or tissue stiffness measurement.

Over the past decade, various microfluidic fluorescence-activated cell sorter (FACS) systems have been demonstrated aiming to provide a fully enclosed environment for sterile, contamination and infectious free sorting, and better downstream microfluidic integration for further analysis after sorting. The biggest challenge of μFACS systems, however, is the orders of magnitude lower sorting throughput and purity than commercial aerosol based FACS (90% purity at 70,000 cells/sec). To solve this problem, the key innovation is to find a faster switching mechanism. In this dissertation, the importance of the cell sorting in academic research and clinical practice will be discussed at first. Among the commercial FACS machines, the state-of-the-art performance is achieving above 90% purity at 70 000 cells/sec. Other reported μFACS presents 1~2 order of magnitude lower in throughput.

In this context, we attempt to use the phenomena of the pulsed laser induced cavitation bubble to switch particles/cells in high speed. The first version simply excited the bubble in the microfluidic channel that is running parallel to the sample channel. The bubble expansion deformed the elastic PDMS channel wall and induced fluid swing in the sample channel which carried target particle/cell flowing into the collection channel. This first trial demonstrated the fast dynamics of this switching mechanism and the strong power the cavitation bubble can affect the flow. However, the deformation of the channel wall extended over hundreds of microns in length, resulting in low post-sort purity. To overcome this, the second version designed a connecting nozzle between the sample channel and the bubble channel. During the expansion of bubble, a liquid jet was created through the nozzle to the sample channel, which limited the disturbance range to tens of microns. With 2D hydrodynamic focusing, the pulsed laser activated cell sorter (PLACS) demonstrated performance with above 90% post-sort purity at 3 000 particles/sec. The drop of post-sort purity at higher throughput was due to the lack of the third dimension focusing which created large time variation in cell arrival time between the detection and switching zones. Then 3D hydrodynamic focusing that utilized multilayer PDMS with vertical vias solved the synchronization issue but also allows efficient particle switching using a smaller bubble with a smaller perturbation volume, and a shorter on-off switching cycle. As a result, the 3D PLACS was able to sort at 23 000 cells/sec with ~ 90% purity or at 45,000 cells/sec with 45% purity within a single channel. However, the large amount of diluting sheath fluid needed for tight 3D focusing requires a high initial cell concentration in samples (>10⁷ cells/ml), which can result in cell clogging in channels, and high pressure pumps to drive fluid through microchannels at high speeds. Then inertial focusing was employed as a substitute to hydrodynamic focusing. The integrated system achieved sorting at 10 000 particles/sec with >90% sort purity or 6 000 cells/sec with >80% sort purity and used 10 times lower initial concentration cell samples than that in sheath-based PLACS.

The achieved PLACS not only outperformed other μFACS but also achieved performance at a level comparable to conventional aerosol-based FACS. It is anticipated that by integrating with upstream and downstream microfluidic cell analysis functions, the developed PLACS will greatly facilitate biomedical research and clinical diagnostics.

The field of mechanobiology seeks to study the interplay between cellular biophysical properties and cellular health. Of particular interest to scientists is cellular stiffness, which has been shown to play a critical role in determining cellular state. For instance, researchers have discovered that cancer cells are less stiff than their benign counterparts, and they believe that this lower stiffness enables cancerous cells to invade benign tissues and adapt to new environments. As a result, profiling cellular biomechanical state can aid in disease diagnosis, making it a crucial research area in the biomedical field.Several methods have been proposed for measuring cellular stiffness, but most of them have limitations in terms of measuring a small number of cells or having low throughput. While isolated single cell mechanical property sensing can provide informative clues for detecting changes in cellular states or diseases, most disease developments involve collective cell migration and tissue reorganization. Therefore, measuring collective cellular stiffness or even at the tissue-level is crucial for gaining a more comprehensive understanding of the process. Here, my PhD research project aims to develop a device that we name “Optomagnetic Micromirror Arrays” (OMA) for steady state and real-time stiffness mapping for biomimetic materials with similar stiffness range to real biological tissues on a 5.1 mm x 7.2 mm field of view and with cellular resolution. This innovative device enables us to extract a large area of biomimetic material stiffness with single cellular resolution from the color spectra of an array of magnetic micromirrors, which are fabricated with sub-micron grating structures embedded in an elastic PDMS substrate. By applying a magnetic field at a specific orientation, the magnetic micromirrors align themselves with the field due to magnetic shape anisotropy. The color spectra of these micromirrors undergo changes, which allows us to detect the tilting angle changes of these micromirrors. This information enables us to deduce the local stiffness of the biomimetic material located on top of the micromirror array. By harnessing the power of OMA, we aim to contribute to the advancement of toolboxes in the mechanobiology field and drive forward our understanding of biological processes.

Intracellular delivery of diverse biomolecules, such as protein, nucleic acids, nano-devices, has been of great importance and interest in biomedical fields like cancer therapy, gene editing and intracellular environment probing. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to meet the emerging requirements of the cutting-edge research. In this thesis, I focused on three major hurdles in the current intracellular delivery, which are suspension cell delivery, complexity of incorporating nanotechnology, and large cargo delivery.

Photothermal mechanism is the underlying physics throughout all the work to be introduced here. It utilizes the light energy and transforms it into thermal energy and then into mechanical energy, serving for different functions in delivery. Nanosecond laser was chosen as the original power tool due to its high energy density, remote operation capability, and selective absorption. The combination of laser and micro/nano structure has been extensively explored to develop various delivery capabilities.

The first problem tackled in this thesis is to deliver materials into suspension cells with high efficiency, viability, and throughput. Suspension cells, especially lymphocytes, which represent 25-30% of immune cells, are of great interest in cancer immunotherapies and known as hard-to-transfect cells. To achieve effective delivery, the microwell structure with metallic sharp tips were designed to provide both cell anchoring and controllable membrane disruption on each cell. Suspension cells self- position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme β-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.

The second problem raised from the notice of the huge potential of nanostructures, especially combined with photothermal mechanism, in contrast with their current limited applications in this field. Nanostructures, such as nanoneedle array, have been adopted in the intracellular delivery field due to its unique scale advantages, including minimal damage of the cell membrane and large cargo loading capacity from high surface-to-volume ratio. However, nanotechnologies have suffered from its complexity of high-precision fabrication and are limited to small area. Thus, we demonstrate the fabrication of large-area plasmonic gold (Au) nanodisk arrays that enable photothermal intracellular delivery of biomolecular cargo at high efficiency. The Au nanodisks (350�nm in diameter) were fabricated using chemical lift-off lithography (CLL), a high-throughput and low-cost for nanoscale chemical patterning. This technique is applied to produce Au nanostructures on a variety of substrates (e.g., silicon, glass, and plastic), which facilitate in situ intracellular delivery in laboratory cell culture environments, enabling integration with existing medical devices. Nanosecond laser pulses were used to excite the plasmonic nanostructures, thereby generating transient pores at the outer membranes of targeted cells that enable the delivery of biomolecules via diffusion. We studied nanodisks of various sizes and found that an increase in delivery efficiency correlated with decreasing disk radius, which we attribute to higher density of pores per cell. Delivery efficiencies of >98% were achieved with 1-μm Au plasmonic disk arrays, using the cell impermeable dye Calcein (0.6�kDa) as a model payload, while maintaining cell viabilities at >98%. The highly efficient intracellular delivery approach demonstrated in this work will facilitate translational studies targeting molecular screening and drug testing that bridge laboratory and clinical investigations.

Despite that major problems were nicely solved in the prior two projects, an apparent drawback appears, as the delivery efficiency drops significantly when cargo size increases. Photothermal energy was adopted, in both projects, to generate bubble explosion near the adjacent cell membrane so as to disrupt the membrane. Cargoes had to passively diffuse into the membrane, which posed the hardship to large cargoes. Thus, in the third project, the integration of membrane disruption and active pumping was studied to facilitate large cargo delivery with precise control and large-area uniformity. We utilized the high initial pressure of the laser-induced bubbles as the pump source for high-speed fluidic jet, which cuts the cell membrane and delivers cargos into the cytosol and nucleus. The fabrication processes of the devices are designed to be conventional and simple with large-area uniformity. The penetration was demonstrated by injecting 140 nm polystyrene beads into Agarose hydrogel which was prepared to have similar Young’s Modulus as cells. With delicate device designs, we achieved penetration depths from tens of microns to a hundred microns, indicating the capability of three-dimensional tissue delivery and epidermal in vivo delivery, besides intracellular delivery into single layer of cells.

Implantable neural probes are one of the most important technologies for neuroscientists to detect and stimulate neural activities inside the brain to advance the understanding of brain function and behavior, especially in the deep brain regions where other non-invasive approaches cannot reach. With the development of microelectromechanical systems (MEMS) in the past few decades, a great amount of efforts has been made to improve the neural probes to provide reliable and versatile tools to enable neuroscience studies that otherwise impossible to conduct.

In this dissertation, I focused on three important objectives to improve the current neural probe platform: 1) to incorporate optical stimulation capability on top of the current silicon-based microelectrode arrays (MEAs) with electrochemical sensing function for optogenetics applications; 2) to integrate microfluidic channels with the current silicon platform for chemical deliveries; and 3) to develop a soft neural probe to reduce the brain damage and foreign body response. All the probes developed in this work are capable of deep brain implantation (>5 mm) and equipped with various electrochemical biosensors to study neural activities in deep brain regions.

For the first objective, an ultra-thin silicon nitride waveguide was integrated on top of the silicon probe using grating couplers to couple light in and out of the waveguide. PECVD silicon nitride film was optimized to reduce the waveguide propagation loss to ~ 3dB/cm at 450 nm wavelength. The probe is capable of delivering ~40 μW light power to the probe tip with ~2 mW input light power from a 3 μm polarization maintaining optical fiber coupled with a pigtailed laser diode. The average light intensity is ~200 mW/mm2 which is sufficient for Channelrhodopsin-2 excitation. Using this probe, we have detected optically-evoked glutamate release in the rat nucleus accumbens several weeks after injection of channelrhodopsin-expressing AAV into the above region.

For the second objective, PDMS microfluidic channels were transferred to the front and backside of the current Si probes using a novel PDMS thin-film transfer process. With this process, microfluidic channels can be easily bonded to Si probes as an add-on module to provide a simple solution to integrate chemical delivery functions into existing silicon-based probes, providing another level of control of the brain activities. Local injection of drug solution with nanoliter precision can be controlled by the pumping pressure and duration. The system was validated in vivo by local glucose injection through the fluidic channel and detection by biosensors in rat striatum.

For the third objective, a multifunctional neural probe with Ultra-Large Tunable Stiffness (ULTS) was developed for chemical sensing, delivery and deep brain implantation, whose stiffness can be tuned by 5 orders of magnitude before and after brain implantation. ULTS probe is stiff enough to penetrate 2 cm deep into a gel with similar mechanical properties as the brain tissue and becomes soft and flexible within few minutes to minimize brain damage due to the brain movements in all directions. With appropriate coatings, the electrodes on ULTS were converted into different biosensors which exhibits similar sensing performance as previous Si probes. The integration of microfluidic channels permits delivery of chemicals in the local vicinity of the sensing sites. Acute stimulation and sensing experiments were conducted in rats, in which potassium induced glutamate release was recorded demonstrating the capability of in vivo application. The chronic immune response was compared between ULTS and silicon probes with similar geometry, which shows a reduced immune response indicated by the lower astrocytes density around ULTS.

Techniques for processing nanoscale metallic and semiconductor structures with spatial order and tunable physical characteristics, such as size and shape, are important for realizing broad applications in areas such as magnetism, optics, electronics, biosensing, and drug delivery. Laser annealing is widely applied in modern semiconductor manufacturing processes for crystallizing amorphous structures into polycrystalline structures using thermal energy from laser illumination. This project aims to develop nanosecond laser assisted photothermal annealing technologies for high throughput nanofabrication of metal nanostructures on both planar and nonplanar surfaces using pulse laser induced rapid heating and nanomorphology evolution processes. This technology allows direct printing/transfer of functional devices on rigid substrates such as glass, silicon, as well as on flexible, low melting temperature substrates such as plastic and polymer. By controlling optical patterns through means of using phase-shifting photomask or pre-patterning the metal film to take advantage of the structured substrate, as well as selecting desired laser illumination duration, light wavelength, pulse number and dosage energy, thermal energy can be selectively deposited at target locations to melt materials within that area followed by nanomorphology evolution driven by surface tension. Such a low cost, high throughput and high spatial resolution nanofabrication technology could provide numerous applications in various areas including photonic devices, nanoelectronics, bio/chemical sensors, and devices for improved energy capturing and conversion in large area.

We further developed a novel gold nanoparticle embedded PDMS micropillar array to measure cell force in a large scale. The gold nanoparticles serve as point source like scattering hot spots in dark field images, where PDMS pillars and cells are almost invisible. By fitting the gold nanoparticle image with Gaussian point spread function, 30 nm pillar localization precision could be achieved even using a low N.A. 20x objective lens. This precision is comparable to prior reported fluorescent pillars imaged by a high magnification optical system with 60x or 100x objective lenses. Our plasmonic pillar sensor allows for monitoring collective cell behavior in a much larger area without sacrificing the force resolution. The plasmonic pillar also has the potential for directly delivering molecules into live cells through transient holes on the cell membrane opened by rapidly expanding cavitation bubbles, which are photothermally generated by gold nanoparticles sitting on pillar tip under laser triggering pulses.

The manipulation of biological objects is essential in various biomedical applications, such as single-cell analysis, cell-cell interactions, spheroid fabrication, drug delivery, and tissue engineering. Traditional methods of cell manipulation—including optical, magnetic, dielectrophoresis (DEP), and hydrodynamic techniques—each offer distinct advantages and limitations. In comparison, acoustics has been demonstrated to have superior biocompatibility and wide range of operable sizes of target, making it an attractive option to be widely utilized in many applications. However, traditional acoustic manipulation methods like BAWs and SAWs, which rely on standing waves, are constrained by limitations such as spatial resolution, periodic patterning, and restricted operational area. To overcome these limitations, the Compliant Membrane Acoustic Patterning (CMAP) mechanism was developed. Unlike BAWs and SAWs, CMAP does not rely on standing acoustic waves. Instead, it utilizes large acoustic impedance mismatches to generate a near-field acoustic potential gradient, enabling the precise patterning of microparticles and cells at sub-wavelength resolution with complex, non-periodic acoustic potential profiles over a large operational area. While CMAP offers high-resolution, reconfigurable patterning in complex, non-periodic shapes, which is challenging for traditional acoustic methods like BAWs and SAWs to achieve, several limitations remain. The fabrication process for CMAP devices is still complex and hard to control, with the operational area and patterning resolution restricted by the constraints of current fabrication methods. Moreover, CMAP is only currently capable of trapping particles and cells in groups, falling short of achieving single-cell level trapping and manipulation. Additionally, once microparticles and cells are trapped, these platforms lack the ability to selectively release specific particles or cells for downstream collection and further analysis. In this dissertation, two innovations are explored to address the limitations described. The first is a rapid prototyping method for fabricating Compliant Membrane Acoustic Platform (CMAP) devices using laser-assisted manufacturing. This direct-write approach not only offers a large area manufacturing capability but also eliminates the need for photolithography, significantly reducing the cost and time associated with the fabrication process. This new manufacturing method allows the transfer of a thin membrane onto the acoustic device reliably. The devices created using this approach achieve sub-wavelength, complex, and non-periodic patterning of microparticles and biological objects, with a spatial resolution of 60 μm across a large active manipulation area of 10 × 10 mm2—which is nearly 10 times larger than the priorly demonstrated CMAP device (CMAP 3mm x 3mm). The second innovation involves the development of a novel single-cell CMAP device that integrates a photothermal mechanism, enabling precise single-cell trapping and selective release. This advancement allows for the isolation and collection of individual cells, which is crucial for applications in single-cell analysis. The device utilizes spherical air cavities embedded in a PDMS substrate to create localized acoustic potential wells for trapping cells, enabling the individual trapping of both synthetic microparticles and biological cells across a 1 cm2 with more than 75,000 traps, accommodating a broad size range from 8 to 30 µm. The integration of a near-infrared laser facilitates selective release, achieving a release rate of approximately 40 cells per minute without compromising cell viability or proliferation.

Optoelectronic tweezers (OET), combining the advantages of optical and dielectrophoretic (DEP) manipulation, has been widely used in laboratories around the world. Despite being a useful platform for single-cell manipulation, OET still suffers from two limitations, the difficulty of being integrated with microfluidic components and the lack of large-area real-time detection function. The two issues restrain OET from complicated single-cell analysis and large-area automated manipulation. To integrate the PDMS-based microfluidic components with OET, two techniques are purposed in this research, SWNT-embedded electrodes and 3D PDMS membranes. The first technique uses SWNT thin films as transparent and conductive electrodes for OET inside multilayer PDMS channels. 3D microfluidic technique makes PDMS membranes to be a multilayer network between two rigid OET substrates. Based on these two new fabrication methods, novel microfluidic OET platforms have been developed to enable single or multiple cell manipulation and to accomplish multi-steps tasks. Therefore, off-chip and on-chip single-cell RT-qPCR are achieved on these two platforms respectively.

To address the issue of large-area real-time detection, a new technology, holographic on-chip imaging microscopy, is employed. This integrated platform has optical detection area equal to the size of an optoelectronic sensor array. Recording holographic images or digital reconstruction images, the automated image analysis in the system can keep the real-time positions of thousands of objects in field of view. Based on the real-time position information, the system simultaneously transports the target objects to the desired destinations. Particles or cells can be patterned or sorted on the platform which enabling simultaneous automated imaging, image analysis, real-time position feedback and interactive manipulation of thousands of micro-objects over an area of 240 mm2.

Manipulation of biological micro-objects in vitro is essential for many biomedical applications, such as the study of cells’ or molecular interaction, single-cell analysis, drug delivery, and tissue engineering. There are various physical mechanisms employed to achieve the manipulation; electrokinetic, optical, magnetic, hydrodynamic, and acoustic mechanisms are all conventional examples that have been deeply researched. In comparison, acoustics has been demonstrated to have superior biocompatibility and wide range of operable sizes of target, making it an attractive option to be widely utilized in many applications. On the other hand, acoustics suffers from limited Degree-of-Freedom (DOF) in objects’ manipulation due to the fundamental constraint stemmed from the principle that conventional acoustic devices built upon. Furthermore, acoustic patterning of micro-objects that allows not only real-time, versatile adjustment but also re-configuration to the patterning profile has not yet been realized. This re-configurable feature is particularly crucial in taking the acoustic manipulation technology from the current research phase to the next stage for broader applications.

For this dissertation, two important concepts are investigated and realized to overcome the acoustic limitations described. The first concept explores a new field of acoustofluidics where a novel manipulation platform is developed, making a breakthrough to the conventional approach in objects’ manipulation in achieving highly complex and non-periodic patterning shapes. The second concept involves integration of a photothermal mechanism with the developed acoustic platform which allows the acoustic potential field to be adjusted and re-configured, leading to a programmable platform for highly complex patterning of micro-objects.

In the first concept, a new acoustofluidic field using deep, sub-wavelength approach is exploited. Different from the traditional techniques that rely on using standing waves to generate the acoustic potential wells only spaced periodically at half the wavelength, the new approach can generate the acoustic wells spaced arbitrarily within half the wavelength. To achieve such approach, we developed a “Complaint Membrane Acoustic Patterning” (CMAP) platform utilizing an air-embedded, viscoelastic Polydimethylsiloxane (PDMS) structure to precisely control the wave fronts by providing barriers to incoming waves using acoustic impedance mismatch between air and PDMS, creating a non-uniform energy field and thus the acoustic wells. The embedded air cavities dictate the shape of the wells. Since the cavities can be fabricated into any geometry, complicated profiles of the wells can be realized. As experiments have demonstrated, we succeeded in the patterning of micro-polystyrene beads and HeLa cells into various numeric-letter shapes with resolution one tenth of the wavelength across a large 3 x 3 mm2 area.

In the second concept, a photothermal mechanism is integrated into the CMAP platform to achieve re-configurable patterning of micro-objects. A layer of light absorbing hydrogenated amorphous silicon (a-Si:H) is incorporated into the bottom of CMAP’s PDMS structure, where the cavities are initially fluid-filled. By focusing a laser light onto the a-Si:H layer to evaporate the fluid within the cavities selectively, we can modify where in the PDMS structure would contain fluid or air. Cavities containing air can also be reverted to contain fluid. Since the acoustic impedance of PDMS matches closely to that of water, acoustic waves could pass through easily. This way, we have the versatility in shaping and re-shaping an array of air cavities, permitting the generation of adjustable potential profile of the acoustic wells which leads to field-programmable functionality for highly complex and non-periodic patterning.

Enabling technologies that transfer micron-sized objects into mammalian cells are needed to advance key applications in cell engineering. High-throughput delivery of organelles, modified intracellular pathogens, enzymes, proteins, and other types of cargo is required to obtain reliable data in settings of cellular heterogeneity. Delivered objects must also avoid entrapment in cell vacuoles or endosomes to retain functionality. Here, we report a massively parallel photothermal microfluidic platform for high-throughput delivery of large cargo directly into the cytosol of mammalian cells. Micron-sized cargo is reproducibly delivered in up to 100,000 cells on the platform in a minute, with parameters for cargo delivery tunable for each cell and cargo type. The delivery platform is a compact silicon chip on which hundreds of thousands of micron-sized cavitation bubbles expand and collapse within nanoseconds in response to pulsed laser excitation. Cavitation bubbles are formed by rapid heat transfer from nearby metallic nanostructures via a photothermal process enhanced by the lightning-rod effect. High speed, localized fluid flows near cavitation bubbles puncture contacting cell membranes with precision, resulting in micron-sized transient membrane pores. Applied pressure provides an active driving force to speed slow diffusing large cargo through these transient pores before membrane repair and resealing. We have reproducibly delivered large cargo including micron-sized bacteria, enzymes, antibodies, and functional nanoparticles into a variety of cell lines, including three different types of primary cells, with high efficiency and high cell viability. Our experiments involving delivery of functional enzymes, antibodies and vacuole escape-incompetent bacteria prove that cargo is delivered directly into the cell cytosol on this platform, bypassing endocytosis. Massively parallel and nearly simultaneous delivery of cargo into cells under the same physiological conditions enables reliable statistical measurements of cargo interactions with cells over time. To demonstrate its utility, 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 vacuolar escape into the cell cytosol. Our photothermal platform approach, termed BLAST (Biophotonic Laser Assisted Surgery Tool), provides a reliable mechanism for high-throughput engineering of cells with micron-sized natural and man-made materials.