Luke P. Lee

Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.

Tunable liquid-filled microlens array integrated with microfluidic network

An elastomer-based tunable liquid-filled microlens array integrated on top of a microfluidic network is fabricated using soft lithographic techniques. The simultaneous control of the focal length of all the microlenses composing the elastomeric array is accomplished by pneumatically regulating the pressure of the microfluidic network. A focal length tuning range of hundreds of microns to several millimeters is achieved. Such an array can be used potentially in dynamic imaging systems and adaptive optics.

Nanowire-based single-cell endoscopy

One-dimensional smart probes based on nanowires and nanotubes that can safely penetrate the plasma membrane and enter biological cells are potentially useful in high-resolution and high-throughput gene and drug delivery, biosensing and single-cell electrophysiology. However, using such probes for optical communication across the cellular membrane at the subwavelength level remains limited. Here, we show that a nanowire waveguide attached to the tapered tip of an optical fibre can guide visible light into intracellular compartments of a living mammalian cell, and can also detect optical signals from subcellular regions with high spatial resolution. Furthermore, we show that through light-activated mechanisms the endoscope can deliver payloads into cells with spatial and temporal specificity. Moreover, insertion of the endoscope into cells and illumination of the guided laser did not induce any significant toxicity in the cells.

Microfluidics-based systems biology

Systems biology seeks to develop a complete understanding of cellular mechanisms by studying the functions of intra- and inter-cellular molecular interactions that trigger and coordinate cellular events. However, the complexity of biological systems causes accurate and precise systems biology experimentation to be a difficult task. Most biological experimentation focuses on highly detailed investigation of a single signaling mechanism, which lacks the throughput necessary to reconstruct the entirety of the biological system, while high-throughput testing often lacks the fidelity and detail necessary to fully comprehend the mechanisms of signal propagation. Systems biology experimentation, however, can benefit greatly from the progress in the development of microfluidic devices. Microfluidics provides the opportunity to study cells effectively on both a single- and multi-cellular level with high-resolution and localized application of experimental conditions with biomimetic physiological conditions. Additionally, the ability to massively array devices on a chip opens the door for high-throughput, high fidelity experimentation to aid in accurate and precise unraveling of the intertwined signaling systems that compose the inner workings of the cell.

Inspirations from Biological Optics for Advanced Photonic Systems

Observing systems in nature has inspired humans to create technological tools that allow us to better understand and imitate biology. Biomimetics, in particular, owes much of its current development to advances in materials science and creative optical system designs. New investigational tools, such as those for microscopic imaging and chemical analyses, have added to our understanding of biological optics. Biologically inspired optical science has become the emerging topic among researchers and scientists. This is in part due to the availability of polymers with customizable optical properties and the ability to rapidly fabricate complex designs using soft lithography and three-dimensional microscale processing techniques.

Quantized plasmon quenching dips nanospectroscopy via plasmon resonance energy transfer

We observed quantized plasmon quenching dips in resonant Rayleigh scattering spectra by plasmon resonance energy transfer (PRET) from a single nanoplasmonic particle to adsorbed biomolecules. This label-free biomolecular absorption nanospectroscopic method has ultrahigh molecular sensitivity.

Remote Optical Switch for Localized and Selective Control of Gene Interference

Near infrared-absorbing gold nanoplasmonic particles (GNPs) are used as optical switches of gene interference and are remotely controlled using light. We have tuned optical switches to a wavelength where cellular photodamage is minimized. Optical switches are functionalized with double-stranded oligonucleotides. At desired times and at specific intracellular locations, remote optical excitation is used to liberate gene-interfering oligonucleotides. We demonstrate a novel gene-interfering technique offering spatial and temporal control, which is otherwise impossible using conventional gene-interfering techniques.

Human iPSC-based Cardiac Microphysiological System For Drug Screening Applications

Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity. Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC50/EC50) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.

Tunable microdoublet lens array

We report a tunable microdoublet lens capable of creating dual modes of biconvex or meniscus lens. The microdoublet lens consists of a tunable liquid-filled lens and a solid negative lens. It can be tuned either by changing the shape of the liquid-filled lens into biconvex or meniscus or by changing a filling media with different refractive index. The microfabrication is based on photopolymer microdispensing and elastomer micromolding methods. The microdoublet lens can provide a solution for minimizing optical aberrations and maximizing the tunability of focal length or field of view by controlling variable and fixed lens curvatures.

Selective and sensitive detection of metal ions by plasmonic resonance energy transfer-based nanospectroscopy

Highly selective and sensitive optical methods for the detection of metal ions have had a substantial impact on molecular biology, environmental monitoring and other areas of research. Here we demonstrate a new method for detecting metal ions that is based on selective plasmonic resonance energy transfer (PRET) between conjugated metal-ligand complexes and a single gold nanoplasmonic probe. In addition to offering high spatial resolution due to the small size of the probe, our method is 100 to 1,000 times more sensitive than organic reporter-based methods. Moreover, it can achieve high selectivity owing to the selective formation of Cu(2+) complexes and selective resonant quenching of the gold nanoplasmonic probe by the conjugated complexes. We expect that PRET-based metal ion sensing could have applications in cellular imaging, systems biology and environmental monitoring.