Chau-Hwang Lee

Noninterferometric differential confocal microscopy with 2-nm depth resolution

Abstract By utilizing the sharp slopes of the axial response curve of confocal imaging, we demonstrate a differential confocal technique for surface imaging with depth resolution as great as 2 nm. Neither servo feedback loop nor lock-in detection are used, hence measurements can be performed at high speed with long working distance. Because the technique uses no interferometric effects, it offers large open-loop dynamic range and is compatible with fluorescence microscopy.

Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device.

Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

Fabrication of spatial transient-density structures as high-field plasma photonic devices

Fabrication of periodic transient-density structures in a gas jet with a boundary scale length approaching 10μm was demonstrated. This was achieved by passing an ultrashort high-intensity laser pulse through a patterned mask and imaging the mask onto the target plane. Gas/plasma density at the laser-irradiated regions drops as a result of hydrodynamic expansion following ionization and heating by the laser pulse. The fabrication of gas/plasma density structures with such a scheme is an essential step in the development of plasma photonic devices for applications in high-field physics.

The ability of LCRMP-1 to promote cancer invasion by enhancing filopodia formation is antagonized by CRMP-1

Metastasis is a predominant cause of death in patients with cancer. It is a complex multistep process that needs to be better understood if we are to develop new approaches to managing tumor metastasis. Tumor cell invasion of the local stroma is suppressed by collapsin response mediator protein-1 (CRMP-1). Recently, we identified a long isoform of CRMP-1 (LCRMP-1), expression of which correlates with cancer cell invasiveness and poor clinical outcome in patients with non-small-cell lung cancer (NSCLC). Here, we report that LCRMP-1 overexpression in noninvasive human cell lines enhanced filopodia formation, cancer cell migration, and invasion via stabilization of actin. This effect required a highly conserved N-terminal region of LCRMP-1 as well as the WASP family verprolin-homologous protein-1/actin nucleation pathway (WAVE-1/actin nucleation pathway). Furthermore, LCRMP-1 appeared to act downstream of Cdc42, a Rho family protein known to be involved in actin rearrangement. In addition, LCRMP-1 associated with CRMP-1, which downregulated cancer cell metastasis by interrupting the association of LCRMP-1 and WAVE-1. Finally, we found that high-level expression of LCRMP-1 and low-level expression of CRMP-1 were associated with lymph node metastasis and poor survival in patients with NSCLC. In sum, we show that LCRMP-1 and CRMP-1 have opposing functions in regulating cancer cell invasion and metastasis and propose that this pathway may serve as a potential anticancer target.

A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.

Efficient generation of extended plasma waveguides with the axicon ignitor-heater scheme

By using an axicon lens in conjunction with the ignitor-heater scheme, a 1.2-cm-long high-quality plasma waveguide is generated efficiently, which can extend the range of laser-plasma interaction much beyond the limit of Rayleigh range

Optical measurement of the viscoelastic and biochemical responses of living cells to mechanical perturbation

We have developed an optical method for real-time monitoring of cellular motion in a natural environment with nanometer resolution. From the motion driven by small optical forces, we measured dynamic viscoelastic responses of living cells in the linear reversible region. Cytoplasmic gel-to-sol transition that was due to the disruption of the actin-filament framework was detected, and a linear release of Ca(2+) from intracellular storage that was related to submicrometer cell deformation was observed. The method was shown to be a powerful tool for studying the natural response of cells to mechanical perturbation.

Deconvolution of local surface response from topography in nanometer profilometry with a dual-scan method

In profilometric measurements, by scanning the sample twice with a fixed vertical offset, one can separate the signal that comes from surface heterogeneity from the topographic signal. Using differential confocal microscopy, a newly developed open-loop nanometer profilometric technique, we demonstrated this dual-scan method on composite samples and obtained 10-nm depth resolution. This technique can also be applied to other profilometric techniques such as atomic force microscopy and scanning tunneling microscopy.

Membrane ripples of a living cell measured by non-interferometric widefield optical profilometry

We measured the membrane topography and dynamics on a living fibroblast by using the non-interferometric widefield optical profilometry (NIWOP) technique. With a water-immersion objective of a 0.75 numerical aperture, our NIWOP system provides depth resolution about 20 nm. The imaging speed could be as high as 5 frames/min. We directly observed and profiled the inward propagation of membrane ripples near the cell edge. To verify if the membrane activity was driven by the underlying cytoskeleton, we changed the structure of the cell cortex while observing the membrane topography. After dissolving the actin cortex by cytochalasin D, we found that the propagation of the membrane ripples disappeared and the edge of the cell shank. The non-contact NIWOP technique does not affect the motility and viability of cells and therefore is suitable for the studies on cell physiology related to membrane motions.

Migration and vascular lumen formation of endothelial cells in cancer cell spheroids of various sizes

We developed a microfluidic device to culture cellular spheroids of controlled sizes and suitable for live cell imaging by selective plane illumination microscopy (SPIM). We cocultured human umbilical vein endothelial cells (HUVECs) within the spheroids formed by hepatocellular carcinoma cells, and studied the distributions of the HUVECs over time. We observed that the migration of HUVECs depended on the size of spheroids. In the spheroids of ∼200 μm diameters, HUVECs migrated outwards to the edges within 48 h; while in the spheroids of ∼250 μm diameters, there was no outward migration of the HUVECs up to 72 h. In addition, we studied the effects of pro-angiogenic factors, namely, vascular endothelial growth factor (VEGF) and fibroblast growth factor (β-FGF), on the migration of HUVECs in the carcinoma cell spheroid. The outward migration of HUVECs in 200 μm spheroids was hindered by the treatment with VEGF and β-FGF. Moreover, some of the HUVECs formed hollow lumen within 72 h under VEGF and β-FGF treatment. The combination of SPIM and microfluidic devices gives high resolution in both spatial and temporal domains. The observation of HUVECs in spheroids provides us insight on tumor vascularization, an ideal disease model for drug screening and fundamental studies.