Mechanical circuits in single cells

C.-L. Guo, M. Ouyang, J.-Y. Yu, J. Maslo, A. Price and C.-Y. Shen, "Long-range mechanical force enables self-assembly of epthelial tubular patterns," Proc. Natl. Acad. Sci. USA 109, 5576-5582 (2012). Link. PDF.

Scaffold-free self-assembly of tissues, through both experimental and modeling approaches

Plane-projection multiphoton microscopy (PPMP)

PPMP is a wide-field (potentially scanningless), multiphoton fluorescence microscopy that aims to provide 3D imaging of biological sample at high acquisition rates. Owing to our innovative integration of temporal focusing and structured illumination, PPMP can be expected to achieve finer axial resolution than confocal microscopy.

J.-Y. Yu, D. B. Holland, G. A. Blake and C.-L. Guo, "The wide-field optical sectioning of microlens array and structured illumination-based plane-projection multiphoton microscopy," Opt. Express. 21, 2, 2097-2109 (2013). Link. PDF.

J.-Y. Yu, C.-H. Kuo, D. B. Holland, Y. Chen, M. Ouyang, G. A. Blake, R. Zadoyan and C.-L. Guo, "Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy," J. Biomed. Opt. 16, 116009 (2011). Link. PDF.

The mechanism for spontaneous cell polarization and migration

Eukaryote cells can spontaneously polarize and migration. Spontaneous migration also plays an important role in immune response and cancer cell metastasis. Using the mating process of budding yeast and the epithelial cells as model systems, we are studying how the dynamics of cytoskeleton involve in these processes.

Figure: Using Cdc10-YFP as the polarity marker, we showed that budding yeast cells create an internal flux before spontaneous polarization. Time is in hours and minutes. Scale bar: 5 mm

The spontaneous patterns and intermediate states in cell morphogenesis

One long-standing challenge in biomedicine is to understand how the individual cells control their differentiation and assembly in tissue development and regeneration as a function of space and time. While most research efforts to date have been focused on either the initial or the final state of tissue morphogenesis, we are interested in the development and transition of the intermediate states, as well as how they regulate the development of the entire system. We use optical characterization, biochemical perturbation, and mathematical modeling to quantify these states. Unraveling the dynamics and control mechanisms of intermediate morphogenetic patterns will advance the understanding of fundamental developmental biology, prevent abnormality and cancer formation, and improve tissue engineering for regenerative medicine.

Figure: A) Schematic phase diagram for the distribution of single-cell morphologies. B, C) The intermediate morphogenetic patterns at multi-cell level. D) Model to explain how single cell pattern formation facilitates tissue assembly. E) Zoomed window in D).

Nonlinear optical modality for the study of membrane dynamics

Mammalian cells dynamically interact with the surrounding microenvironments through the surface molecules. Thus, it is important to detect the subtle changes of the membranes, thereby understanding how the membrane dynamics lead to the profoundly different classes of differentiation behavior and enable a cell to acquire the robustness as well as longevity engendered through tissue formation.  However, membranes are continuous sheets and the image signal contains the information of the inter-membrane distances, as well as the membrane positions, orientations, and local topographic variations. Segregating these data from one another has been extremely challenging. Using non-interferometric optical profilometry (NIOP) and high harmonic generation (HHG) optical modality, we are trying to develop a technique to visualize the 3-d membrane dynamics. This is an international collaboration with Prof. Shi-Wei Chu at National Taiwan University, Physics, and Prof. Chau-Hwang Lee at Academia Sinica, Taiwan, Applied Sciences.

Figure 1: The schematics of how intracellular signaling and cytoskeleton dynamics are regulated in response to epithelial cell-cell adhesion and apicobasal polarity formation.

Figure 2: The z-series of cell membrane images obtained from living epithelium through multi-photon excitation. Red arrows indicate lateral and the yellow arrow indicate the apical domain. This series indicates the difficulty to discriminate each membrane domains from one another, each of which is responsible for different biological process. The z-plane number is indicated from 1 to 15 with a step size of 500 nm.

Figure 3: The schematics of our NIOP-HHG system.

Nonlinear optical modality for the study of cytoskeletal dynamics

Through interaction with the surrounding microenvironments, mammalian cells reorganize their cytoskeleton and signaling activities for proper tissue development and homeostasis. However, current understanding of cytoskeleton dynamics is mainly contributed from in vitro tissue culture on 2-dimensional platforms. The study of in vivo cytoskeleton dynamics has been extremely challenging. Using HHG and optical modality, we are trying to develop a technique to visualize the filamentous structure of cytoskeleton in vivo. This is in collaboration with Prof. Colin Jamora at UCSD, Biology.

Figure: The z-series of microtubule images obtained from the tissue sample (upper panel) and cell culture (lower panel) through multiple photon excitation. While the in vitro sample shows clear formation and distinct separation of microtubules (yellow arrows), the filamentous structure of microtubule is not clear in the tissue sample (red arrows). The z-plane number is indicated from 1 to 5 with a step size of 500 nm.

The morphogenetic patterns in the pseudo 3-dimensional microenvironments

Mammalian cells are mostly living in 3-dimensional environments, where their behavior can be totally different from 2-d platforms. However, the understanding of how cells behave in 3-d microenvironment is limited. Collaborating with Prof. Poling Kuo at National Taiwan University, Electrical Engineering and Medicine, we are fabricating a series of microenvironments by which cells encounter an intermediate matrix dimensionality and surface topology between 2-dimensional and 3-dimensional matrix environments.

Figure: A) Fabricated pseudo 3-d islands and trenches. Cells preferentially migrate to B) the edges or C) corners of the trenches. Size of trench width in B) and C): 20 mm.