This project focused on the development of a photonic crystal enhanced microscope (PCEM) as a powerful multimode imaging approach for quantification and visualization of the interactions between cells and surfaces. The PCEM is designed to utilize the properties of a photonic crystal (PC) biosensor surface that is designed to produce narrow bandwidth optical resonances for two distinct purposes. First, the resonant coupling condition of the PC is modified by cell attachment, and the PCEM is capable of producing images of the spatial distribution of cell attachment with sufficient resolution to quantify variations in the strength of attachment within individual cells. Second, the PC surface enables efficient coupling to laser illumination, resulting in the ability to enhance the output of fluorescent molecules in close proximity to the PC by two orders of magnitude, using a technique called "PC Enhanced Fluorescence." The PCEM will be an enabling tool for a broad range of life science research and pharmaceutical screening applications that require characterization of the interaction between extracellular matrix materials and cell membrane-expressed proteins in response to cell growth, pro-apoptotic stimuli, forced differentiation protocols, chemotaxis, ion channel activation, transmembrane protein activation, and proliferation. PCEM offers the ability to perform assays with a limited number of available cells for applications involving primary cells or stem cells. The proposed system builds upon the successful implementation and demonstration of the first PCEM system, which was designed and built for applications involving DNA microarrays and protein microarrays. The proposed instrument incorporates objective-coupled illumination from above and below the PC for simultaneous label-free imaging, bright field imaging, and enhanced fluorescence imaging with optics designed to optimize the sensitivity and resolution of each modality when the PC surface is immersed in cell media. The system also incorporates a CO2 environmental chamber to facilitate continuous monitoring of cells for extended periods of time.
Biologists are developing a more sophisticated understanding of how cell membrane interactions with surfaces, chemical stimuli, and other cells are modulated, and the role of integrins, ion channels, G-coupled transmembrane proteins, and filapodia in fundamental processes such as migration, wound healing, differentiation, and apoptosis. Yet, there are few tools currently available that allow visualization and quantification of these processes. While the initial set of demonstrated applications for PCEM will include cancer cell cytotoxicity, T-cell induced apoptosis, stem cell differentiation, and cardiac cell stimulation, the system's capabilities extend to any cell type and any process that can be carried out upon a surface. The environment at Illinois, in which the PI is an investigator within the NSF-sponsored Center for Cellular Mechanics (CCMB) IGERT, and the NIH-sponsored Cancer Nanotechnology training grant, and the location of the system within the Micro and Nanotechnology Laboratory's BioNanotechnology Laboratory assures that the system will be included in multidisciplinary training programs. Users of the system will be comprised of faculty, graduate students, and undergraduate students from Engineering, Pharmacology, Animal Sciences, Microbiology, and Immunology. The project developed general-purpose methods for studying cell-surface interactions that can be used broadly in pharmaceutical screening and life science research - extending the applicability of photonic crystal biosensors, and incldued an education plan that is closely linked with the program's technical goals with impact upon graduate student research, undergraduate research, undergraduate classroom/ laboratory learning, and teaching biosensing/photonics concepts to a University-based all-girls middle school.
Y. Zhuo and B.T. Cunningham. (2015). Label-Free Biosensor Imaging on Photonic Crystal Surfaces. IEEE Sens. J. 15, 21613.
Y. Zhuo, H. Hu, W. Chen, M. Lu, L. Tian, H. Yu, K.D. Long, E. Chow, W.P. King, S. Singamaneni, and B.T. Cunningham. (2014). Single nano particle detection using photonic crystal enhanced microscopy. Analyst, 139, 1007.
W. Chen, K.D. Long, M. Lu, V. Chaudhery, H. Yu, J.S. Choi, J. Polans, Y. Zhuo, B.A. Harley, and B.T. Cunningham. (2013). Photonic crystal enhanced microscopy for imaging of live cell adhesion. Analyst, 138, 5886.
W. Chen, K.D. Long, H. Yu, Y. Tan, J.S. Choi, B.A. Harley, and B.T. Cunningham. (2014). Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy. Analyst, 139, 5955.