Imaging Cellular Responses to Mechanical Stimuli Within Three-Dimensional Tissue Constructs WEI TAN, 1 CLAUDIO VINEGONI, 1 JAMES J. NORMAN, 2 TEJAL A. DESAI, 2 AND STEPHEN A. BOPPART 1,3–5 * 1 Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Illinois 2 Department of Biomedical Engineering, Boston University, Boston, Massachusetts 3 Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 4 Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 5 Department of Internal Medicine, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois KEY WORDS three-dimensional microscopy; optical coherence tomography; multi-photon microscopy; tissue engineering; scaffolds; cellular biomechanics ABSTRACT The cellular response to environmental cues is complex, involving both structural and functional changes within the cell. Our understanding of this response is facilitated by micros- copy techniques, but has been limited by our ability to image cell structure and function deep in highly-scattering tissues or 3D constructs. A novel multimodal microscopy technique that combines coherent and incoherent imaging for simultaneous visualization of structural and functional prop- erties of cells and engineered tissues is demonstrated. This microscopic technique allows for the si- multaneous acquisition of optical coherence microscopy and multiphoton microscopy data with par- ticular emphasis for applications in cell biology and tissue engineering. The capability of this tech- nique is shown using representative 3D cell and tissue engineering cultures consisting of primary fibroblasts from transgenic green fluorescent protein (GFP) mice and GFP-vinculin transfected fibroblasts. Imaging is performed following static and dynamic mechanically-stimulating culture conditions. The microscopy technique presented here reveals unique complementary data on the structure and function of cells and their adhesions and interactions with the surrounding microen- vironment. Microsc. Res. Tech. 70:361–371, 2007. V V C 2007 Wiley-Liss, Inc. INTRODUCTION Microscopic techniques have been used to investigate the fundamental roles that mechanical forces and cell- material interactions play in cell culture and in the fields of cell biology, tumor biology, and tissue engineer- ing. To maintain proper functionality, cells rely on adhesions to and interactions with the surrounding substrate, structure, or extracellular matrix. The sur- rounding microenvironment provides a construct in which cells move, orient, organize, and differentiate to form cultures and tissues. Mechanical forces trans- duced through the microenvironment also alter both the morphology and genetic expression patterns of the cells. Cell-material interactions are one of the key com- ponents in tissue engineering (Desai, 2000; Griffith, 2002). While the molecular bases of the cell–substrate interactions have been extensively studied, much less is understood about the dynamic response of cellular and subcellular structure to the microenvironment, especially for cells in artificial, 3D tissue-like con- structs. One primary reason for this limited investiga- tion is because of the inadequate imaging technology for high-resolution, real-time, noninvasive imaging deep within highly scattering tissues or tissue con- structs. Furthermore, simultaneous 3D visualization of both cell morphology and material structures is prob- lematic using current microscopic methods. Conventional microscopy techniques at visible wave- lengths such as light and fluorescence microscopy, though widely applied for imaging cellular and molecu- lar activities, have disadvantages mainly because of poor light penetration depth in highly-scattering tis- sues, and the potential of severe photodamage to living cells. More advanced technologies for imaging engi- neered tissues, including high-field strength magnetic resonance imaging and microcomputed tomography, have been pursued for the assessment of cell or scaffold structure, with limited success. These techniques, with long data acquisition rates, hazards associated with high-energy radiation, and relatively high costs, are less suitable for both real-time and long-term imaging of living, dynamic, 3D cultures (Constantinidis et al., 2002; Lin et al., 2003). Confocal microscopy has been an important advance in microscopy, and has enabled the imaging of intact, optically nontransparent specimens to produce high- resolution (submicron) images of tissue structure with the use of fluorescent probes (Breuls et al., 2003; Gar- eau et al., 2004; Stephens and Allen, 2003). For a thick *Correspondence to: Stephen A. Boppart, M.D., Ph.D., Biophotonics Imaging Laboratory, Beckman Institute for Advanced Science and Technology, 405 N. Mathews Avenue, Urbana, IL 61801. E-mail: boppart@uiuc.edu Received 7 September 2006; accepted in revised form 7 November 2006 Contract grant sponsor: National Institutes of Health; Contract grant number: 1 R01 EB00108; Contract grant sponsor: National Science Foundation; Contract grant number: BES 05-19920. DOI 10.1002/jemt.20420 Published online 29 January 2007 in Wiley InterScience (www.interscience. wiley.com). V V C 2007 WILEY-LISS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 70:361–371 (2007)