[Frontiers in Bioscience E2, 1028-1041, June 1, 2010]

[Frontiers in Bioscience E2, 1028-1041, June 1, 2010]

AFM and fluorescence imaging of nanomechanical response in periodontal ligament cells

Liang Shi ^1.2,3, Shenggen Shi², Jing Li¹, Quanmei Sun¹, Kai Feng², Peipei Chen¹, Shaoyan Si², Long Chen¹, Ye Li², Ping Dang², Chuhua Tang², Dong Han¹

1Lab for Biological Imaging and Nanomedicine, National Center for Nanoscience and Technology, Beijing, 100190, China, ²Department of Stomatology, 306 Hospital, Beijing, 100101, China, ³Faculty of Stomatology, Capital University of Medical Sciences, Beijing, 100050, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Methods and materials 3.1. Cell culture 3.2. BIO-AFM combined system and imaging 3.3. Attaching a micro-bead to the tipless AFM cantilever and exerting nanoNewton forces onto the cells 3.4. Elasticity detection >3.5. Fluorescence imaging of nitric oxide integrated with force stimulation 4. Results 4.1. Cantilever probe with the attached micron-sized glass bead 4.2. AFM Imaging of PDLCs 4.3. Elasticity detection of PDLCs 4.4. Nitric oxide production in the response of PDLCs to mechanical stimulation 5. Discussion 6. Conclusion 7. Acknowledgment 8. References: 3.1. Cell culture; 3.2. BIO-AFM combined system and imaging; 3.3. Attaching a micro-bead to the tipless AFM cantilever and exerting nanoNewton forces onto the cells; 3.4. Elasticity detection; >3.5. Fluorescence imaging of nitric oxide integrated with force stimulation
4. Results 4.1. Cantilever probe with the attached micron-sized glass bead 4.2. AFM Imaging of PDLCs 4.3. Elasticity detection of PDLCs 4.4. Nitric oxide production in the response of PDLCs to mechanical stimulation: 4.1. Cantilever probe with the attached micron-sized glass bead; 4.2. AFM Imaging of PDLCs; 4.3. Elasticity detection of PDLCs; 4.4. Nitric oxide production in the response of PDLCs to mechanical stimulation
5. Discussion
6. Conclusion
7. Acknowledgment

1. ABSTRACT

Most biologists think that AFM has only a limited use in biological research due to its inability to study other than surface structures. Therefore, a BIO-AFM system has been developed to combine both AFM imaging and fluorescence detection, which acts as a powerful tool for a better understanding of dynamic cell processes. In this study, based on a custom-made BIO-AFM system, the elasticity and ultrastructure of living periodontal ligament cells (PDLCs) were investigated. The cantilever probe with a micron-sized bead was used to exert nano-loading force onto the PDLCs. The related signal of NO was then recorded simultaneously. The results show that PDLCs hold strong networks of stress fibers as well as high elastic modulus value, exhibiting the ability for better counteracting the external forces. In the mechano-transduction studies, an initial increase and subsequent drop in intracellular NO response was found. Furthermore, NO may diffuse from a stimulated cell to adjacent cells. In conclusion, our single-cell nano-mechanical study provides a significant advancement in elucidating the magnitude, location, time scale, and biomolecular mechanisms underlying cell mechano-transduction.