Investigating the Ultrastructure of Tympanic Membrane in Rats March 26 th , 2013 Gordon Locke

1. Investigating the Ultrastructure of Tympanic Membrane in Rats March 26th, 2013 Gordon Locke MedBio 3970 6 Week Project

2. Acknowledgements Dr. Jian Liu Dr. HanifLadak MedBio 3970 – 6 Week Project

3. Objectives • Image the Tympanic Membrane (TM) using Scanning Electron Microscopy (SEM) • Analyze the collagen fiber diameters using ImageJ and qualitatively look at the images. • Apply knowledge to improve computer modeling MedBio 3970 6 Week Project

4. Introduction • The Middle Ear • The Tympanic Membrane • Scanning Electron Microscopy (SEM) MedBio 3970 6 Week Project

5. The Middle Ear (Human) (Grey, 2000) MedBio 3970 6 Week Project

6. The Middle Ear (Human) (Grey, 2000) MedBio 3970 6 Week Project

7. The Tympanic Membrane • Transforms sound waves into mechanical vibrations • Acts as impedance matching transformer • The TM has multiple layers and non-uniform thickness • 2 layers of collagen: a radial layer and a circumferential layer MedBio 3970 6 Week Project

8. The Tympanic Membrane Daphalapurkar et al., 2009 MedBio 3970 6 Week Project

9. Scanning Electron Microscopy MedBio 3970 6 Week Project

10. Scanning Electron Microscopy • TMs were coated with Osmium • Metal coating is required because an electrically conductive surface is required • Allowed for individual fibers to be observed MedBio 3970 6 Week Project

11. Methods • TMs removed from adult rats • Incubated in a 1% trypsin phosphate buffer solution to remove epidermal and mucosal layers • Washed in phosphate buffer saline then exposed to a 2.5% glutaraldehyde solution • Specimens were then dehydrated, coated with Osmium and imaged with SEM MedBio 3970 6 Week Project

12. Fiber Diameter • Measured using SEM Images and ImageJ • Improved the contrast and found individual fiber diameters with imageJ Presentation Title Here

13. Banding Frequency • Collagen fibers are composed of smaller subunits • When combined there is a visible “band” between each subunit • The frequency of each fiber is related to the type of collagen it is composed of Presentation Title Here

14. Results Radial fibers MedBio 3970 6 Week Project

15. Results Radial fibers MedBio 3970 6 Week Project

16. Results Radial fibers MedBio 3970 6 Week Project

17. Results Radial fibers MedBio 3970 6 Week Project

18. Results Fibers at the Manubrium MedBio 3970 6 Week Project

19. Analysis ImageJ MedBio 3970 6 Week Project

20. Results • The average diameter of the radial fibers found is 36.1nm with SD =3.6nm with a sample size of n=30 • The subunit frequency in the radial fibers was 52.2nm with a SD of 5.7 with a sample size of n=26 MedBio 3970 6 Week Project

21. Discussion • Fibers in the center and at the tympanic ring (outer edge) were highly disorganized and randomly oriented • Fibers that extended radially were highly consistent in their orientation • The organization of collagen fibers can be incorporated into models and biomaterials for medical applications • Literature indicates that the radial fibers are type 2 and 4 collagen MedBio 3970 6 Week Project

22. Conclusions • Individual fibers can be imaged and have their diameters measured • The subunit frequency and fiber diameter can be used to identify the types of collagen in the radial fibers MedBio 3970 6 Week Project

23. References • Gray, Henry. Anatomy of the Human Body. Philadelphia: Lea & Febiger, 1918; Bartleby.com, 2000. • I. Kawabata and H. Ishii, “Fiber arrangement in the tympanic membrane. Scanning electron microscope observations,” ActaOtolaryng, 72: 243-254, 1971. • T. Shimada and D.J. Lim, “The fiber arrangement of the human tympanic membrane. A scanning electron microscopic observation,” Annals of Otology Rhinology and Laryngology, 80: 210-217, 1971 • D.J. Lim, “Human tympanic membrane. An ultrastructural observation,” ActaOtolaryngol, 70: 176-186, 1970 • D.J. Lim, “Tympanic membrane. Electron microscopic observation. Part I: pars tensa,” Acta. Oto-laryngologica., 66: 181–198, 1968 MedBio 3970 6 Week Project