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

2. Acknowledgements Dr. Jian Liu Dr. Hanif Ladak 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) MedBio 3970 6 Week Project (Grey, 2000)

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

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 MedBio 3970 6 Week Project Daphalapurkar et al., 2009

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 MedBio 3970 6 Week Project Radial fibers

15. Results MedBio 3970 6 Week Project Radial fibers

16. Results MedBio 3970 6 Week Project Radial fibers

17. Results MedBio 3970 6 Week Project Radial fibers

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

19. Analysis MedBio 3970 6 Week Project ImageJ

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,” Acta Otolaryng, 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,” Acta Otolaryngol, 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