Investigating the Ultrastructure ofTympanic Membrane in RatsMarch 26th, 2013Gordon Locke

MedBio 3970 6 Week Project

Acknowledgements

Dr. Jian LiuDr. Hanif Ladak

MedBio 3970 – 6 Week Project

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

Introduction• The Middle Ear

• The Tympanic Membrane

• Scanning Electron Microscopy (SEM)

MedBio 3970 6 Week Project

The Middle Ear (Human)

MedBio 3970 6 Week Project

(Grey, 2000)

The Middle Ear (Human)

MedBio 3970 6 Week Project

(Grey, 2000)

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

The Tympanic Membrane

MedBio 3970 6 Week Project

Daphalapurkar et al., 2009

Scanning Electron Microscopy

MedBio 3970 6 Week Project

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

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

Fiber Diameter• Measured using SEM Images and ImageJ

• Improved the contrast and found individual fiber diameters with imageJ

Presentation Title Here

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

Results

MedBio 3970 6 Week Project

Radial fibers

Results

MedBio 3970 6 Week Project

Radial fibers

Results

MedBio 3970 6 Week Project

Radial fibers

Results

MedBio 3970 6 Week Project

Radial fibers

MedBio 3970 6 Week Project

Results

Fibers at the Manubrium

Analysis

MedBio 3970 6 Week Project

ImageJ

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

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

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

ReferencesGray, 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