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

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


Introduction• The Middle Ear

• The Tympanic Membrane

• Scanning Electron Microscopy (SEM)


The Middle Ear (Human)


(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


The Tympanic Membrane


Daphalapurkar et al., 2009

Scanning Electron Microscopy


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


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


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


Radial fibers


Results

Fibers at the Manubrium

Analysis


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


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


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


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


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

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