investigating the ultrastructure of tympanic membrane in rats march 26 th, 2013 gordon locke medbio...
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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
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Introduction• The Middle Ear
• The Tympanic Membrane
• Scanning Electron Microscopy (SEM)
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The Middle Ear (Human)
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(Grey, 2000)
The Middle Ear (Human)
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(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
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The Tympanic Membrane
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Daphalapurkar et al., 2009
Scanning Electron Microscopy
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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
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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
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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
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Radial fibers
Results
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Radial fibers
Results
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Radial fibers
Results
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Radial fibers
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Results
Fibers at the Manubrium
Analysis
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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
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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
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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
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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
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