Investigating the
Ultrastructure of
Tympanic Membrane
in Rats
March 26th, 2013
Gordon Locke
MedBio 3970 6 Week Project
Acknowledgements
Dr. Jian Liu
Dr. 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)
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The Middle Ear (Human)
(Grey, 2000)
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The Middle Ear (Human)
(Grey, 2000)
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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
Daphalapurkar et al., 2009
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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
Radial fibers
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Results
Radial fibers
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Results
Radial fibers
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Results
Radial fibers
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Results
Fibers at the Manubrium
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Analysis
ImageJ
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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
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
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: 243254, 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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