Livecell Fluorescent Imaging

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Live-cell Fluorescence Imaging
Explorations in Science Research Awareness
Program
Jeannie S. Orlando Graduate Program/Track
(??????) University of Maryland School of
Medicine March 7, 2007
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Living organisms are made up of cells, that work
together to allow growth, reproduction, movement,
and survival.

• Cells are small.
• How can we see a cell?
• How can we see inside a cell?
• How can we see whats happening inside a living
  cell?

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Light Microscopy

• Microscopes magnify images so that we can observe
  small objects with our eyes
• Cameras can be attached to microscopes to allow
  us to capture magnified images
• Different sources of light enable us to visualize
  different aspects of cells

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Fluorescence Microscopy is the most common
approach for studying dynamic cellular events in
live cells

• What is Fluorescence?
• certain molecules emit light from electronically
  excited states
• the excited states can be created from physical,
  mechanical, or chemical processes
• Fluorescence
• The property of certain atoms and molecules to
  absorb light at one wavelength and then emit
  light at a longer wavelength

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Light

• A form of electromagnetic radiation that has
  properties of waves and particles
• light travels as a wave and has a wavelength
  (?)
• light carries energy in packets, called
  photons
• Different colors of light have different ?, and
  have different amounts of energy associated with
  them
• Longer ? of light have less energy than shorter ?

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Absorption and Emission of Light
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Stokes Shift
Sir George G. Stokes Mid-19th century, observed
that a certain mineral, when illuminated with
ultraviolet light (purple, short ?), emitted
light at a longer ? (visible light) Called this
fluorescence Fluorescence microscopy is a
method of studying material that can be made to
fluoresce
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Fluorescent Techniques

• Fluorescent probes (fluorophores) can be used
  to localize within a specific region of a
  biological specimen or to respond to a certain
  stimulus
• For example
• Certain dyes bind only
• to DNA
• Certain dyes fluoresce only
• Why en binding calcium ions
• Most fluorophores are molecules with multiple
  rings of carbon atoms that have many electrons
  that are excitable

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Visualizing the cell
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Importance of Ions in the Cell

• Ions are charged particles
• A number of ions are critical to maintaining the
  survival and function of the cell
• Calcium (Ca2) , Potassium (K), Sodium (Na)
• Ions maintain chemical and electrical gradients,
  activate and deactivate other molecules, and
  carry signals within the cell

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Visualizing changes in ion concentrations within
the cell

• Fluorescent Indicators
• Molecules that emit light when excited by light
• Some indicators will emit more light when they
  bind to certain ions
• Calcium indicators
• Calcium ion concentrations can change inside a
  cell in response to a signal
• Calcium ions are important transmitters of
  signals from outside the cell to inside the cell

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My Project Examining changes in calcium
dynamics of epithelial cells of the cornea

• A number of diseases and injuries of the cornea
  affect many people
• Pain is associated with most of these problems
• By studying the way that the cornea interacts
  with the nerves that supply it, treatments can be
  developed to remedy these problems

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Cornea
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Properties of the Cornea

• Greatest density of peripheral sensory nerve
  innervation of any tissue
• Almost exclusively innervated by nociceptive
  nerve fibers
• Nerve terminals approach within a few ?m of
  surface
• Evolutionarily adapted for optical imaging
  (transparency)

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Structure of the Cornea
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The Nerve Cell
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Nerves carry messages
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Corneal Nerve Terminal
L
L
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Fluorescent indicators of calcium concentration
can be put into the cells of the cornea of the rat
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Nerves of the cornea can also be labeled with
fluorescent indicators
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Questions

• In what way do the epithelial cells of the cornea
  respond to chemical messages from outside the
  cell?
• In what way do the epithelial cells of the cornea
  respond to stimulation of the nerves that supply
  the cornea?

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Changes in calcium concentration in epithelial
cells can be seen

• In response to application of chemicals that
  mimic those the cells encounter naturally
• In response to stimulation of nerves, that are
  thought to release certain chemicals that act on
  epithelial cells

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Changes in calcium concentration in the nerve
cells can be seen
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The Bottom Line

• Using fluorescent imaging of calcium changes in
  the living cell, it is possible to see how
  messages are transmitted within the cell in
  response to various stimuli
• By understanding the specific ways in which the
  nerves and epithelial cells of the cornea
  communicate with each other and respond, it is
  possible to understand diseases and malfunctions
  of the cornea
• New therapies can be developed to treat corneal
  problems if the underlying mechanisms are
  understood