Total Internal Reflection Fluorescence TIRF

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Total Internal Reflection Fluorescence - TIRF

• BMEN 489/689
• Spring 2006

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Evanescent Wave Reveiw

• To create evanescent waves
• Interface between high index and low index
  material
• Angle of incidence greater than the critical
  angle
• Properties of the evanescent wave
• Propagates along the interface
• Exponentially decays in the lower index material
  in about 100nm

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TIRF Microscopy

• Uses evanescent wave to selectively excite
  fluorophores within about 100nm of a glass/water
  interface
• Produces an image with very little background
  fluorescence
• Minimizes the exposure of the biological sample
  to the excitation light
• Good for looking at biological samples adhered to
  a surface

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TIRF Microscopy

• Because the evanescent wave illuminates only
  100nm, specific visualization of features near
  the surface is possible
• Stephens et al., Science 300 33-40 (2003).

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TIRF Microscopy
Images taken of actin-labeled and
phalloidin-labeled cells using TIRF and standard
(EPI) fluorescence techniques. The TIRF images
show only that part of the plasma membrane near
or attached to the substrate. The EPI images
show fluorophores throughout the cell. Sund et
al., Biophysical Journal 79, 1655 (2000).
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TIRF Microscopy

• Can eliminate background fluorescence
• Sako, Nature Cell Biol. 2, 168 (2000).

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Creating the Evanescent Wave

• Light must come in at an angle greater than the
  critical anglelarge angle as measured from the
  surface normal
• Two general categories to perform this optically
• Light coupled through a prism
• Light coupled directly through a high numerical
  aperture lens

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TIRF with a Prism Inverted Scope

• Examples of prism coupling to evanescent modes.
• easier to set up than epi
• purer evanescent wave
• prism used to attain critical angle
• laser focused to spot size about equal to field
  of view
• Glass cover slip is used to save the prism and
  is coupled to prism with matching fluid

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TIRF with a Prism Upright Scope
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Prism-less TIRF
Lens normally oil coupled to coverslip nglass
noil 1.52 N.A. noil sin? ? gt
?critical nsample 1.33 nsample noil
sin?c Thus N.A. 1.4

• Coupling to evanescent modes without a prism.
• beam must pass through periphery of high NA
  objective
• higher NA will allow confinement closer to
  surface
• laser focused off-axis at back focal plane of
  the objective
• not as pure an evanescent wave as prism

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TIRF Comparison

• Characteristics of Prism Method
• easier to set up than prism-less system
• purer evanescent wave
• prism used to attain critical angle
• laser focused to spot size about equal to field
  of view
• Characteristics of Prism-less Method
• beam must pass through periphery of high NA
  objective
• higher NA will allow confinement closer to
  surface
• laser focused off-axis at back focal plane of
  the objective
• not as pure an evanescent wave as prism

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Applications

• Single molecule detection
• Takes advantage of the low background signal
• Cell surface events
• Signaling
• Endocytosis
• Exocytosis

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TIRF Examples
Actin filaments polymerizing in buffer.
Filaments grow from the barbed end labeled
(a,b,c,d). Kuhn et al., Biophysical Journal 88,
1387 (2005).
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TIRF Examples

• EGF receptor signaling on the cell surface
• Sako, Nature Cell Biol. 2, 168 (2000).

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TIRF Examples

• TIRF is very good for looking near a surface
• Can be used with epi-fluorescence to see dynamics
• Movies show fusion of single transport containers
  (TCs) was clearly observed and gave a distinct
  analytical signature. TCs approached the
  membrane, appeared to dock, and later rapidly
  fuse, releasing a bright fluorescent cloud into
  the membrane. Observation and analysis provided
  insight about their dynamics, kinetics, and
  position before and during fusion.

Toomre et al, J. Cell Biol 149 33-40 (2000)
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Comparison

• Confocal
• Entire sample excited, but spatially filtered
• Axial resolution on the order of 500nm
• Slow image acquisition
• Entire cell imaged

• TIRF
• Illuminates only near surface
• Axial resolution on the order of 50nm
• Fast image acquisition
• Images cell surface