Total Internal reflection Fluorescence
Microscopy: Instrumentation and
Applications in Cell biology
Background
Basic concept
Instrumentation
Modifications and developments
Applications in cell biology
What is TIR?
Snell’s law and TIR
n(1) • sinq(c) = n(2)
Or q(c) =sin-1n(2)/n(1)
For TIR , n(2)/n(1) < 1
Basic points
TIR can be achieved at all angles greater than critical
angle.
When TIR occurs, there is always small amount of
light penetration across the interface.
This light generates an evanescent wave within the
limited region of interface
Diagrammatic representation
Properties of evanescent field
Its intensity is given by:
I(z) = I(0)exp(-z/d)
Where d = (i)/4 • (n(1)2sin2q(1) - n(2)2)-1/2
Field strength falls of exponentially normal to interface (z) and so
extends upto ~100 nanometers.
Higher signal to noise ratio because of confinement of secondary
fluorescence to thin region.
Requirements for Instrumentation
Illumination: laser preferred: it is coherent, polarized, and well
collimated, so that it can be easily directed.
Beam expanders, Mirrors, and Focusing lenses.
Objective with High NA or
Prism to obtain total internal reflection.
Basic Setup
Objective lens method
Objective is employed to introduce
light
Prism method
Prism is employed to introduce light
Disadvantages of each
Prism method
Objective lens method
Geometric constraints
Expensive setup
In Inverted microscopes, imaging
of evanescent field is through
bulk of specimen
NA of Objective should be
greater than Refrective index
of medium at interface
Advantage over Epifluorescence
TIRF
Epifluorescence
Tokunaga M., Yanagida T.,, Biochemical and Biophysical Research
Communications: 235, 47–53 (1997).
Comparison of Signal to noise ratio
TIRFM
Normal fluorescence
http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html
Measurement of distance from the surface
Distance ‘z ‘ of the fluorophore from the surface can be
calculated from fluorescence intensity that , in turn, is
proportional to evanescent intensity I(z).
If fluorophore moves from z1 to z2,
z= z1- z2 = dln (I2/I1)
This relationship is valid even in those cases where there are
multiple fluorophores attached to the same structure or
irregularly shaped fluorophores.
Distance measurements for biologists
Distance of fluorophore from the membrane is of importance
rather than from surface.
Membrane impermeant fluorescent dye + fluorescent dye in cell organelle
TIR illumination
Offcell fluorescence is uniformly bright ; in cell substrate contact, dye is confined to thin layer
So, fluorescence
f
separation distance
in contact region will be darker by a factor that can be converted to
h:
h = -dln[1-f/foffcell]
Daniel Axelrod, Methods in enzymology, (2003) Vol 361, p1-33
Monitoring Amyloid fibril growth with TIFR
• Amyloid fibril : a protein that has self-assembled into an insoluble
antiparallel β-pleated sheet.
• These fibrils give rise to the amyloid plaques that are seen in a number of
pathological processes (eg Alzheimer's disease)
• To know the mechanism of amyloid fibril formation, it should be observed at
single fibril level.
• Thioflavin (ThT) binds to amyloid fibril with increase in fluorescence at 455
nm ; em 485 nm
• Combination of ThT fluorescence and TIRFM can be used to monitor Amyloid
growth
.
-Amyloid fibril observed through TIRFM
• Penetration depth of evanescent
field upon excitation at 455 nm is
150 nm
• Amyloid fibrils have diameter of
10-15 nm, so fibrils lying in
parallel with slide glass surface is
observed.
Ban et. al. J Biol Chem. 2003 May 9;278(19):16462-5. Epub 2003 Mar 18
Images showing growth of Amyloid fibril
Amyloid fibrils in test tubes
Amyloid fibril growth on slides
Ban et. al. J Biol Chem. 2003 May 9;278(19):16462-5. Epub 2003 Mar 18.
Visualizing membrane trafficking using TIFR
Example : imaging of insulin vesicle membrane and cargo
during exocytotic event.
1.
2.
Target flurophore (EGFP) to secretory vesicle membrane
Use of some dye (acridine orange) that can go inside vesicle
3.
Monitoring colocalization of the two fluorophores.
4.
Visualizing the movement by the help of TIFR
Tsuboi et al Current Biology (2000) Vol 10 No 20
Colocalization of Acridine
orange and EGFP-phogrin
Time course of fluorescence
intensity of acridine orange
(1,2) and EGFP-Phogrin
(1’,2’).
Tsuboi et al Current Biology (2000) Vol 10 No 20
Structural details revealed by combination of TIFR and
epifluorescence
• Cells were immunocytochemically labeled for the protein tubulin
and observed under microscope.
epifluorescence
TIFR
Overlay
http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html
TIRF versus Confocal microscopy
The depth of optical section ~ 100-150 nm for TIRF whereas in
confocal microscopy it is ~600 nm
TIRF can be adapted to standard microscopic optics with less
expense; confocal microscopy is very expensive
Unlike confocal microscopy, TIRF can be applied to macroscopic
applications
Best suited for applications where illumination as well as
detected emission is restricted to a thin section.
Tsuboi et al Current Biology (2000) Vol 10 No 20
Future work
Analyze molecular mechanisms of exocytosis and endocytosis;
study process of synaptic vesicle fusion.
Study the interactions ‘in situ’ in living cells.
Thank you