Total Internal reflection Fluorescence Microscopy: Instrumentation and

Applications in Cell biology

Background

Basic conceptInstrumentationModifications and developmentsApplications in cell biology

What is TIR?

Snell’s law and TIR

n(1) • sin(c) = n(2)

Or (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)2sin2(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 is employed to introduce light

Prism method

Disadvantages of each

Geometric constraints

In Inverted microscopes, imaging of evanescent field is through bulk of specimen

Expensive setup

NA of Objective should be greater than Refrective index of medium at interface

Prism method Objective lens method

Tokunaga M., Yanagida T.,, Biochemical and Biophysical Research Communications: 235, 47–53 (1997).

EpifluorescenceTIRF

Advantage over Epifluorescence

                                                                                                                 

TIRFM

Normal fluorescence

http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html

Comparison of Signal to noise ratio

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 in contact region will be darker by a factor that can be

converted to separation distance 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

Ban et. al. J Biol Chem. 2003 May 9;278(19):16462-5. Epub 2003 Mar 18

-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

Visualizing membrane trafficking using TIFR

Example : imaging of insulin vesicle membrane and cargo during exocytotic event.

1. Target flurophore (EGFP) to secretory vesicle membrane

2. 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

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’).

Structural details revealed by combination of TIFR and epifluorescence

epifluorescence TIFR Overlay

• Cells were immunocytochemically labeled for the protein tubulin and observed under microscope.

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