1. Total Internal reflection Fluorescence Microscopy: Instrumentation and Applications in Cell biology

2. Background Basic concept Instrumentation Modifications and developments Applications in cell biology

3. What is TIR?

4. Snell’s law and TIR n(1) sin  (c) = n(2) Or  (c) =sin -1 n(2)/n(1) For TIR, n(2)/n(1) < 1

5. 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

6. Diagrammatic representation

7. Properties of evanescent field Its intensity is given by: I (z) = I (0 )exp(- z/d) Where d = (i)/4 (n(1) 2 sin 2  (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.

8. 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.

9. Basic Setup Objective lens method Objective is employed to introduce light Prism is employed to introduce light Prism method

10. 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 methodObjective lens method

11. Tokunaga M., Yanagida T.,, Biochemical and Biophysical Research Communications: 235, 47–53 (1997). Epifluorescence TIRF Advantage over Epifluorescence

12. TIRFM Normal fluorescence http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html Comparison of Signal to noise ratio

13. 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 z 1 to z 2,  z= z 1 - z 2 = dln (I 2 /I 1 ) This relationship is valid even in those cases where there are multiple fluorophores attached to the same structure or irregularly shaped fluorophores.

14. 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/f offcell ] Daniel Axelrod, Methods in enzymology, (2003) Vol 361, p1-33

15. . 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

16. 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.

17. 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 tubesAmyloid fibril growth on slides

18. 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

19. Colocalization of Acridine orange and EGFP-phogrin Time course of fluorescence intensity of acridine orange (1,2) and EGFP-Phogrin (1’,2’).

20. 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

21. 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

22. Future work Analyze molecular mechanisms of exocytosis and endocytosis; study process of synaptic vesicle fusion. Study the interactions ‘in situ’ in living cells.

23. Thank you