FRET, FRAP, TIFR MICROSCOPY

ANALYITICAL TECHNIQUES on FRET FRAP TIFR MICROSCOPY

1. -presented by Baishali Tamuli (BBI17008) Jyotishman Sarma (BBI17009) FRET, FRAP and TIRF Microscopy 1/11/19 1 Principles and Applications of TEZPUR UNIVERSITY

2. FRET stands for Förster resonance energy transfer or fluorescence resonance energy transfer , one of the modern advancements in microscopic techniques crucial for understanding various biological processes. What is FRET ? 1/11/19 2

3. FRET is named after German physicist Theodor Förster who was pioneer in discovering the Förster distance or radius and interaction of molecules which are in close proximity. His theory of FRET was first published in 1946 . Discovery of FRET 1/11/19 3

4. FRET involves the transfer of energy from an excited molecular fluorophore ( donor ) to another fluorophore ( acceptor ) non radiatively whenever the distance between the donor and the acceptor is smaller than Förster radius. The efficiency of FRET is dependent on the inverse sixth power of intermolecular separation making it a sensitive technique for investigating a variety of biological phenomena that produce changes in molecular proximity. Principle 1/11/19 4

5. Principle (a) Protein flurophore complexes (b) Excitation and emission wavelengths of Donor and Acceptor (c) FRET between the complexes 1/11/19 5

6. Principle (a) Donor (b) Acceptor (c) Donor- Acceptor complex Fig.-(a),(b)(c)Excitation and Emission spectra in FRET Em Em Ex Ex Ex- Excitation Em - Emission Em Ex 1/11/19 6

7. Types of FRET (i) Intermolecular FRET ( interaction between two different molecules) (ii) Intramolecular FRET (interaction within the same molecule) Fig.- Schematic representation of Intermolecular and intramolecular FRET ((a) Intermolecular FRET in protease activity assay, (b) Intramolecular FRET in PKG protein activation) (a) (b) ( Source- MORITOSHISATO AND YOSHIO UMEZAWA, ANALYTICAL CHEMISTRY 72:5924,2000 . © 2000 AMERICAN CHEMICAL SOCIETY) 1/11/19 7

8. Instrumentation Fig.- Schematic diagram of instrumentation in FRET (source- Henry Mühlpfordt Fluoreszenzmikroskopie_2008-09-28.svg ) 1/11/19 8

9. Sample preparation In 1960s Osamu Shimomura discovered that a certain species of jellyfish ( Aequorea victoria ) owes its luminescent character to the presence of fluorescent proteins, such as aequorin and the green fluorescent protein (GFP ). In GFP, the light-absorbing/emitting chromophore is formed by self modification (i.e ., by an autocatalytic reaction) of three of the amino acids that make up the primary structure of the GFP polypeptide Live-cell imaging studies can often be made more informative by the simultaneous use of GFP variants that exhibit different spectral properties. Variants of GFP that fluoresce in shades of blue (BFP), yellow (YFP), and cyan (CFP ) were generated by Roger Tsien of the University of California , San Diego, through directed mutagenesis of the GFP gene. 1/11/19 9

10. How FRET helps? Major applications of FRET: Molecular interactions ( eg .- Protein-protein interactions ) Structure elucidation of biomolecules Ligand receptor binding Molecular colocalization ( eg .- Studying lipid rafts ) Developing biosensors and chemosensors 1/11/19 10

11. Future aspects Better understanding of signalling pathways Cellular interaction with the environment Hormone receptor binding Trsansport of biomolecules within a system Drug designing 1/11/19 11

12. FRAP stands for Fluorescence recovery after photobleaching , is a method for determining the kinetics of diffusion through living tissues, cells or a system . What is FRAP? 1/11/19 12

13. Principle The general method is to label a specific cell component with a fluorescent molecule, image that cell, photobleach a small portion of the cell, then image the recovery of fluorescence over time. Diffusion or active movement of molecules within the cell replace bleached fluorophore with unbleached molecules that were located in a different part of the cell. Over time fluorescence in the bleached region recovers. 1/11/19 13

14. Instrumentation and sample preparation The basic apparatus comprises an optical microscope, a light source and some fluorescent probe. Fluorescent emission is contingent upon absorption of a specific optical wavelength or color. The technique begins by saving a background image of the sample before photobleaching. Next , the light source is focused onto a small patch of the viewable area either by switching to a higher magnification microscope objective or with laser light of the appropriate wavelength. 1/11/19 14

15. Instrumentation and sample preparation The fluorophores in this region receives high intensity illumination which causes their fluorescence lifetime to quickly elapse (limited to roughly 10 5 photons before extinction ). Now the image in the microscope is that of a uniformly fluorescent field with a noticeable dark spot . As Brownian motion proceeds, the still-fluorescing probes will diffuse throughout the sample and replace the non-fluorescent probes in the bleached region . The initial and final photograph gives the extent of mobility which can be calculated by the equation, where , D is diffusion constant , ω is the radius of the beam and t D is the characteristic diffusion time 1/11/19 15

16. Fig.- Lipid m embrane movement with the help of FRAP (a) (b) 1/11/19 16

17. Applications Major applications of FRAP are , To study brownian motion which is dependent on molecular size, local environment and binding interactions. Understanding molecular trafficking . Intracellular transport Continuity of compartments Stability of molecular complexes 1/11/19 17

18. Drawbacks of FRAP FRAP can only follow the average movement of a relatively large number of labeled molecules (hundreds to thousands) when they diffuse over a relatively large distance (e.g . 1 µ m ). As a result, researchers using FRAP cannot distinguish between proteins that are truly immobile and ones that can only diffuse over a limited distance in the time allowed . 1/11/19 18

19. What is TIRF Microscopy ? TIRF stands for Total Internal Reflection Fluorescence . A thin region of a specimen, usually less than 200 nanometers can be observed. It is a powerful technique for selectively imaging fluorescent molecules usually in an aqueous environment that are very near a solid substance with a high refractive index (e.g. coverglass ). 1/11/19 19 Fig source: onlinephysicstuition.com.my

20. The idea of using total internal reflection in microscope was first described by E.J. Ambrose in 1956. This idea further extended by Daniel Axelrod at the University of Michigan. Ann Arbor in the early 1980s introduced TIRFM . THE STORY BEHIND 1/11/19 20

21. What is the principle involved? Based on the principle of total internal reflection of light . By Snell’s Law , θ critical  = sin ­-1  (n 1 /n 2 ) where, n1 = refractive index of air(less dense) n2 = refractive index of glass (more dense) Fig source: onlinephysicstuition.com.my (c) (b) (a) 1/11/19 21

22. The evanescent field intensity decays exponentially with increasing distance from the interface into the low-index medium as where I is the intensity at the interface, z is the perpendicular distance from the interface ,and d is the penetration depth The penetration depth (d) is determined by: d = λ /4 π (n 1 2 sin 2 θ – n 2 2 ) 1/2 fig source: ncbi.nlm.nih.gov I z = I e (-z/d) 1/11/19 22

23. (Components- 1. Specimen 2 . Evanescent wave range 3 . Cover slip 4 . Immersion oil 5 . Objective 6 . Emission beam ( signal) 7 . Excitation beam) (Components- 1 . Objective 2 . Emission beam ( signal) 3 . Immersion oil 4 . Cover slip 5 . Specimen 6 . Evanescent wave range 7 . Excitation beam 8 . Quartz prism) Based on the type of objective used, two types of TIRFM are available. (i) Objective based (ii) Prism based 1/11/19 23 Basic Instrumental Approaches Fig source: microscopy.com ncbi.nlm.nih / pmc /articles

24. Epifluorescence versus TIRF Microscopy Epifluorescence TIRF microscopy 1/11/19 24 Fig A: H ela cells recorded by standard epifluorescence microscopy Fig B: Hela cells recorded by TIRF micoscopy Fig source: ncbi.nlm.nih.gov/ pmc /articles

25. Applications Excellent technique for combining kinetic studies with spatial information in live samples or even in vitro. localization of single molecules is achievable with a precision of 1 nm . Examination of membrane-fusion processes such as vesicle trafficking . Useful in studying cellular signaling at the level of plasma membrane. 1/11/19 25 Source: leica-microsystems.com

26. Prospects for future development Other source of light can also be used if modifications are done to block light in the central region. Acquisition of image data at multiple wavelengths is an area of great promise for TIRFM. Improvement of Single molecule studies. Refinement of genetic and molecular manipulation techniques combined with optical detection. 1/11/19 26

27. FRET What is FRET? What is the principle underlying FRET technology? What are the different types of FRET? What is the basic Instrumentation involved? How is the sample prepared for FRAP? Mention some of the applications and future aspects of FRET Questions 1/11/19 27

28. FRAP what is FRAP? What is the principle involved? What is the basic instrumentation and sample preparation of FRAP? Describe the study of lipid membrane movement using FRAP. What are the drawbacks of FRAP? Mention some applications of FRAP. Questions 1/11/19 28

29. TIRF What is TIRF? What is the basic principle underlying the TIRF technology? What are the conditions for total internal reflection? What is evanescent wave? What is critical angle? On what factors do the penetration depth of evanescent wave depend? Mention two basic instrumental approaches involved in TIFR microscopy. Write the advantages of TIRFM over standard fluorescence microscope. Mention some applications and future prospects of TIRFM. Questions 1/11/19 29

30. Thank you 1/11/19 30