The document discusses three fluorescence microscopy techniques: FRET, FRAP, and TIRF microscopy. It explains the principles, instrumentation, sample preparation, applications, and future aspects of each technique. FRET involves energy transfer between fluorophores and is used to study molecular interactions and structure. FRAP examines fluorophore diffusion by photobleaching a region and monitoring recovery. It provides information about molecular mobility. TIRF microscopy uses evanescent waves to selectively excite fluorophores very near a surface and is applied to studies of cellular processes at membranes. All three techniques provide insights into biological phenomena at the molecular level.

1. -presented by
Baishali Tamuli (BBI17008)
Jyotishman Sarma (BBI17009)
FRET, FRAP and TIRF
Microscopy
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?

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

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

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

6. • Principle
(a) Donor (b) Acceptor
(c) Donor- Acceptor complex
Fig.-(a),(b)(c)Excitation and Emission spectra in FRET
Em
EmEx
Ex
Ex- Excitation
Em- Emission
EmEx

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)

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

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.

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

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

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?

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.

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.

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 105
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 tD is the
characteristic diffusion time

16. Fig.- Lipid membrane movement with the help of FRAP(a)
(b)

17. • Applications
Major applications of FRAP are,
 To study brownian motion which is dependent on molecular
size, local environment and binding interactions.
 Understandingmoleculartrafficking.
 Intracellular transport
 Continuity of compartments
 Stability of molecular complexes

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 µm).
 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.

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).
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

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

22. The evanescent field intensity decays exponentially with increasing distance
from the interface into the low-index medium as
where I0 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 /4π(n1
2sin2θ – n2
2)1/2
fig source: ncbi.nlm.nih.gov
Iz = I0e(-z/d)

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
Basic Instrumental Approaches
Fig source: microscopy.com
ncbi.nlm.nih/pmc/articles

24. Epifluorescence versus TIRF Microscopy
 Epifluorescence  TIRF microscopy
Fig A: Hela 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.
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.

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

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

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

30. Thank
you