Fluorescence Resonance

Energy Transfer
Deluck Sajjad malik E19
 Overview
• What is FRET
• History
• Definition
• properties
• Example
• Condition that influenced FRET
• Principal of FRET
• Mechanism of FRET
• Instrumentation
• Application of FRET
• Advantage of FRET
• Future Aspects
 What is FRET?

FRET stands for forester Resonance energy transfer or

fluorescent resonance energy transfer, one of the
modern advancement in microscopic techniques crucial
for understanding various biological processes.
FRET is a process that shift energy from an
electronically excited molecular (donor) to a
neighboring molecule (acceptor).
• If the two fluorophore are close enough, then
excitation of the first molecule (donor) results in
fluorescence emission of the second molecule
(acceptor).
 The Problem

The use of fluorescent probes permits direct

observation of the dynamic properties of specific
proteins in live cell.
But, it is difficult to observe functional information like
protein-protein interactions using florescent probes.
When protein are labelled with different fluorophore,
the optical resolution of light microscopes limits the
detection of protein proximities to about 0.2 m
The addition of probes and dyes to a membrane
system can potentially interfere with the properties of
the liposomal delivery system.
 History of FRET

1918, J.perrin proposed the mechanism of resonance energy

transfer
1922, G.Cario and J. Franck demonstrated that excitation of a
mixture of mercury and thallium atomic vapors with 254nm.
1928, H.kallman and F.London developed the quantum
theory of resonance energy transfer between various atoms
in the gas phase. The dipole dipole interaction are used for
the first time.
1932, F.perrin published a quantum mechanical theory of
energy transfer between molecules of same specie in
solution.
1946-1949, forester develop the first quantative theory of
molecules resonance energy transfer
 Definition

Fluorescence- The property of absorbing light of shorter

wavelength and emitting light of longer wavelength.
Resonance Energy-total energy emitted to attain
stability by excited molecules to come back to ground
state. Transfer of this energy between two fluorophore
is called resonance energy.
FRET is the non radiative of energy from an excited
fluorophore(donor) to a nearby fluorophore (acceptor)
• FRET efficiency relies on proximity of donor and
acceptor fluorophore, being most effective at short
distance 1-10nm
 properties

Fluorescence properties that can be

measured :
• Spectra
• Fluorescence life times
• Polarization(orientation and dynamics
• Excitation transfer
• Location of fluorescence
• No FRET signal
• FRET signal
 Example

• Detecting interaction of two molecules e.g. Drug

binding to active site or antibody binding to
target
• Detecting a compound that blocks interaction of
two molecules.
• Detecting Co-location of molecules on or with in
cell
• Protein cleavage detected by loss of FRET signal.
• Detecting of conformational change.
• Construction of tandem dyes for flow cytometry
 Condition that influencing FRET

• Distance
Donor and Acceptor molecules must be in
close proximity to each other, generally
between 10-100Ḁ
• Spectra
The absorption spectrum of the acceptor
must overlap the emission spectrum of the
donor
• Dipole Orientation
Donor and Acceptor molecule dipole
orientation should be approximately parallel
 Principle

• FRET involves the transfer of energy from an excited

molecular fluorophore (donor) to another fluorophore
(acceptor) non radiatively
• The distance between the donor and the acceptor is
smaller than the forester 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 a changes in
molecular proximity.
• Investigate donor and acceptor fluorophore emission
spectrum.
 Mechanism

Selection of pairs
• selection of appropriate donor and acceptor pairs based
both on their inherent sustainability as a pair, and your
equipment
• choose suitable filters to maximize excitation of your donor,
whilst minimizing any direct excitation of the acceptor
molecule
 Donor excitation(CFP)
The FRET pair selected so that absorption spectrum of the
acceptor(YFP)overlaps with emission spectrum of donor.
• protein interact with each other when proximity between
them below 10nm
 Mechanism

 CFP emit light

• CFP excite with the help of UV light and emit blue light
 Forester energy transfer from CFP to YFP
• Energy transfer only occur when the FRET dye pair are in
close proximity in order of 10-100nm. Energy that is
transferred from the excited donor and the acceptor not
actual photons. YFP emit light.
 Emission spectrum of CFP and YFP
 No FRET signal
• when CFP is more than 10nm distant from YFP
• YFP is not excited and does not emit light
 Instrumentation

• The instrumentation for FRET typically involves the following

components.
 Light source
• In FRET, light source is crucial for exciting the donor
fluorophore, thereby allowing the transfer of energy to the
acceptor fluorophore.
 Fluorophore
• Two types of fluorophore are required a donor which absorb
light and transfer energy and an acceptor which receives the
energy.
 Instrumentations

 Emission filter
• In FRET, an emission filter is a crucial component.
• It works by selectively allowing only the light emitted by the
acceptor fluorophore to pass through while blocking the light
emitted by the donor fluorophore.
 Excitation filter
• The excitation filter help in selecting the wavelength of light
that is used to excite the donor fluorophore.
• The excitation filter essentially act as special filter allow
specific wavelength of light to pass through and reach the
sample.
 Instrumentation

 Dichroic Mirror
• A dichroic mirror is used to separate donor and acceptor emission.
 Detector
• . Photomultiplier tube
• Avalanche photodiodes are used to detect the emitted light from
the acceptor fluorophore
 Data Analysis Software
• specialized software is employed to analyze the FRET efficiency
which is influenced by factors such as the distance and orientation
between the donor and acceptor as well as spectral properties of
fluorophores
 Applications

 Protein-protein interaction
• FRET help in studying protein-protein
interaction allowing researchers to understand
the dynamics and functions of various proteins
with in biological systems.
 Cellular Signaling
• It aids in investigating cellular signaling
pathways, providing insights into how different
molecules communicate with in cells and
regulate various biological processes.
 Applications

 Biosensor
• FRET based biosensor are employed to detect various
biomolecule and ions in biological samples.
• These biosensor offer high sensitivity and specificity
making them useful in application such as ions, ph.,
and enzymatic activity in living cell and tissues.
 Drug Discovery and Development
• FRET is valuable tool in drug discovery, especially in
screening compound libraries for their interaction with
target proteins.
 Applications

 Nanotechnology and material science

• FRET is utilized in nanotechnology of biosensors, molecular
probes, and nanomaterials
 Fluorescence Microscopy and imaging
• FRET is widely used in fluorescence microscopy to visualize
molecular interactions and events with in cells.
• This enables the tracking of specific molecular processes in real
time and visualize of protein localization and dynamic with in
cell
 Applications

 Gene Expression Analysis

• FRET based probes can be used to monitor gene expression
and splicing events in living cells, allowing researchers to
study transcriptional and post Transcriptional regulation in
real time
 Molecular Conformational changes
• By using FRET, researchers can monitor changes in molecular
conformations, such as protein folding or unfolding
providing a deeper understanding of protein structure
function relationship.
• Nucleic Acid analysis
 Advantages

 Proximity Measurement
• Fret is used to measure molecular distance providing
insights into molecular interaction and
conformational change
 Quantitative Analysis
• FRET measurement can be quantitively analyzed to
measure of molecular interactions and structural
changes.
 Live cell imaging
• FRET is compatible with live cell imaging, allowing
researchers to observe and analyze dynamic cell
processing.
 Advantages

 Fluorescent probe development

• FRET is instrument in the development of new
fluorescent probes, facilitating advancement in
molecular imaging and diagnostics.
 Real time monitoring
• Fret enables real time monitoring of dynamic
processes with in living cells .
 Multicolor imaging
• Using different fluorophore with distinct
emission spectra researcher can study various
molecular interaction in real time.
 Future Aspects

 Improved sensitivity
 Advanced molecular imaging
 Multicolor FRET
 Single Molecule FRET
 In vivo Application
 FRET biosensor
 Nanotechnology integration
 High throughput screening
 MCQ’s
1. What does FRET stand for in the context of molecular interactions?
a. Fluorescent Radiative Energy Transfer
b. Fluorescence Resonance Energy Transfer
c. Fundamentally Radiant Electron Transmission
d. Fast Resonant Energy Transition
2. In FRET, what is the primary mechanism by which energy is transferred
between fluorophores?
e. Electron donation b. Photon emission
c. Resonant energy transfer d. Chemical bonding
3. Which phenomenon is essential for FRET to occur between two fluorophores?
a. Fluorescence b. Absorption
c. Scattering d. Reflection
4. What is the typical range of distances over which FRET can effectively operate?
b. Nanometers to micrometers b. Millimeters to centimeters
c. c. Micrometers to millimeters d. Meters to kilometers
5. Which factors affect the efficiency of FRET?
d. Temperature and pressure b. pH and ionic strength
c. Wavelength and intensity d. All of the above
6. What is the role of donor and acceptor fluorophores in FRET?
a. Both emit light b. Donor absorbs and acceptor emits
b. Donor emits and acceptor absorbs d. Neither emit light
7. What is the significance of FRET in drug discovery?
a. Identifying new drug targe b. Monitoring drug interactions in real
c. Enhancing drug delivery systems d. All of the above
8. What is the advantage of using FRET over traditional methods in studying
molecular interactions?
c. Higher cost b. Lower sensitivity
c. Real-time monitoring d. Limited applicability
9. Which fluorophores are commonly used as FRET pairs?
a. Cyan and yellow fluorescent proteins b. Red and blue quantum dot
c. Green and orange organic dyes d. Ultraviolet and infrared pigments
10. Which Nobel Prize was awarded for the development of the principles of
FRET?
b. Physics b. Chemistry
c. Medicine d. Peace
11. Which biological processes can FRET be employed to investigate?
c. Protein-protein interactions b. DNA replication
c. Cellular respiration d. All of the above
12. FRET is commonly employed in the study of:
a) Astrophysics b) Cell biology
b) c) Geology d) Economics
13. FRET can be quantified by measuring changes in:
c) Absorbance b) Fluorescence intensity
c) Refractive index d) Electrical conductivity
14.The effiency of FRET is influenced by:
a). Moon phase b) spectral overlap
c) Ocean currents d) none of them
15. Which relation describe the FRET efficiency concerning distance :
a) Linear b) Exponential c) inverse sixth power d) none
 Short Question
Q1.What is Fluorescence Resonance Energy Transfer (FRET) and how
does it work at a molecular level?
Q2.Can you explain the key role of donor and acceptor fluorophores in
a FRET pair?
Q3.How is FRET used to study molecular interactions and
conformational changes in biological systems?
Q4.What are the factors that influence the efficiency of Fluorescence
Resonance Energy Transfer?
Q5.In what scientific applications or fields is FRET commonly employed
for studying molecular dynamics?
 Long Question
Q1. How does Fluorescence Resonance Energy Transfer contribute to
our understanding of molecular interaction, and what are the key
principal and application that make it a powerful tool in different fields?