Fluorescent methods in plant biology

an INTRODUCTION

Attila Ördög
 Schedule
Lecture (Fridays, 10.00–11.30) Practical (Fridays, 12.00-13.30)
1. (Feb 15) 1. (Feb 15)
Introduction to fluorescence, fluorescent probes and devices for 2. Introduction to microscopy – History and types of microscopes
fluorescence measurements (Ördög A) (Szőllősi R)
2. (Feb 22) 2. (Feb 22)
Fluorescent probes for investigation cell division, organelles, Fluorescent probes for investigation cell division, organelles,
intracellular pH or ions (Feigl G) intracellular pH or ions – from practical aspects (Feigl G)
3. ( March 1) 3. (March 1)
Detecting ROS: fluorescent and other techniques (Szepesi Á) Detecting ROS (Feigl G)
4. (March 8) 4. (March 8)
Detecting RNS: fluorescent and other techniques (Kolbert Zs) Detecting RNS (Feigl G)
5. (March 15) 5. (March 15)
National holiday National holiday
6. (March 22) 6. (March 22)
Detecting PCD (Poór P) Detecting cell death (Poór P)
7. (March 29) 7. (March 29)
Spring Break Spring Break
8. (April 5) 8. (April 5)
Chlorophyll fluorescence (Ördög A) Chlorophyll fluorescence measurements (Ördög A)
9. (April 12)
9. (April 12)
Advanced applications of confocal microscopy (bleaching techniques,
Microscope techniques (F Ayaydin)
photo conversion, protein-protein interactions) (Ördög A)
10. (April 19) 10. (April 19)
National holiday -
11. (April 26) 11. (April 26)
Immunofluorescence (Kolbert Zs) Nucleic acid detection using fluorescent probes (Horváth E)
12. ( May 3) 12. (May 3)
Genetically encoded fluorescent probes (Csiszár J) roGFP detection using fluorescent microscopy (Horváth E)
13. (May 10) 13. (May 10)
Investigating protein activity with fluorescent probes (Gémes K) Written exam (Ördög A)
14. (May 17)
Written exam (Ördög A)
 Fluorescent methods Lab Books

General:
 Record all lab notes in a lab notebook! Do not use loose paper or handouts.
 Lab book should not be spiral bound.
 Keep a detailed description of your lab activities, including description of all steps, materials, general observations, results and
conclusions.
 Use new site for every practices.
 Number each page.
 Be as specific as you can.
 Use units for measurements: 12 µl, or 5 minutes, etc.

Sections:
 Table of Contents: Title and page number of each lab exercise.
 For each lab experiment:
a. Introduction: What are you going to do, general background information from lecture, PowerPoint’s or handouts. What is
the main objective (hypothesis)
b. Materials and Methods: What supplies and reagents are needed? This should include a description of all the steps carried
out in the experiment.
c. Results: Provide the results from you experiment. These include:
i. All general observations! Color of solutions, viscosity, mistakes! (Ex: left tube in incubator for 5 minutes instead of 2)
ii. Actual Pictures (preferred) or drawings of gels, tubes, graphs, etc.
iii.Any data collected
d. Discussion and Conclusion: Explain the significance of your results.
Introduction to fluorescent methods
What is fluorescence?
The phenomenon put simply: emission of light by a molecule that had been triggered by illumination
Photoluminescence is the term for the general process when specimens, living or non-living, organic or inorganic,
absorb and subsequently re-radiate light. It incorporates:
• Phosphorescence, when the emission of light persists for up to a few seconds after
the excitation energy (light) is discontinued.
• Fluorescence, when light emission continues only during the absorption of the excitation light.
(The time interval between absorption and emission is very short: usually less than a millionth of a second.)
The method: Studying the characteristics of the light emitted by a molecule that had been triggered by illumination.
Fluorescence belongs to the science of spectroscopy.

Fluorescence of aragonite
Introduction to fluorescent methods
What is spectroscopy?

The science that deals with the analysis of electromagnetic spectrum. It studies the interaction of
electromagnetic radiation with matter.

It studies the intensity and energy (frequency, wavelength) of the waves in the spectrum.

(λ)

(Hz)
Introduction to fluorescent methods
Light and the electromagnetic spectrum

Visible light: the portion of the electromagnetic spectrum that is visible

to humans.
(And incidentally the portion that is also used in photosynthesis!)

Light has characteristics of both wave and a particle:

• It’s an electromagnetic wave, in which both electric and magnetic
components oscillate perpendicularly to the direction of propagation
of the wave and at 90° with respect to each other.
• Wavelength (λ), frequency (ν) and speed (c) are related as follows:
c=λ×ν

• It is also a particle called a photon. Each photon contains a

particular amount of energy: E = h × ν
(where h is Planck’s constant: 6.626 × 10–34 J s)
c
Energy is inversely proportional to wavelength, as E = h × —
λ
Introduction to fluorescent methods
Chromophore – fluorophore

A chromophore is a molecule or part of a molecule which is

responsible for the absorption of the light.
(e.g., the chromophore of hemoglobin is heme)

A fluorophore is a molecule or part of a molecule which is

responsible for the emission of the light.
(e.g., the fluorophore of chlorophyll is the Mg-porphyrin ring)

Prerequisites of fluorescence:
Not all substances are capable of emitting photons.
(In fact, there are very few of them!)
In the outermost electron shell of the molecule there must be an
“excitable” electron.
The energy of the absorbed photon must equal the energy
difference between the excited and ground states of the
fluorophore.
Introduction to fluorescent methods
The interaction of light and matter

When a photon meets a molecule it may be:

• scattered (bounce off the molecule);
• transmitted (pass through the molecule), or
• absorbed:
The molecule acquires the energy of the photon.
It is thereby raised from ground state to excited state: an electron is
boosted to a molecular orbital farther from the nucleus.
(This increases the chemical reactivity of the molecule!)

The excited state is unstable: the molecule returns to ground state

and energy is released:
• non-radiatively as heat or
• radiatively, via photon emission: fluorescence, or phosphorescence

Excited state
Energy of electron

Heat

Photon emission
(fluorescence, or phosphorescence)
Photon
Ground state
Molecule
Introduction to fluorescent methods
The fluorescence process

Fluorescence is the result of a multi-stage process that occurs in certain molecules

(generally polyaromatic hydrocarbons or heterocycles)

S1
1. Excitation: the fluorophore absorbs a
photon of energy (hνex) and becomes excited
due to the energy of the absorbed photon
(excited electronic singlet state).
2. In the unstable excited state,
the fluorophore loses a part of its excitation energy
due to molecular interactions (internal conversion).
3. Fluorescence emission:
a photon of energy (hνem) is emitted, which
returns the fluorophore from the hνex
excited state to the ground state. hνem
Due to partial energy dissipation in the excited state:
hνex > hνem, therefore λex < λem (Stokes shift)

S0
Introduction to fluorescent methods
The fluorescence process – in more detail
Introduction to fluorescent methods
The advantages of fluorescence in biology

1. Non-invasive: it can be applied in living cells and tissues

2. Highly sensitive: we can detect 1 molecule among 1 billion (the one which emits light, the
others are invisible).

3. Highly selective: we can detect exactly 1 molecule among 1 billion because it emits light
(whereas the others don’t and so they are invisible).

4. Easy to detect: nowadays there are lots of high-quality light-sensitive detectors.

5. Quantifiable: the intensity of the emitted light is proportional to the concentration of the
fluorophore.

6. User-friendly: it is relatively easy to perform and can be quickly acquired.

7. Fast: it provides information during and immediately after the experiment.

Introduction to fluorescent methods
Fluorescence in biology
Expression of phototropin1-GFP in the epidermis of an
Arabidopsis leaf.
Red fluorescence arises from chloroplasts in underlying
mesophyll cells.

Quinine solutions
under visible

... and UV light.

Aequoria victoria (GFP) Deep sea fish

Introduction to fluorescent methods
Fluorescence and fluorophores
Primary or autofluorescence: emission of fluorescent light by a
material that can be made to fluoresce in its natural form.
Secondary fluorescence: emission of fluorescent light by a
material treated with chemicals (called fluorophores) capable of
fluorescing

Autofluorescence is usually too faint and/or non-specific (thus non-

informative) in biological samples.
(Well-known counter example: chlorophyll fluorescence!)

Advantages of secondary fluorescence (fluorophores)

• higher emission-absorption ratios (quantum yield)
• sensitivity: custom designed fluorophores for modification of
spectral characteristics.
• specificity: specific binding of fluorochromes ensures specific
labeling of certain materials: nucleus, cytoskeleton
components, Ca2+, pH, ROS, NO, etc. (immunofluorescence!)

In fluorescence devices, filters are used for optimal (specific)

excitation and emission of fluorescence.
(crystal of fluorspar)
Introduction to fluorescent methods
Fluorophore characteristics
Spectrum: A graph showing the wavelength-dependence of a given
parameter. (E.g. the absorption and emission of a fluorophore.)

Absorption spectrum: a graph showing the wavelength-dependence of

the exciting light absorbed by a fluorophore.
It shows what proportion of the light of a given colour (wavelength) is
absorbed the fluorophore.
(The absorption spectrum is similar to the fluorescence excitation
spectrum, which shows the wavelength-dependence of the number of
photons generated by the fluorophore.)

Emission spectrum: a graph showing the wavelength-dependence of light

emitted by a fluorophore.
It shows what proportion of the light of a given colour (wavelength) is
emitted by the fluorophore.

Molar extinction coefficient (ε):

The efficiency with which the fluorochrome
absorbs the excitation light
(at a specific wavelength).

Quantum yield (Φ): The yield of emitted light,

that is, the ratio of the number of quanta
("packets" of energy, or photons) emitted
compared to the number of quanta absorbed
(usually between 0.1 and 0.9).
It may depend on environmental factors,
such as metallic ion concentration, pH, and solvent polarity.
Introduction to fluorescent methods
Recording absorbance and fluorescence

The essentials for recording absorbance

A spectrophotometer records absorption spectra
1. Light source,
2. A chromophore,
3. A detector that registers and compares
the intensity of light entering and leaving
the sample and produces a
recordable output
(usually as an electrical signal)

The essentials for recording fluorescence

A spectrofluorometer records fluorescence spectra
1. An excitation light source,
2. A fluorophore,
3. Wavelength filters to isolate emission
photons from excitation photons,
4. A detector that registers emission
photons and produces a recordable
output (usually as an electrical signal).
Introduction to fluorescent methods
Fluorescence instrumentation

1. Spectrofluorometers and 2. Fluorescence microscopes resolve fluorescence as a

microplate readers measure function of spatial coordinates in two or three dimensions for
the average properties of bulk microscopic objects (less than ~0.1 mm diameter).
(µL to mL) samples.

3. Fluorescence scanners resolve fluorescence 4. Flow cytometers measure fluorescence per cell in a flowing
as a function of spatial coordinates in two stream, allowing subpopulations within a large sample to be
dimensions for identified and
macroscopic objects, quantitated
such as (and sorted).
electrophoresis gels,
blots and
chromatograms
(e.g. microarray
readers).
Introduction to fluorescent methods
Fluorescence instrumentation
1: FSC detector
2: SSC detector
Flow cytometer: used specifically for studying cell suspensions 3: Fluorescence detector
4: Filters/mirrors
Large number of cells (10 to 20 thousand) can be 5: Charged deflection plates
measured in minutes.
The light source (a laser) illuminates every single cell
separately and excites the fluorophore in them.
The detector measures the intensity of the light emitted by
every single cell.
Histogram: a graph showing the cell number in the
function of fluorescence intensity.
Flow cytometry is capable of sorting cells based on
almost any of the features it measures.

Fluorescence Activated Cell Sorter (FACS)

It can separate the cells based on the
intensity or wavelength (colour) of the emitted light.
Additional optical information:
• Forward scatter (FSC): cell size
• Side scatter (SSC): cell granularity
The separated cells are collected in different vials.
They are a special subpopulation of the original cell mass
that can be used for further studies.
Introduction to fluorescent methods
Fluorescence instrumentation: Fluorescence microscope

Enables visualization of single molecular species: localization,

diffusion coefficients, transport characteristics
and interactions with other biomolecules
Excitation and emission wavelengths are
selected and isolated by filters
to optimize sensitivity
Light sources: UV/vis
The detector: eye, digital camera(s)
Thermo-regulatable object stage
Computerised image recording, stage manipulation,
lightsource/filter manupulation and image processing
Introduction to fluorescent methods
Fluorescence instrumentation: Confocal microscope

Makes a series of optical „sections” from one single cell by

confocal point scanning:

The result is a higher resolution image compared to “traditional”

(widefield) microscopy:
Introduction to fluorescent methods
Fluorescence instrumentation: Confocal microscope
Makes a series of optical „sections” (x-y scans) from one single
cell by confocal point scanning:
↓
The focal distance (z-axis) can be changed after every shoot
↓
It carries out the „sectioning” in several parallel planes
↓
Finally, it creates a 2D or 3D picture from the intensity values
recorded.
A colour scale can be fitted to the fluorescence intensities:
pseudocolour technique

The 2D cell contour can be

superimosed onto the
light microscope image
of the same cell,
thereby the intracellular
location of the fluorophore
can be identified:
Introduction to fluorescent methods
Monitoring cellular events with fluorescence

Fluorescence is an important imaging tool in biology, as it is sensitive and able to

specifically target structural components and dynamic processes.

Why is fluorescence capable of monitoring cellular events?

There are practically no (or only weakly) fluorescent molecules in the cells
Fluorescent probes can be easily detected
They report on a specific state or phenomenon taking place in the cells
Toxicity is usually low due to the low dye concentration (specificity)

What kind of states/phenomena can be studied by fluorescence?

Many fluorescent may (be designed to)
• specifically bind with a biological macromolecule (for example, a protein or nucleic acid) or
• localize within a specific structural region, such as the cytoskeleton, mitochondria, Golgi
apparatus, endoplasmic reticulum, and nucleus.
Monitoring dynamic processes and localized environmental variables, including concentrations of
inorganic metallic ions, pH, reactive oxygen or nitrogen species, and membrane potential.
Monitoring cellular integrity (live versus dead and apoptosis), endocytosis, exocytosis, membrane
fluidity, protein trafficking, signal transduction, and enzymatic activity.
Applied to genetic mapping and chromosome analysis in the field of molecular genetics.
Introduction to fluorescent methods
Brief history of fluorescent probes

The first synthetic fluorescent probes were developed in the late

19th century. Many of them were only weakly fluorescent, but some
proved to be highly fluorescent and provided a foundation for the
development of modern synthetic fluorescent probes.
Most notable early fluorescent dyes: are the substituted xanthenes
fluorescein, rhodamine B, and acridine derivative, acridine orange. Acridine orange

Xanthene

Fluorescein Rhodamine
Introduction to fluorescent methods
Brief history of fluorescent probes

Early 20th cent: fluorochromes as vital stains for bacteria, protozoa, and trypanosomes
(used in fluorescence microscopy)

Crystal violet Trypanosome staining with

(used in Gram staining) acridine orange

1920s: fluorescence microscopy was first used to study dye binding in fixed tissues and living cells.

Simultaneous vizualization of F- and G-actins in a Multiple labelling in lung artery endothelial cells:
lung artery endothelial cell F-actin (phalloidine + fluorescein: magenta pseudocolor),
F-actin: phalloidin + Oregon green endomembrane system (DiOC6(3): green),
G-actin: DNase I + Texas red nucleus (DAPI: blue)
Introduction to fluorescent methods
Brief history of fluorescent probes

Early 1940s: the birth of immunofluorescence (Albert Coons) the technique for labeling antibodies with
fluorescent dyes
Since then: development of a wide spectrum of secondary antibodies and design of fluorescent
probes targeted at specific regions within macromolecular complexes.
Introduction to fluorescent methods
Brief history of fluorescent probes

Early 1940s: the birth of immunofluorescence (Albert Coons) the technique for labeling antibodies with
fluorescent dyes
Since then: development of a wide spectrum of secondary antibodies and design of fluorescent
probes targeted at specific regions within macromolecular complexes.

Immunofluorescent analysis of Phalloidin (orange)

and alpha-Tubulin (green) in NIH 3T3 (fibroblast)
cells. Actin was stained with DyLight 550
Phalloidin and nuclei (blue) were stained with
Hoechst. Images were taken on a Zeiss Axio
Observer Z1 microscope at 20X magnification.
Introduction to fluorescent methods
Brief history of fluorescent probes

The discovery of green fluorescent protein (GFP) from jellyfish and the
development of mutant spectral variants opened the door to non-invasive
fluorescence multicolor investigations of subcellular protein localization,
GFP
intermolecular interactions, and trafficking using living cell cultures.
Chromophore
(Ser65–Tyr66–Gly67)

Despite the numerous advances made in fluorescent dye synthesis during the past few decades, there is very little solid
evidence about molecular design rules for developing new fluorochromes, particularly with regard to matching
absorption spectra to available confocal laser excitation wavelengths.
As a result, the number of fluorophores that have found widespread use in confocal microscopy is a limited subset of the
many thousands that have been discovered.
Introduction to fluorescent methods
Common fluorescent probes

Fluorophores must be compatible with available laser (excitation) systems and detectors.
The confocal microscope itself is responsible for emission/signal loss
(pinhole aperture, efficiency of photomultiplier tubes etc.)
It is important to adjust excitation intensity so as not to saturate fluorescent probes.
Current lasers used in confocal microscopy produce discrete lines in the ultraviolet, visible, and near-infrared portions of
the spectrum (10 to 12 specific lines between 400 and 650 nm), but these lines do not always coincide with absorption
maxima of popular fluorophores (this, however, is not necessary, but it decreases fluorescent yield).
Introduction to fluorescent methods
Common fluorescent probes

Fluorophore molecules could be generally classified into four categories: proteins and peptides, small organic compounds,
synthetic oligomers and polymers, and multi-component systems.
The confocal microscope itself is responsible for emission/signal loss
(pinhole aperture, efficiency of photomultiplier tubes etc.)
It is important to adjust excitation intensity so as not to saturate fluorescent probes.
Current lasers used in confocal microscopy produce discrete lines in the ultraviolet, visible, and near-infrared portions of
the spectrum (10 to 12 specific lines between 400 and 650 nm), but these lines do not always coincide with absorption
maxima of popular fluorophores (this, however, is not necessary, but it decreases fluorescent yield).

Chemical family Example

Xanthene derivatives
Cyanine derivatives
Coumarin derivatives
Oxadiazole derivatives
Anthracene derivatives
Pyrene derivatives
Oxazine derivatives
Acridine derivatives
Arylmethine derivatives
Tetrapyrrole derivatives
fluorescein, rhodamine, Oregon green, eosin, and Texas red
cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine
Hydroxycoumarin, Aminocoumarin
pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole
anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange
cascade blue etc.
Nile red, Nile blue, cresyl violet, oxazine 170 etc.
proflavin, acridine orange, acridine yellow etc.
auramine, crystal violet, malachite green
porphin, phthalocyanine, bilirubin
Introduction to fluorescent methods
Common fluorescent probes

Fluorescein and derivatives (e.g. fluorescein isothiocyanate,

FITC)
• Amax = 495 nm (excitable with many commercially
available lasers and xenon-arc lamps)
• High quantum yield
• Fluorescence emission is pH-dependent and wide
(potential interference in multiple labeling experiments)
Tetramethyl rhodamine (TMR) and the isothiocyanate
derivative (TRITC)
• Widely used in multiple labeling experiments in wide-field
microscopy (excitable with mercury-arc lamp)

• Emission less sensitive to environment (compared to

fluorescein)

Acridine dyes (acridine orange): dsDNA (green

fluorescence),
RNA, ssDNA (red fluorescence) binding dye with broad
absorption spectrum

Propidium iodide (closely related to ethidium bromide): Endothelial cells under the microscope. Nuclei are stained blue
DNA binding dye (intercalation, like acridines) with DAPI, microtubules are marked green by an antibody bound
to FITC and actin filaments are labeled red with phalloidin bound
to TRITC.
4',6-diamidino-2-phenylindole (DAPI) and the bisbenzimide
Hoechst dyes: DNA and chromatin staining (bind to minor
groove of DNA) with vivid blue emission. It is a popular
counterstain.
Introduction to fluorescent methods
Common fluorescent probes

Alexa Fluor® dyes (Molecular probes trademark) are good examples

of advances in modern fluorophore technology.
• sulfonated rhodamine derivatives
• exhibit higher quantum yields
(for more intense fluorescence emission)
than spectrally similar probes
• enhanced photostability (resistance to photobleaching)
• absorption spectra matched to common laser lines
• pH insensitivity
• and a high degree of water solubility

Because of the large number of available excitation

and emission wavelengths in the Alexa Fluor® series,
multiple labeling experiments can often be conducted
exclusively with these dyes.
Introduction to fluorescent methods
Common fluorescent probes

Cyanine Dyes® (Molecular Probes)

These probes exhibit fluorescence excitation and
emission profiles that are similar to many of the traditional
dyes, such as fluorescein and tetramethylrhodamine, but
with
• enhanced water solubility
• enhanced photostability
• higher quantum yields
• enhanced environmental stability

The cyanine dyes generally have broader absorption

spectral regions than members of the Alexa Fluor family,
while emission profiles are comparable in spectral width
to the Alexa Fluor series.

Dylight Dyes® (Pierce)

• enhanced water solubility
• enhanced photostability
• higher quantum yields
• enhanced pH stability
Introduction to fluorescent methods
Common fluorescent probes

Fluorescent Environmental Probes are designed to probe the internal environment, including: metals,
inorganic ions, thiols and sulfides, nitrite, ROS, RNS, pH, solvent polarity, and membrane potential.
The binding induces changes in the wavelength and/or intensity of absorption and emission spectra
(ratiometric or intensity dyes).

Drawbacks of intensity-dyes:
• The intensity of fluorescence does not depend
exclusively on the binding of the dye to the ligand
• It is also affected by the concentration of the dye within
the cell and by the interactions between the dye and
other intracellular molecules (e.g., proteins)
Ratiometric dyes eliminate the drawbacks of intensity dyes:
• The dye can be present in two (mutually exclusive) forms
(bound or free).
A ratiometric response to calcium ion fluxes
• The two forms of the dye have different spectral properties, can be obtained when a mixture of fluo-3 and
fura red is excited at 488 nanometers and
as they either absorb or emit at a different wavelength. fluorescence is measured at the emission
The two forms can be probed simultaneously maxima (525 and 650 nanometers, respectively)
of the two probes. Because the emission intensity
• The ratio of two fluorescence intensities is independent of fluo-3 increases monotonically while that of
of the dye concentration and is directly proportional to fura red simultaneously decreases, an isosbestic
the ligand concentration point is obtained when the dye concentrations are
constant within the localized area being
Fluo-3: a commonly used Ca2+-sensitive intensity-dye investigated

Fura-2: Ca2+-sensitive, ratiometric fluorescent probe.

Introduction to fluorescent methods
Common fluorescent probes

Fluorescent Organelle Probes

Fluorophores targeted at specific intracellular organelles, such as the mitochondria, lysosomes, Golgi apparatus,
and endoplasmic reticulum, are useful for monitoring a variety of biological processes in living cells.
In general, organelle probes consist of a fluorochrome
nucleus attached to a target-specific moiety that
assists in localizing the fluorophore through
covalent, electrostatic, hydrophobic or
similar types of bonds.
Many of the fluorescent probes designed for
selecting organelles are able to permeate or
sequester within the cell membrane
(and therefore, are useful in living cells),
while others must be installed using
monoclonal antibodies with
traditional immunocytochemistry techniques.
MitoTracker: mitochondria
LysoSensor: lysosomes
BODIPY fluorophore: Golgi apparatus
ER-Tracker: ER

Fluorescent Proteins: see GFP

Quenching and Photobleaching:
reversible and irreversible loss of fluorescence,
respectively
Introduction to fluorescent methods
Techniques for the analysis of protein kinetics in living cells
Fluorescence recovery after photobleaching (FRAP):
The fluorescent signal (blue circle) is bleached in a small
intracellular area and the recovery is measured inside this
region as a function of time.

Using this technique, diffusion coefficients (D) and the

proportion of molecules that are mobile can be measured.

Red curve: 100% of molecules are diffusing

(D = 0.5 m2 s-1); green curve: 80% of molecules are
diffusing (D = 0.2 m2 s-1) and 20% are immobile.

FRAP can also be used to quantify two dimensional

lateral diffusion in membranes
Introduction to fluorescent methods
Techniques for the analysis of protein kinetics in living cells

Fluorescence loss in photobleaching (FLIP):

A small area (blue circle) is repeatedly photobleached
and the loss in fluorescence intensity is recorded
as a function of time in another region of the cell.
FLIP provides measurements of the number of
populations of a particular molecule and their
relative proportion:
Green curve: a molecule that is present as
a single population.
Red curve: two different populations of a molecule
are present, each of which has a different rate constant.

Inverse-FRAP (i-FRAP):
The total fluorescent signal in a cell is bleached (blue circle) except for one area in which the
signal is recorded as a function of time.
This technique is useful for studying small organelles as it gives a direct readout of the
residency time of different factors.
One of the main limitations of this approach lies in the time that is needed to photobleach the
total fluorescent signal that is present in a cell; this makes this technique unsuitable for
detecting fast translocations as the signal is lost during the bleaching phase.
Red curve: the fluorescent molecule has a half residency time of 15 seconds in the
compartment that is being studied.
Introduction to fluorescent methods
Techniques for the analysis of protein kinetics in living cells

Photoactivation:
A photoactivable version of green fluorescent protein
(GFP), which has a decreased absorbance at 488 nm
and therefore does not behave as wild-type GFP, is
activated in a cell (purple circle) and fluorescence is
recorded at the activation site.
Although similar to i-FRAP, the advantage of
photoactivation is that it requires less energy than
bleaching.
Sub-populations that move rapidly and have a short
residence time can be detected, whereas in i-FRAP
they are bleached and undetectable.
Photoactivation allows for the spatial detection of the
successive translocations of proteins in a cell, and
therefore serves as a visual, pulse-chase, real-time
view of protein dynamics.
Red curve: the fluorescent molecule has a half
residency time of 15 seconds in the activated
compartment 1.
Green curve: the molecules translocate to compartment
2 after leaving compartment 1.
Introduction to fluorescent methods
The interaction of light and matter

https://www.youtube.com/watch?v=dkARLSQWHH8
 Thank You
for your attention!