Molecular Biology of the Cell: Questions for the Oral Exam
1) GFP/Green fluorescent protein
GFP consists of 238 amino acids that form 11 β-strands in a barrel formation. In the middle of the barrel, 3
amino acids form a chromophore.
GFP is isolated from a jellyfish. It was
• discovered in 1962
• cloned in 1992
• brought to expression in 1994
GFP can be used for targeting as a fluorescent probe. Probes = molecules targeting something specific. There
are different ways of targeting:
• GFP fused with protein of interest.
• Direct targeting => used on proteins, nucleic acids, ions, and Specific organelles.
• Immunofluorescence = Fluorescent molecule conjugated to antibody => used on primary antibody
and secondary antibody (secondary antibody is for signal amplification).
• Fluorescent molecule conjugated to DNA.
Fluorescent probes are not dyes. Dyes are used for subcellular labeling during sample preparation, and there
are different types:
• Dyes with an affinity to specific sub-cellular components => hematoxylin used for DNA, RNA, and
acidic proteins, because it has an affinity for negative charge.
• Nucleic acid labels => used in hybridization situations of mRNA and for gene expression.
• Fluorescent hybridization => used on chromosomes for chromosome study.
• Fluorescent antibodies => used for the localization of specific molecules/organelles.
GFP can also be used in live-cell imaging and FRAP.
Live-cell imaging:
= study of living cells by using time-lapse microscopy. It allows to obtain a better understanding of biological
functions by observing cellular dynamics in real time.
This requires a high-resolution visualization across both space and time within the organism. GFP tagging is
the best way of showing the distribution and dynamics of a protein in a living organism.
For live imaging in Drosophila => a GFP gene was joined to a fly neuron subset promoter, and this gene is
only active in a specialized set of neurons.
,2) FRET & FRAP
These 2 techniques use fluorescence.
FRET/Fluorescence Resonance Energy Transfer:
FRET is a mechanism that describes the energy transfer between 2 chromophores.
Chromophores = light-sensitive molecules that absorb light, and after the excitation they will emit a photon
at a longer wavelength (longer wavelength = less energy).
A fluorochrome is a chromophore capable of emitting a fluorescent light when excited.
In this technique, 2 molecules of interest are labeled with different
fluorochromes. Each fluorochrome is chosen so that the emission
spectrum of one, overlaps the absorption spectra of the other. If the
2 proteins interact closer than 5nm, the excitation of 1
fluorochrome results in the emission of the other fluorochrome.
FRET is one of the 3 techniques to uncover a cell's kinetic properties
and determine its interactions. This technique is often used to study
interactions between:
• Protein
• Signaling molecule and its receptor
• Macromolecular complex
FRAP/Fluorescence Recovery After Photobleaching
FRAP is another way of using GFP fused to a protein of interest. It is a technique
used in fluorescence microscopy to measure the rate of molecular diffusion.
This technique can also be applied in the field of live imaging.
How it works:
1. A small region of the cell is illuminated with a high-intensity laser beam
2. This high-intensity laser beam will bleach the fluorescent molecules in
that region.
3. Fluorescence recovery = unbleached fluorescent molecules from the
surrounding areas diffuse into the bleached region.
The rate of fluorescence recovery is related to the rate of molecular diffusion,
this provides info about the mobility of molecules and their kinetic parameters
within the cell.
This technique can deliver valuable data about:
• Diffusion coefficient
• Active transport rate
• Binding/dissociation rates
, 3) How to eliminate out-of-focus fluorescence in microscopy & how expansion microscopy works
Out-of-focus fluorescence = Light emitted by fluorophores that aren’t in the plane of focus. This out-of-focus
light create blurry backgrounds, which makes it harder to identify structures of interest.
In a fluorescence microscope, a beam of light illuminates the whole field of view, and this excites all the
fluorophores. The rays from the in-focus fluorophore are focused in the primary image plane, producing a
sharp image of the fluorophore. The unfocused rays from the out-of-focus fluorophore produce a blurry
background.
There are 2 techniques to eliminate out-of-focus fluorescence:
• Confocal microscopy => Eliminates the out-of-focus light, which allows us to see the in-focus objects
clearly.
• Image deconvolution => Computational method that distinguishes the in- and out-of-focus light, and
removes the out-of-focus light from the image.
Optical microscope + image deconvolution => More sensitive cameras, better for weak staining, better for
sensitive to light specimens, and has a max depth of 40µm.
Confocal microscopy:
The results of confocal microscopy are superior to
those obtained by ‘classical’ light microscopy. It is
better to use it for thick specimens and lots of out-of-
focus light, and it's faster and easier to use.
In confocal microscopy, the specimen is stained with a
fluorochrome. This microscopy uses a focused beam of
light, to illuminate 1 small point of the specimen at a
time. The beam of light passes through a pinhole. This
pinhole allows only the in-focus light to pass, while the
out-of-focus light is blocked. This reduces background
noise.
It does optical sectioning = capturing multiple 2D
images at different depths to reconstruct the 3D structure of the specimen
There are also multiphoton confocal microscopes, which can obtain sharp images at a depth of 0,5mm within
a specimen. This is valuable for studies of
• living tissues
• the dynamic activity of synapses and neurons just below the surface of living brains
Multiphoton confocal microscopes to take advantage of the ‘2-photon’ effect => Fluorescent molecules are
usually excited by the absorption of 1 single high-energy photon, but they can also be excited by the
absorption of 2 or more lower energy photons, as long as they all arrive within a femtosecond of each other.
Lower energy = longer wavelength, and this longer-wavelength excitation has some important advantages
such as reducing background noise, and helping with the deeper penetration of red or near-infrared light in a
specimen.
1) GFP/Green fluorescent protein
GFP consists of 238 amino acids that form 11 β-strands in a barrel formation. In the middle of the barrel, 3
amino acids form a chromophore.
GFP is isolated from a jellyfish. It was
• discovered in 1962
• cloned in 1992
• brought to expression in 1994
GFP can be used for targeting as a fluorescent probe. Probes = molecules targeting something specific. There
are different ways of targeting:
• GFP fused with protein of interest.
• Direct targeting => used on proteins, nucleic acids, ions, and Specific organelles.
• Immunofluorescence = Fluorescent molecule conjugated to antibody => used on primary antibody
and secondary antibody (secondary antibody is for signal amplification).
• Fluorescent molecule conjugated to DNA.
Fluorescent probes are not dyes. Dyes are used for subcellular labeling during sample preparation, and there
are different types:
• Dyes with an affinity to specific sub-cellular components => hematoxylin used for DNA, RNA, and
acidic proteins, because it has an affinity for negative charge.
• Nucleic acid labels => used in hybridization situations of mRNA and for gene expression.
• Fluorescent hybridization => used on chromosomes for chromosome study.
• Fluorescent antibodies => used for the localization of specific molecules/organelles.
GFP can also be used in live-cell imaging and FRAP.
Live-cell imaging:
= study of living cells by using time-lapse microscopy. It allows to obtain a better understanding of biological
functions by observing cellular dynamics in real time.
This requires a high-resolution visualization across both space and time within the organism. GFP tagging is
the best way of showing the distribution and dynamics of a protein in a living organism.
For live imaging in Drosophila => a GFP gene was joined to a fly neuron subset promoter, and this gene is
only active in a specialized set of neurons.
,2) FRET & FRAP
These 2 techniques use fluorescence.
FRET/Fluorescence Resonance Energy Transfer:
FRET is a mechanism that describes the energy transfer between 2 chromophores.
Chromophores = light-sensitive molecules that absorb light, and after the excitation they will emit a photon
at a longer wavelength (longer wavelength = less energy).
A fluorochrome is a chromophore capable of emitting a fluorescent light when excited.
In this technique, 2 molecules of interest are labeled with different
fluorochromes. Each fluorochrome is chosen so that the emission
spectrum of one, overlaps the absorption spectra of the other. If the
2 proteins interact closer than 5nm, the excitation of 1
fluorochrome results in the emission of the other fluorochrome.
FRET is one of the 3 techniques to uncover a cell's kinetic properties
and determine its interactions. This technique is often used to study
interactions between:
• Protein
• Signaling molecule and its receptor
• Macromolecular complex
FRAP/Fluorescence Recovery After Photobleaching
FRAP is another way of using GFP fused to a protein of interest. It is a technique
used in fluorescence microscopy to measure the rate of molecular diffusion.
This technique can also be applied in the field of live imaging.
How it works:
1. A small region of the cell is illuminated with a high-intensity laser beam
2. This high-intensity laser beam will bleach the fluorescent molecules in
that region.
3. Fluorescence recovery = unbleached fluorescent molecules from the
surrounding areas diffuse into the bleached region.
The rate of fluorescence recovery is related to the rate of molecular diffusion,
this provides info about the mobility of molecules and their kinetic parameters
within the cell.
This technique can deliver valuable data about:
• Diffusion coefficient
• Active transport rate
• Binding/dissociation rates
, 3) How to eliminate out-of-focus fluorescence in microscopy & how expansion microscopy works
Out-of-focus fluorescence = Light emitted by fluorophores that aren’t in the plane of focus. This out-of-focus
light create blurry backgrounds, which makes it harder to identify structures of interest.
In a fluorescence microscope, a beam of light illuminates the whole field of view, and this excites all the
fluorophores. The rays from the in-focus fluorophore are focused in the primary image plane, producing a
sharp image of the fluorophore. The unfocused rays from the out-of-focus fluorophore produce a blurry
background.
There are 2 techniques to eliminate out-of-focus fluorescence:
• Confocal microscopy => Eliminates the out-of-focus light, which allows us to see the in-focus objects
clearly.
• Image deconvolution => Computational method that distinguishes the in- and out-of-focus light, and
removes the out-of-focus light from the image.
Optical microscope + image deconvolution => More sensitive cameras, better for weak staining, better for
sensitive to light specimens, and has a max depth of 40µm.
Confocal microscopy:
The results of confocal microscopy are superior to
those obtained by ‘classical’ light microscopy. It is
better to use it for thick specimens and lots of out-of-
focus light, and it's faster and easier to use.
In confocal microscopy, the specimen is stained with a
fluorochrome. This microscopy uses a focused beam of
light, to illuminate 1 small point of the specimen at a
time. The beam of light passes through a pinhole. This
pinhole allows only the in-focus light to pass, while the
out-of-focus light is blocked. This reduces background
noise.
It does optical sectioning = capturing multiple 2D
images at different depths to reconstruct the 3D structure of the specimen
There are also multiphoton confocal microscopes, which can obtain sharp images at a depth of 0,5mm within
a specimen. This is valuable for studies of
• living tissues
• the dynamic activity of synapses and neurons just below the surface of living brains
Multiphoton confocal microscopes to take advantage of the ‘2-photon’ effect => Fluorescent molecules are
usually excited by the absorption of 1 single high-energy photon, but they can also be excited by the
absorption of 2 or more lower energy photons, as long as they all arrive within a femtosecond of each other.
Lower energy = longer wavelength, and this longer-wavelength excitation has some important advantages
such as reducing background noise, and helping with the deeper penetration of red or near-infrared light in a
specimen.
