Fluorescence Flashcards
What does FACS stand for?
Fluorescence-Activated Cell Sorter
What does FLIM stand for?
Fluorescence Live Imaging Microscopy
What does FRAP stand for?
Fluorescence Recovery After Photobleaching
What does FIONA stand for?
Fluorescence wIth One Nanometer Accuracy
What does TIRF stand for?
Total Internal Reflection Fluorescence microscopy
What is fluorescence?
Fluorescence is the radiative return (photon emission) of an excited electronic state (singlet) to the ground electronic state.
It is the emission of light from an atom or molecule and occurs from electronically excited states.
What is a fluorophore?
A fluorophore is a substance capable of displaying fluorescence
Give some applications of fluorescence in biological research
Protein-protein and protein-ligand interactions
Intracellular Ca2+ (Ca2+ spikes and sparks)
DNA sequencing and gene expression
Enzymatic assays
Molecular organisation: FRET as a molecular ruler
All the other microscopies
(FACS, FLIM, FRAP, FIONA, TIRF)
Why are fluorescent probes commonly used to detect rapid biochemical changes in single living cells?
1) they can be designed to give an essentially instantaneous report (within nanoseconds) on the changes in intracellular conc. of a second messenger or in the activity of a protein kinase
2) fluorescence microscopy has sufficient resolution to reveal where in the cell such changes are occurring
Which organism is the naturally occurring fluorescent protein GFP derived from?
Aequorea victoria
How does GFP fluoresce? Which of its residues are involved in this fluorescence?
When excited by the absorption of a photon of light, it emits a photon of light (fluoresces) in the green region of the spectrum.
The light-absorbing/emitting center of GFP (its chromophore) comprises an oxidised form of the tripeptide -Ser65-Tyr66-Gly67-
Which residues constitute the chromophore in GFP?
An oxidised form of the tripeptide:
Ser65-Tyr66-Gly67 (SYG)
What catalyses oxidation of the tripeptide making up the GFP chromophore? Why is this important?
GFP itself. This means it can be cloned into virtually any cell where it can serve as a fluorescent marker for any protein to which it is fused.
How are the variants of GFP (YFP, BFP and CFP) produced? give an example.
Genetic engineering:
YFP- Ala206 is replaced with a Lysine
An excited fluorescent molecule such as GFP or YFP can dispose of the energy absorbed from a photon in one of two ways. What are they?
1) fluorescence - emitting a photon of slightly longer wavelength
2) nonradiative FRET (fluorescence resonance energy transfer) in which the energy of the excited molecule (donor) passes directly to a nearby molecule (acceptor) WITHOUT EMISSION OF A PHOTON, exciting the acceptor
What is FRET? How does it work?
Fluorescence resonance energy transfer.
A fluorescent molecule (eg GFP/YFP) absorbs a photon of light and becomes excited. The energy of the excited molecule (DONOR) passes directly to a nearby molecule (ACCEPTOR) without emission of a photon, exciting the acceptor. The acceptor can now decay to its ground state by fluorescence; the emitted photon has a longer wavelength (lower energy) than both the original exciting light and the fluorescence emission of the donor.
When can FRET occur?
When the donor and acceptor molecules are close: within 1 to 50 amstrongs
How close must the donor and acceptor molecules be for FRET to occur?
between 1 and 50 amstrongs
How does the efficiency of FRET relate to the distance between the donor and acceptor molecules?
It is INVERSELY PROPORTIONAL to the SIXTH POWER of the distance between donor and acceptor.
The efficiency of FRET is inversely proportional to the sixth power of the distance between donor and acceptor. What is the significance of this?
Very small changes in the distance between donor and acceptor register as very large changes in FRET, measured as the fluorescence of the acceptor molecule when the donor is excited. With sufficiently sensitive light detectors, this fluorescence signal can be located to specific regions of a single, living cell.
How has FRET been used to measure [cAMP] in living cells?
The gene for GFP is fused with that for the regulatory subunit (R) of cAMP-dependent protein kinase (PKA), and the gene for BFP is fused with that for the catalytic subunit (C).
When these two hybrid proteins are expressed in a cell, BFP and GFP in the inactive PKA (R2C2 tetramer) are close enough to undergo FRET.
Wherever in the cell [cAMP] increases, the R2C2 complex dissociated into R2 and 2 x C and the FRET signal is lost, because the donor and acceptor are now too far apart for efficient FRET.
Viewed in the fluorescence microscope, the region of higher [cAMP] has a minimal GFP signal and higher BFP signal. Measuring the ratio of the emission from each gives a sensitive measure of the chance in [cAMP]. By determining this ratio for all regions of the cell, the investigator can generate a false colour image of the cell in which ratio (relative [cAMP]) is represented by the intensity of colour.
When examining [cAMP] using FRET, which fluorescent proteins are fused to which protein?
GFP - fused to the regulatory subunits of PKA (R)
BFP - fused to the catalytic subunits of PKA (C)
When measuring [cAMP] using FRET, which fluorescent protein is used as a donor, and which as an acceptor? What is the excitation and emission wavelengths of each?
BFP is used as the donor (fused to the CATALYTIC subunit of PKA). Excitation at 380 nm, emission at 460 nm.
GFP is the acceptor (fused to the REGULATORY subunit of PKA).
Excitation at 475 nm, emission at 545 nm.
What are the excitation and emission frequencies for BFP?
380nm
460nm
What are the excitation and emission frequencies of GFP?
475 nm
545 nm
How is [cAMP] detected by FRET in cells?
Labelling PKA with BFP (fused to C subunits) and GFP (fused to R subunits).
Excite BFP with 380 nm photon light. If [cAMP] high, it will bind to the R subunits and cause the PKA tetramer to dissociate into R2, and 2 x C. Therefore FRET won’t occur between BFP and GFP, and so there will be a higher BFP signal and a minimal GFP signal. In regions lacking cAMP, PKA will be in its tetrameric form and FRET will occur as the regulatory and catalytic subunits will be between 1 and 50 angstroms from each other
Do shorter or longer wavelengths have higher energy?
Shorter
What is the approximate range of wavelengths in the visible light part of the spectrum?
350 nm to 750 nm
Which of the following techniques involves the shortest (therefore most energetic) wavelengths?
X-ray crystallography
Absorption/ fluorescence spectroscopy
NMR spectroscopy
X-ray crystallography (0.01nm to 10nm)
What happens when a molecule absorb a photon of light?
Transition (electrons gain energy and move from ground state to excited state)
What is the fundamental spectroscopy selection rule (as depicted in the Jablonski diagram)?
The wavelength of light absorbed (or emitted) must have an energy that matches an energy gap of energy states in a molecule.
Give three properties of the excited state
1) reactive: proton transfer, excimer formation, photobleaching
2) directional: fluorophores preferentially absorb photons whose electric vectors are parallel to their transition moment (a property exploited in polarisation measurements)
3) exchange (or transfer) with the excited state of another molecule: the excitation energy can be transferred to another molecule
What is meant by ‘singlet state’ and ‘triplet state’ with respect to electrons?
Singlet state - all electrons are spin-paired
Triplet state - one set of electrons is NOT spin-paired
How does the lifetime of triplet states compare to that of singlet states? How does this affect phosphorescence?
Lifetimes of excited triplet states are much longer. Thus phosphorescence is quite rare since internal conversion and other processes provide competing non-radiative mechanisms that lead to the release of energy
With regard to fluorescence, give three processes involving photons and three radiationless transitions
Photons: 1) absorption, 2) fluorescence, 3) phosphorescence
Radiationless: 1) vibrational relaxation, 2) intersystem crossing, 3) internal conversion
All fluorescence measurement done in laboratories will fall into one of which 6 categories?
1) emission spectrum
2) excitation spectrum
3) quantum yield and quenching
4) FRET
5) polarisation (anisotropy)
6) lifetime measurement
How is a fluorescence emission spectrum collected?
The excitation wavelength is fixed and the intensity of the emission is measured at different wavelengths
Early examination of a large number of emission spectra resulted in the formulation of 3 general rules that govern fluorescence. What are these?
1) the fluorescence spectrum is invariant and independent of the excitation wavelength
2) the fluorescence spectrum lies at longer wavelengths than the excitation (Stokes shift)
3) the fluorescence spectrum is approximately a mirror image of the excitation spectrum
What is shown on an emission spectrum if a fluorescent molecule is excited with 2 different wavelengths?
The SHAPE of the emission spectra is the same; the amplitude is determined by the intensity and is thus different
What is Stokes shift?
The fluorescence (emission) spectrum lies at longer wavelengths than the excitation
Which two observations explain the Stokes shift?
1) fluorescence emission mainly occurs from the lowest vibrational level of the first excited electronic state (E1) and may reach any vibrational level of the electronic ground state (E0)
2) in the ground state fluorophores exist predominantly in the lowest vibrational level
“the fluorescence spectrum is approximately a mirror image of the excitation spectrum” …what is the explanation for this?
1) absorption is always from the lowest vibrational level in the ground state
2) emission is always from the lowest vibrational level in the excited state
3) the spacings of the energy levels in the vibrational manifolds of the ground state and the first excited states are usually similar
Why is the fluorescence spectrum invariant and independent of the excitation wavelength?
Because emission takes place from the lowest vibrational level of the first excited electronic state
How is a fluorescence excitation spectrum collected? What are the: a) intensity, and b) wavelength related to?
The emission wavelength is fixed and the intensity of excitation is followed at different wavelengths.
Intensity is related to the PROBABILITY of the event
Wavelength is related to the ENERGY of the light absorbed
Is there competition between the fluorescence process and other nonradiative processes that leave the excited state?
Yes
Quantum yield = ?
no. photons emitted / no. photons absorbed
or Kf / Kf + Knr, where Kf = rate constant for fluorescence decay and Knr = rate constant for radiationless decay
What is the term for the quantity of energy contained in a photon?
Quantum
What is fluorescence? (re emission?)
Light emission accompanying decay of excited molecules
What happens to a molecule’s electrons (chromophores) when a photon is absorbed?
The electron is lifted to a higher energy level
Decay of excited molecules may not always result in light emission called fluorescence. Give two forms of radiationless decay that may occur.
Internal conversion
Intersystem crossing
If a molecule has a quantum yield of 0, what does this mean?
The molecule is non fluorescent
What is quenching?
A process that leads to the reduction of fluorescence intensity (or the quantum yield of the fluorophore)
What type of loss of energy is quenching a result of?
Non-radiative (as opposed to fluorescence)
Fluorescence quenching can take place via two distinct processes. What are these?
1) collisional/ dynamic quenching
2) static quenching
What is meant by collisional/dynamic quenching? (also known as Dexter electron transfer) Give an example of a process involving dynamic quenching.
Quenching that results from the interaction of the quenching molecule with the fluorophore in the excited state.
FRET is a dynamic quenching mechanism as energy transfer occurs while the donor is in the excited state.
What is static quenching?
Quenching that results from the interaction of the quenching molecule with the fluorophore in the ground state.
When were molecular beacons invented?
1996 (by Tyagi and Kramer)
What do molecular beacons consist of?
They are specifically designed DNA hairpin structures (ssDNA) with:
1) an internal complementary sequence (STEM)
2) a LOOP that anneals to the target
3) a FLUOROPHORE at one end of the DNA sequence
4) a QUENCHER at the other end of the DNA sequence
Give 5 applications of molecular beacons?
1) genetic screening
2) biosensor development
3) biochip construction
4) detection of single-nucleotide polymorphisms
5) mRNA monitoring in living cells
What conformation do molecular beacons conform to in the absence of a target sequence? How does this affect the fluorescence?
Stem-loop structure. This holds the fluorophore and quencher in close proximity. As a result, the fluorescence emission of the fluorophore is strongly suppressed.
What happens when a molecular beacon is in the presence of the target sequence?
The target sequences hybridises with the LOOP domain of the MB and forces the stem helix to open and form a hybrid helix which is more stable than the stem helix, whereupon fluorescence is restored because of the spatial separation of the fluorophore from the quencher
Many nucleic acid probes employ FRET as their signal-transduction mechanisms. Give some examples of such probes.
Adjacent probes
TaqMan probes
What does static quenching involve?
The fluorescent and quenching moieties are brought into close proximity (1-50A) by the stem helix, and most of the energy absorbed is dissipated as heat- only a small amount of energy is emitted as light
MBs use different energy-transfer mechanisms for signal transduction. What are the two major categories?
Dynamic quenching
Static quenching
What does dynamic quenching involve? What do the energy-transfer rates depend upon?
Forster transfer (RET or FRET) and Dexter transfer (collisional/ electron-transfer quenching). This occurs without the release of a photon and is the result of long-range dipole-dipole interactions between the donor and the acceptor.
The energy-transfer rates depend upon the extent of spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor, and the distance between the donor and acceptor.
In dynamic quenching, what 4 factors does the rate of energy-transfer depend on?
1) extent of spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor
2) the quantum yield of the donor
3) the relative orientation of the donor and acceptor
4) the distance between the donor and acceptor
What is the Forster distance?
The distance at which RET occurs with 50% efficiency (typically in the range of 20-50A)
What does static quenching require?
The formation of ground-state complexes
Single nucleotide polymorphisms (SNPs) make up about 90% of human genetic variation, and are regarded as potent molecular genetic markers. Why are molecular beacons (MBs) useful for their detection?
Their inherent signaling mechanism by energy transfer and their high selectivity allow them to simply, rapidly and sensitively detect SNPs
HOW can MBs be used to detect SNPs?
During PCR, MBs with sequences complementary to those of the wild type and variant alleles respectively can be introduced to a homogeneous solution.
How do MBs compare to other FRET-based homogeneous hybridisation methods for SNP scoring (eg TaqMan)?
They provide more reliable genotyping results as well as more flexible fluorescence detection for multiplexes analyses
…homogeneous and simultaneous signal amplification and target detection - MB-based SNP assays suitable for high-throughput genotyping studies
What is FRET?
A process by which excitation energy is transferred from one chromophore (the DONOR) to another chromophore (the ACCEPTOR)
Give three features of FRET.
1) FRET is NOT emission and re-absorption of a photon
2) energy is transferred by dipolar coupling before any fluorescence is emitted
3) fluorescence resonance energy transfer is therefore a misnomer (but is in popular usage)
What three parameters does FRET depend on?
1) distance between the donor and acceptor molecules
2) extent of overlap between the absorption spectrum of the acceptor and the emission spectrum of the donor
3) the orientation of the dipoles of the donor and acceptor molecules
Why has FRET been called a ‘spectroscopic ruler’?
Because it only occurs between molecules that are between 1-50 amstrongs (or 20-80, according to diff source)
How is energy transferred in FRET?
Dipole-dipole interaction
How can you measure FRET efficiency (E)?
1) from Donor quenching
- fluorescence lifetime is shortened in the presence of the acceptor - often a more reliable measure of FRET
2) from sensitised emission from the acceptor
- compare acceptor emission via FRET with direct excitation - but fluorescence needs to be corrected for relative absorption of A and D as well as instrument sensitivity
What does the Forster equation link?
Efficiency of energy transfer to the donor-acceptor distance
What does R0 in the Forster equation stand for?
The critical Forster distance, i.e. the distance at which the energy transfer efficiency is 50%
What is the Forster equation?
E = 1/1+(R/R0)^6
What is an assumption of the Forster equation?
That the fluorophores are rapidly tumbling so that orientational effects are averaged out - therefore calculations are NOT ALWAYS VALID
Give the R0 values (ie critical Forster distance/ distance at which energy transfer efficiency is 50%) for the following Donor:Acceptor pairs:
1) Tyrosine:Tryptophan
2) Tryptophan:ANS
3) BFP:GFP
4) Fluorescein:Rhodamine
1) 1.5 nm (15 A)
2) 2.2 nm (22 A)
3) 4.1 nm (41 A)
4) 4.5 nm (45 A)
Why might calculations based on the Forster equation not be valid?
Because they assume that the fluorophores are rapidly tumbling so that orientational effects are averaged out
Give some applications of FRET
1) a signal to quantify interactions (binding, kinetics)
2) dimerisation/oligomerisation/assembly
3) conformational change (distance measurement: a molecular ruler)
4) surface topography of membrane protein
5) protein folding
Give an example of how FRET can be used to observe conformational change.
Calcium-induced movement of tropomyosin in skeletal muscle thin filaments was observed by multi-site FRET
Give an example of how FRET can be used as a signal to quantify interactions
Detection of oestrogen - YFP attached to LBD; CFP attached to LXXLL motif. When LBD and LXXLL motif interact, fluorescence transferred from CFP (emission = 480 nm) to YFP (excitation = 440 nm)
FRET can be used to study the surface topography of a membrane protein… why it is better than some other techniques?
This is difficult to study by NMR or crystallography. Embedded part may not be particularly important to understand the mechanism. Understanding conformational change on the surface is possible to monitor by FRET.
Give an example of how FRET can be used to study the surface topography of a membrane protein
Studying the conformation of an apolipoprotein when bound to lipids.
e.g. ApoA-I is one of the most effective activators of lecithin:cholesterol acyltransferase (LCAT). This interaction leads to the formation of a discoidal form of high density lipoprotein (HDL). In late 1990s there was a debate whether the proteins bind as a belt or a picket fence.
Determining distances with FRET/ distance changes caveats
1) angular dependence
2) environment dependence
3) distance is an average
4) probe is large, linkages can be long
5) construct complications
- free fluorophores
- donors without partner acceptors
- acceptors without partner donors
Light can be considered as oscillations of an electromagnetic field. What is it characterised by?
Electric and magnetic components - perpendicular to the direction of light propogation
What is the difference between polarised and natural light?
In natural light, the electric field vector can assume any direction of oscillation perpendicular or normal to the light propogation direction. Once passed through a ‘polariser’, light is polarised - i.e. the vibrations occur in a single plane
What is a polariser?
Optically active device that can isolate one direction of the electric vector
What are the two most common types of polarisers used today?
1) Dichroic devices - absorb one plane of polarisation
2) Double refracting calcite crystal polarisers - differentially disperse the two planes of polarisation
Give examples of the following types of polarisers:
1) dichroic devices
2) double refracting calcite
1) Polaroid type-H sheets based on stretched polyvinyl alcohol impregnated with iodine
2) Nicol polarisers, Wollaston prisms, Glan-type polarisers
Polarisation (anisotropy) is another parameter commonly used in the biochemical application of fluorescence. What does anisotropy measurement provide information on?
The size and shape of proteins and on the molecular environment
What are anisotropy measurements based on?
The photoselective excitation of fluorophores by polarised light
RE anisotropy/polarisation: What is the value of I1 is molecules are in a crystal and dipoles are oriented in same plane as excitation light (no rotation)?
I1 = 0 Polarisation = Anisotropy = 1
RE anisotropy/polarisation: What is the value of I1 if molecules are free in solution and tumbling rapidly?
I1 = I2
Polarisation = Anisotropy = 0
What is the value of polarisation and anisotropy if molecules are randomly distributed in solution but static?
Polarisation = 0.5; Anisotropy = 0.4
Give an example of how anisotropy can be used to measure interactions of proteins
Transcriptional-activating protein AreA binding to fluorescently-labelled DNA
Give an example of the application of polarisation in clinical assays
FPIA - fluorescence polarisation ImmunoAssay
What is the basic principle of a polarisation immunoassay (FPIA), e.g. the Abbott TDx?
1) add a fluorescent analog of a target molecule - e.g., a drug - to a solution containing antibody to the target molecule
2) measure the fluorescence polarisation, which corresponds to the fluorophore bound to the antibody
3) add the appropriate biological fluid, e.g., blood, urine, etc., and measure the decrease in polarisation as the target molecules in the sample fluid bind to the antibodies, displacing the fluorescent analogs
Polarised fluorescence depletion can be used to measure what aspect of protein domains? What equation describes this?
The ROTATIONAL MOTION of protein domains
…Stokes-Einstein equation
What is meant by the ‘lifetime’ of a fluorophore?
The fluorescence lifetime is the time delay between the absorption of a photon and the emission of another photon by a fluorescent molecule. i.e., it is the mean period for which an electron stays in the excited state before emitting a photon
Knowledge of a fluorophore’s excited state lifetime is crucial for what?
Quantitative interpretations of numerous fluorescence measurements such as quenching, polarisation and FRET
The lifetime and quantum yield for a fluorophore is often dramatically affected by what? Give an example.
Its environment.
e.g., NADH, which in water has a lifetime of ~0.4 ns, can have a lifetime as long as 9 ns when bound to dehydrogenases
Give 4 examples of how a molecule’s environment can affect its fluorescence lifetime.
1) NADH in water has lifetime 0.4 ns, but up to 9 ns bound to dehydrogenases
2) ANS in water has lifetime 100 ps but can be 8-10 ns bound to proteins
3) Ethidium bromide is 1.8 ns in water, 22 ns bound to DNA, 27 ns bound to tRNA
4) tryptophan in proteins has a lifetime ranging from 0.1 ns up to 8 ns
Give two advantages of time-resolved measurements
1) they contain more info than steady state data (e.g., can resolve contribution of two diff amino acids in a protein more readily in binding site of substrate)
2) fluorescence cell imaging is better done using lifetime because it does not depend on intensity (conc.) unlike steady state methods
What is fluorescence anisotropy?
The phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarisation
What are the two main ways of measuring fluorescence lifetime?
1) The intensity (‘direct’) method (TIME DOMAIN)
2) Phase modulation (‘harmonic’) method (FREQUENCY DOMAIN)
How does the TIME DOMAIN (‘direct’/’intensity’) method for measuring the fluorescence lifetime work?
The sample is illuminated with a short pulse of light and the intensity of the emission versus time is recorded. The decay of intensity with time is an exponential process
How does the FREQUENCY domain (‘phase modulation’/’harmonic’) method for measuring the fluorescence lifetime work?
A CONTINUOUS light source is utilised (eg laser/xenon arc) and the intensity of this light source is modulated sinusoidally at high frequency
Give 6 advantages of fluorescence
1) very sensitive
2) provides good info on molecular environment (eg pH, polarity..)
3) good time resolution (ns)
4) flexible: can be used both as a ‘clock’ (lifetime), and a ‘ruler’ (FRET)
5) multidimensional info (possibility of combining multiple probes)
6) multimodal (not only spectroscopy, but also readily combined with microscopy)
Give 3 disadvantages of fluorescence
1) usually requires a fluorescent label
2) excitation light can be damaging (photobleaching)
3) spectroscopic information poor in comparison to NMR, IR…
How does the sensitivity of fluorescence spectroscopy compare to that of absorption spectroscopy?
Fluorescence spectroscopy can detect compounds at nM concentrations (PROVIDED QUANTUM YIELD IS HIGH), whilst absorption spectroscopy detects compounds at only uM concentrations.
Fluorescence is generally more sensitive to macromolecule structure, dynamics and interactions with other molecules than is absorption
The Beer-Lambert law is: A = Log (Ir/Is). For each of the following values of A, and with 1000 photons entering the sample, how many photons leave and are detected? Hence what is the useful range of absorption?
1) A = 0.01
2) A = 0.1
3) A = 1
4) A = 2
5) A = 3
1) 975
2) 800
3) 100
4) 10
5) 1
…useful range of absorption = 0.05-1 OD units
Fluorescent light observed is against a low background (better sensitivity). Why is this?
Because fluorescence wavelength is different from excitation wavelength
Fluorescence is generally much more sensitive to macromolecules structure, dynamics, and interactions with other molecules than is absorption. Why is this?
The lifetime of the excited (singlet) state (1-10ns) is often comparable to the timescale of many processes that occur in proteins, such as protonation/deprotonation, local conformational dynamics, and overall rotation and translation (in contraast, absorption occurs on timescales in the order to 10^-15 sec; hence the molecule is essentially fixed in the course of this spectroscopic measurement)
Give 6 properties fluorescence can measure
1) solvent polarity
2) solvent viscosity
3) pH
4) ions
5) temperature
6) electric field
Factors such as solvent polarity & viscosity, pH, electric field, temp and ions can affect a number of propertied of fluorescence. What are these properties?
Emission wavelength Structural details of spectrum Quantum yield Anisotropy Lifetime
How is fluorescence sensitive to solvent polarity and viscosity?
The excited state has stronger polar interaction with the solvent …and the rate of solvent relaxation can be measured
What happens to the emission spectra of DOS as the solvent polarity is increased from cyclohexane to butanol?
The emission spectra shift dramatically to longer wavelengths as the polarity is increased
Give an example of how fluorescence can be used as a pH sensor
Fluroscein was used in a major ground-water tracing experiment in southern Germany
Give an example of a solvent-sensitive fluorophore
DOS
Give some examples of some fluorescent pH sensors
Fluoroscein
HPTS, a wavelenght-ratiometric pH sensor
SNAFL and SNARF pH probes
Give a medical use of fluorescence energy transfer techniques
Glucose sensing to help control blood glucose in diabetes patients. Based on glucose binding protein, ConA (labelled as donor) and the polysaccharide dextran (labelled as acceptor) which serves as a competitive ligand for glucose
How does change in pH change fluorescence? Use an example.
An extra proton could change excitation energy required. Eg aniline is fluorescent around 310-400 nm, but addition of an extra proton to its zwitterionic form, changing it to an anilinium ion, results in no fluorescence.
The effect of temp on fluorescence properties is often complex because temp affects many factors simultaneously. Give some examples of this.
temp affects:
1) the viscosity of the solvent
2) the vibrational energy states of the fluorophore
3) the kinetic energy of the solvent
Which deactivation processes does temperature favour? What is the result of this?
Those other than fluorescence emission. Therefore there is a decrease in fluorescence intensity, quantum yield and lifetime
How does temperature affect local motion of fluorophores? What is the effect of this on fluorescence anisotropy?
Increases their local motion, thus decreasing fluorescence anisotropy
What is the effect of temperature on:
1) the emission wavelength
2) anisotropy
1) as temp increases, the emission wavelength becomes longer
2) as temp increases, anisotropy decreases
How can tryptophan fluorescence be enhanced?
Through the addition of a chloramphenicol analogue to the CAT transferase enzyme
Give an example of how fluorescence can be used to monitor conformational change
Muscle tropomyosin can be labelled with pyrene iodoacetamide, and the change in fluorescence when it is bound to actin/ myosin/ ATP can be visualised in the fluorescence emission spectra
Give an example of how fluorescence is sensitive to the environment.
Flavin coenzyme in NADH dehydrogenase enzyme: bound flavin has 10% less absorption than free flavin, but 75% less fluorescence because protein side chains quench emission
What is the timescale of fluorescence?
Nanoseconds
Give an example of how fluorescence can be used in different modalities (i.e. how different techniques can be combined)
Fluorescence spectroscopy can be combined with fluorescence microscopy. E.g., FRAP of GFP-myosin IIA using TIRF microscopy
Fluorescence is inherently multidimensional. Give 5 factors it can be characterised by
Position Intensity Wavelength Lifetime Polarisation
In fluorescence microscopy, what does colocalisation refer to?
Observation of the spatial overlap between two (or more) different fluorescence labels, each having a separate emission wavelength
What is the purpose of fluorescence colocalisation?
To see if the different fluorescently labelled ‘targets’ are in the same area of the cell or very near to one another
Give an example of two proteins that co-localise
F-actin and Talin 1
Fluorescence is flexible. Which techniques allow it to be used as a:
1) clock
2) ruler
1) lifetime fluorescence
2) FRET
Which fluorescence techniques give the following information:
1) temporal
2) spatial
3) distance measurement
1) lifetime (e.g., kinetics of a reaction)
2) localisation
3) FRET
What are the different lamp light sources?
1) xenon arc lamp (wide range of wavelengths)
2) high pressure mercury lamps (high intensities but conc. in specific lines)
3) mercury-xenon arc lamp (greater intensities in the UV)
4) tungsten-halogen lamps
5) light emitting diodes (LEDs) (multiple colour LEDs can be bunched to provide a broad emission range)
In what range of wavelengths is the Mercury-Xenon Arc Lamp most intense?
UV
What is an advantage of the Xenon Arc Lamp?
Wide range of wavelengths
What is an advantage of LED lamps?
Multiple colour LEDs can be bunched to provide a broad emission range
What devices can be used for wavelength selection?
Optical filters
Monochromators
What is a longpass optical filter?
An optical interference/ coloured glass filter that attenuates shorter wavelengths and transmits longer wavelengths over the active range of the target spectrum
What are monochromators used for? What form can monochromators be in?
To disperse polychromatic/white light into the various colours or wavelengths. This can be achieved using prisms or diffraction gratings
In a monochromator, how does the size of the slit width affect the signal levels and the resolution?
Wider slit = increased signal levels but decreased resolution
How can the problem of photobleaching of the sample be addressed?
1) decreasing the excitation intensity (i.e. less wide slits in monochromator)
2) gentle stirring of sample
What are the two different types of gratings monochromators may have?
Planar or concave
RE monochromators, what do the following terms mean/describe:
1) slit width (mm)
2) bandpass
3) dispersion
1) the dimension of the entrance/exit slits of the monochromator
2) the FWHM of the selected wavelength
3) the factor to convert slit width to bandpass
What do most fluorometers use as detectors?
Photomultiplier tubes (PMTs)
What are the main components of photomultiplier tubes in a monochromator?
(Photon) Window Photocathode Focusing electrode Voltagedividers Dynode chain Photoelectrons Anode Power supply
What are the 3 main materials used for the cuvettes that hold samples in fluorescence? Which has the highest transmittance %?
Quartz
Glass
Polysteren
…Quartz has the highest % transmittance of all three
Transmittance v. similar for all three once above 300 nm wavelengths, however
What are 5 possible errors in fluorescence measurements due to monochromators?
1) inner filter effect
2) photobleaching
3) linearity of signal
4) fluorescent contaminants
5) Raman and Rayleigh scatter
What is the inner filter effect?
If excitation light is absorbed significantly, then chromophores at ‘far’ side of cuvette will not be fully excited… Rule of thumb = absorbance should be
How might the linearity of fluorescence signal change with concentration?
At higher concentrations the linearity may deviate significantly
What do ‘Rayleigh’ and ‘Raman’ scattering refer to?
scattering of photons - shown on spectra preceding the fluorescence curve
Give definitions for the following:
1) intrinsic probe
2) chromophore
3) fluorophore
1) naturally occuring probe
2) component that absorbs light
3) chromophore that emits light (all fluorophores are chromophores)
The chromophore of fluorphores generally contains which features?
Double bonds/ metal ions to give absorption in region of 200-700 nm (common feature: electrons in low energy orbitals)
What are the absorption ranges for:
1) aromatic amino acids (tryptophan, tyrosine and phenylalanine)
2) coenzymes (NADH and Flavins)
3) autofluorescent proteins (GFP)
1) 260-280 nm
2) NADH = 340 nm, Flavins = 450 nm
3) 390-490 nm + other colours
Give some features of the excitation and emission spectra of aromatic amino acids
Excitation:
Trp absorbs 5x more strongly than Phe, however the abundance in proteins is Phe > Tyr > Trp - a few proteins lack Trp (eg troponin C)
Emission:
Phe has lowest intensity fluorescence
Tyr is insensitive to solvent polarity
Trp is sensitive to solvent polarity
Name an aromatic amino acid that is insensitive to solvent polarity
Tyrosine
Name an aromatic amino acid that is sensitive to solvent polarity
Tryptophan
Name some common synthetic fluorophores
Dansyl chloride FITC ANS NBD Pyrene Ethenoadenosine
Why is artificial labelling, e.g., with FITC, used?
If you label a common amino acid it is difficult to know which protein/ part of protein is giving you a signal
Why is cysteine targetted with synthetic fluorescent labels. Give one that binds non-covalently and one that binds covalently.
It is a rare amino acid.
Non covalent = ANS/ ethidium bromide
Covalent = pyrene
What was the purpose of the Alexa-Fluor series?
“There is a need for probes with high fluorescence quantum yield and high photostability to allow detection of low-abundance biological structures with great sensitivity and selectivity”
Have fluorescent probes been developed for ions?
Yep. Wide variety.
Why is the study of calcium important?
It is a very important intracellular component (2nd messenger).
It plays a role in a lot of cellular events: extensively studied.
The regulation of the intracellular level of calcium is very complex
List some probes used for calcium determination
FURA INDO (Indo-1, Indo 5F) FLUO RHOD CALCIUM GREEN OREGON GREEN 488-BAPTA Quin-2
What are calcium indicators based on?
the BAPTA chelator, which binds Ca2+ with affinities near 100 nM
Calcium probes based on the BAPTA chelator can be used to monitor intracellular calcium. Give an example of such a probe
Fura-2, Indo-1, Fluo-3, Rhod-2, CG-1, OgB-1, Quin-2
The salt forms of calcium probes such as the Fluo-3 dye do not diffuse across cell membranes. How is this problem addressed?
Microinjection
Use of an ester derivative (e.g. fluo-3acetoxymethyl)
Electrophoration
What is the use of acetoxymethyl (AM) esters of calcium probes?
They are able to pass through cell membranes
How are calcium probes like Fluo-3 and Indo-1 moved into cells via esterification?
They are esterified with carboxy groups to form AM-esters. In this form the dyes are less polar and passively diffuse across cell membranes. Once inside the cell, the AM esters are cleaved by intracellular esterases, and the negatively charged probe is trapped in the cells
Name some probes used for pH determination
BCECF
Fluoresceins and carboxyfluoresceins
SNARF indicators
HPTS (pyranine)
How is the chromophore of GFP formed?
SPONTANEOUSLY from Ser-65, Tyr-66 and Gly-67, upon folding of the polypeptide chain
What is the cause of fluorescence in GFP?
Deprotonated phenolate of Tyr66
Fluorophore formation involves 3 steps. What are they?
1) CYCLISATION: nitrogen of Gly-67 attacks carbonyl of Ser-65
2) DEHYDRATION: loss of a water molecule
3) OXIDATION: of C-C bond in Tyr-66 from single bond to a double bond
GFP has active site SYG. What do CFP and BFP have?
SWG
XHG
CFP and BFP are produced by mutating Tyr-66 in GFP. How is RFP obtained?
It is naturally produced in Discosome sp. (a sea anenome). It has a similar structure to GFP (beta barrel) and led to the discovery of several GFP-like proteins
Labelling ‘in vivo’ requires incorporation of the fluorescent dye. Which two main ways can this occur?
1) genetic incorporation
2) mechanical incorporation
Briefly explain how a fluorescent protein, e.g. GFP, can be incorporated in vivo
GFP encoding plasmid (pUG35)
Insert gene of interest, e.g. P2b (in BamH1 site)
Introduce into different organisms
Give three examples of membrane probes
Laurdan
Prodan
Bis ANS
How are labelled nucleotides (labelled dUTP) synthesised?
By chemically coupling ALLYLAMINE-dUTP to SUCCINIMIDYL-ESTER DERIVATIVES of:
1) fluorescent dyes
2) haptenes
e.g. fluorescein-aha-dUTP
Give some examples of fluorescent dyes used to label DNA
DAPI DEAC FITC TAMRA Cy5 BIO
Microscopy is a method that allows biologists to make a large image of a small object. It allows examination of tissue, cell or organelle preparations. List 3 of its uses.
1) evaluation of the integrity of a sample during an experiment
2) mapping the fine details of the spatial distribution of cellular and subcellular structures (organelles, macromolecular complexes, localisation of protein…)
3) directly measuring biochemical processes within living preparations (protein dynamics, protein interactions, protein conformation…)
There are several different types of microscopes based on the physical principle used to make an image. List 7.
Optical microscopes
X-ray microscopes
Scanning acoustic microscope (SAM)
Scanning helium ion microscope (SHIM or HeIM)
Neutron microscope
Electron microscope (TEM/SEM)
Scanning Probe microscopes (Atomic Force microscopes)
Why do X-ray microscopes produce higher resolution images than optical microscopes?
Because they use smaller wavelengths
How do scanning acoustic microscopes (SAMs) generate images? When are they used?
Using sound waves. Used in materials science to detect small cracks or tensions in materials. Can also be used in biology to uncover tensions, stress and elasticity inside biological structure
What are the advantages of scanning helium ion microscopes (SHIMs / HeIMs) over electron microscopes? (how do they work)
These devices use a beam of Helium ions to generate an image. There are several advantages over EM microscopy:
1) sample left mostly intact (due to low energy requirements)
2) high resolution
Possible advantages of experimental-stage NEUTRON microscopes?
May offer better contrast than other forms
Which is the type of microscopy that is still in an experimental stage that has a high resolution and may offer better contrast than other forms of microscopy?
NEUTRON microscopy
How are the different optical microscopes classified?
Based on HOW contrast is achieved
Based on HOW CONTRAST is achieved, what are the different types of optical microscope? What do each of them measure?
Brightfield: absorption Phase contrast: phase interference Normalised interference: variation of phase contrast Darkfield: scattering Fluorescence: fluorescence
Which type of optical microscope measures absorption?
Brightfield microscopes
Which type of optical microscope measures phase interference?
Phase contrast microscopes
Which type of optical microscope measures scattering?
Darkfield microscopes
What are the 4 basic concepts in light microscopy?
1) image
2) magnification
3) resolution
4) contrast
In optical microscopes, what is the image produced by?
Interaction of incident light with the object
How does light interact with objects?
Through wave-like and particle-like properties
An ideal optical system would image an object point perfectly as a point. Why does this not happen in reality?
Because, due to the wave-like nature of radiation, diffraction occurs
In optical microscopy, what is diffraction caused by?
The limiting edges of the system’s aperture stop
What is the result of diffraction in light microscopy?
An image of a point is a blur, no matter how well the lens is corrected. This is the Airy disk
What is the ‘Airy disk’?
The blurred image of a point that results from diffraction
When is the resolution limit reached?
When two point-like objects can not be imaged as two distinct images
What is the term for the distance between two point-like objects at which they can no longer be imaged as two distinct images?
The resolution limit
The resolution of a microscope is the shortest distance two points can be separated and still be observed as 2 points. What is another term for this distance?
Lateral resolution
When is Rayleigh criterion satisfied?
When the first dark ring produced by one Airy disk is coincident with peak of nearby airy disk
What is the Axial resolution?
Depth of FIeld
…defined by NA^2 (i.e. numerical aperture squared)
What is lateral resolution?
The shortest distance two points can be separated and still be observed as two points
What is the numerical aperture of a microscope objective?
A measure of its ability to gather light and resolve fine specimen details at a fixed object distance
How does numerical aperture relate to opening angle?
High numerical aperture have large opening angle
How do higher magnification objectives relate to NAs?
They usually have greater NAs
How does NA relate to resolution?
High NA generates better resolution
How does wavelength length relate to resolution?
Shorter wavelength gives better resolution
