2022-23 WEEK 28 BIOL25012 Fluorescence and FRET.pdf

BIOL25012 Advanced Biochemistry WEEK 28 08/Feb/2023 Fluorescence I ALDO BIOL25012: Advanced Biochemistry WEEK 28 Support asynchronous material Frequency, resonance and energy ALDO Excellent technical manual (Perkin Elmer, 16 pages) PDF copy in NOW Contents Learning outcomes: Fluor...

BIOL25012 Advanced Biochemistry WEEK 28 08/Feb/2023 Fluorescence I ALDO BIOL25012: Advanced Biochemistry WEEK 28 Support asynchronous material Frequency, resonance and energy ALDO Excellent technical manual (Perkin Elmer, 16 pages) PDF copy in NOW Contents Learning outcomes: Fluorescence spectrophotometry After this lecture you should be able to : • Explain fluorescence in terms of “radioactive decay”, and the meaning of the Stokes shift. • Discuss the meaning of a Jablonski diagram. • Explain fluorescence resonance energy transfer (FRET) and its main properties. • Explain the main features of excitation and emission spectra. Absorbance and fluorescence: “There cannot be emission without previous absorbance” Absorption – Emission: two sides of a single event (from last week) A cycle of absorption -emission takes place VERY QUICKLY: ~ 1x10 -12 seconds = 0.0000000000001 second Relation between absorption and fluorescence: There cannot be emission without previous absorption Emission (fluorescence ) Fluorescence happens when an excited electron relaxes back to its ground state in a relatively slow time -scale Fluorescence: An electron’s delayed return to the ground state Duration: 8 minutes Duration: 3 minutes Fluorescence: Understanding the emission spectra Excitation and emission spectra are generally broad •Within each electronic state there are multiple vibrational energy levels (electronic levels are depicted with thicker lines and the vibrational levels are with thinner lines). •Radiative transition is depicted by solid arrows, while the non -radiative transition is shown by squiggly arrows. •Fluorescence usually takes place from the ground vibrational level of the electronically excited state S1. Energy absorbed during emission is emitted back as the electron relaxes to the most stable (i.e. ground) level. Emitted light has less energy ( E = h n ) than the exciting light because some energy has been radiated as heat (kinetic energy) by the electron during its transition across different levels. As a consequence the emitted light has a lower frequency ( n ), [a longer wavelength ( l )]. This results in emission having a different, (i.e. “weaker”) colour. The Stokes shift Stokes shift is the difference (in wavelength or frequency units) between positions of the band maxima of the absorption and emission spectra of the same electronic transition. When a system (be it a molecule or atom) absorbs a photon, it gains energy and enters an excited state. One way for the system to relax is to emit a photon, thus losing its energy (another method would be the loss of energy as heat). When the emitted photon has less energy than the absorbed photon , this energy difference is the Stokes shift. The Stokes shift is the result of two actions: vibrational relaxation and/or dissipation, and solvent reorganisation. Often a fluorophore’s dipole is surrounded by water molecules. When a fluorophore enters an excited state, its dipole moment will change, but water molecules will not be able to adapt this quickly. Only after vibrational relaxation, will there will be a realignment of their dipole moments. The Stokes shift • Excited states have limited life times (typically in the order of a nanosecond) and they can decay via several modes. • Excited state may returns to the ground state without emitting a photon . (radiationless decay) . For example, the excited states of DNA bases are very short -lived thanks to efficient radiationless decay; this protects DNA from photochemical damage. • Electron relaxation to the ground state may be accompanied by vibrational energy emission as during ABSORPTION . The electron gives away the excess energy in the form of kinetic energy, and returns to lowest vibrational level • Alternatively, return to the ground state is via the emission of a photon. Such radiative decay is called FLUORESCENCE . What happens after excitation : Fates of Excited States Relation between absorption and fluorescence: Different relaxation (emission) timescales ABSORPTION: Excitation ➔ Relaxation (emission) : 10 -15 s FLUORESCENCE: Excitation ➔ Relaxation (emission) : 10 -9 - 10 -7 s Radiative: photons are emitted (i.e. electromagnetic wave, E: hv ) Non - Radiative: relaxation to ground state does not involve photon emission Fluorescence happens when an excited electron relaxes back to its ground state (i.e. emits photons, E = hv ) in a relatively slow time -scale. ABSORPTION: Excitation ➔ Relaxation (emission) : 10 -15 s FLUORESCENCE Excitation ➔ Relaxation (emission) : 10 -9 - 10 -7 s If one absorption cycle (excitation ➔ emission) were to take 1 minute , then a fluorescence cycle would take ~ 200 years However, in the timescale we inhabit both appears equally instantaneous If emission takes place within the same timescale than excitation (i.e. all excitation energy is released as kinetic energy) then fluorescence doesn’t take place . Not all chromophores are fluorophores DNA’s nucleotides are good chromophores , but they do not fluoresce Aleksander Jablonski Born: February 26, 1898, Ukraine Died: September 9, 1980, Poland Professor Aleksander Jabłoński was a Polish physicist and member of the Polish Academy of Sciences. Jabłoński initially studied the violin at Warsaw Conservatory, under the virtuoso Stanisław Barcewicz , but later switched to science. He is regarded as the founder of modern fluorescence spectrometry Jabłoński diagram “Fluorescence resonance energy transfer (FRET)” Also known as: Förster resonance energy transfer (FRET ), Resonance energy transfer (RET ) or Electronic energy transfer (EET ). Initial excitation Final emission FRET takes place when the emission frequency of a fluorophore matches the excitation frequency of a nearby second fluorophore. As a result, the final emission is of a much larger wavelength value, making it easy to observe (i.e. not peak overlapping) FRET takes place when the emission frequency of a fluorophore matches the excitation frequency of a nearby second fluorophore. As a result, the final emission is of a much larger wavelength value, making it easy to observe (i.e. not peak overlapping) FRET is extremely sensitive to intermolecular distance (i.e. it greatly decreases/ increases with even minor distance changes). Equation for FRET efficiency For FRET to take place, the second fluorophore must be close enough A happy coincidence: FRET effective range is in the same scale as biological molecules and their interactions (0 -50 Å, amstrongs = 7 nm) http://www.weizmann.ac.il/plants/Milo/images/proteinSize120116Clean.pdf Average monomeric protein 20 -30 Å; 2 -4nm A happy coincidence: FRET effective range is in the scale as biological molecules and their interactions (0 -50 Å, amstrongs = 7 nm) An example: Green -fluorescence proteins (GFP) CLOSE SEPARATED FRET is extremely sensitive to intermolecular distance (i.e. fade away with even minor distance changes). Because of this property, it can be used as a atomic “ruler” to measure the relative dynamics between two molecules. No FRET can take place because the second fluorophore is too far. FRET takes place because the second fluorophore is close enough. Additional self -study material to help understanding FRET PDF file in NOW Additional self -study material to help understanding FRET Postgraduate lecture on FRET and fluorescence Fluorescence spectrophotometry CONCLUSIONS • Stokes shift is the difference (in wavelength or frequency units) between positions of the band maxima of the absorption and emission spectra of the same electronic transition • Fluorescence happens when an excited electron relaxes back to its ground state by emitting energy as photons (E = hv ) in a relatively slow time -scale (radioactive decay). • FRET takes place when the emission frequency of a fluorophore matches the excitation frequency of a nearby second fluorophore . • As a result, in FRET , the final emission is of a much larger wavelength value, making it easy to observe (i.e. not peak overlapping) FRET = Fluorescence resonance energy transfer.