Laura van den End
1. Basics of uorescence
Goals: get familiar with new techniques based on Absorption, emission and Jablonski
fluorescence and fluorescence microscopy
diagram
- Quantification of absorption is done with the
Interaction of light with matter Lambert-Beer law: exponential dependence on the
- Nature of light: wave-particle duality = consists of [ ] and thickness of the material
waves and consists of photons (specific E = hν) - The Franck-Codon energy diagram for absorption
- When light interacts with matter: conservation law and emission spectra → simplified by Jablonski
of energy diagram — transition with the highest probability
- This course: visible light and UV → when using will reveal the max peak
these λ, study systems that have same dimensions
(relevant to the light scale) and choose a technique Stokes shift
that matches the dimensions of the subject - Difference between the absorption and emission
- For fluorescence, a molecule needs to absorb a
maxima of a fluorophore (can fluorescence)
photon: transition E needs to match the photon - When a fluorophore absorbs photons, it transitions
- When an e- is promoted from bonding to anti-
to an excited state and later returns to the ground
bonding orbital, corresponding bonds will be
state by emitting photons. The emitted photons
a bit more relaxed
have a longer λ than the absorbed ones, resulting
- How to know which λ/E will be absorbed? Pass
in the observed shift.
some monochromatic (single λ) light → if the - Useful to separate excitation and emission —
Ephoton correspond to Etransition, it can also absorb
chose filters
that specific λ
- When polychromatic light: pass white light
(combination of all colors) towards the
sample → molecule absorbs some λ ⇒ at a
certain point, max absorption is reached
Types of interactions
- For light to be useful it must interact with matter
- Absorption
- Luminescence incandescence Jablonski diagram
- Emission of light by a substance not resulting
from heat; cold body radiation
- Results from an electronically excited state
- Reflection: instant light beam → specular
reflection via specific angle
- If the reflecting surface is very smooth, the
reflection of ligt is called specular or regular
reflection
- The incident ray, the reflected ray and the
normal to the reflection surface at the point of
the incidence lie in the same plane. - Fluorophores absorb and emit energy between
- The angle of the incident ray with the normal
closely spaced vibrational and rotational energy
is equal to the angle of the reflected ray with
levels of excited states in different molecular
the same normal.
orbitals — visualized with Jablonski energy
- Refraction: from one medium to another →
diagram: illustrates the singlet ground state (S(0)),
changing refraction index
along with the first (S(1)) and second (S(2)) excited
- Change in direction of a wave due to a
singlet states as horizontal lines.
change in its speed - Thicker lines represent electronic energy levels,
- Refractie index of a substance is measured by
while thinner lines represent various vibrational
the speed of light in that medium
energy states, with rotational states ignored.
Advanced Fluorescence and Fluorescence Microscopy
  ↔︎ fl
, Laura van den End
Photophysical processes - Exceptions: pH sensitive HTPS — In low pH,
- After a molecule absorbs light and accesses an the protonation of OH group results in an
electronically- excited state, it deactivates and absorption spectrum with typical vibrational
return to its ground state through different structure. However, the emission spectrum,
pathways characterized by a large Stokes shift, lacks the
vibrational features seen in absorption due to
NON-RADIATIVE ionization of the OH group.
- The excited state species are transformed in other
electric to vibrational states, returning the excess Fluorescence quantum yield and lifetime
energy as heat
- Vibrational relaxation: process in which the Quantum yield
excited molecule decreases its vibrational energy - Absorption can have more transitions but by
within a single electronic state fluorescence we lose some energy. It can either be
- Internal conversion: “allowed” isoenergetic via radiative or non-radiative processes → they
transition between two electronic states with the will all be taken up in this equation
same coin multiplicity, generally followed by
vibrational relaxation ΦF = number of emitted photons via fluorescence
- Intersystem crossing: “forbidden” iso-energetic number of absorbed photons
transition between two electronic states with
different spin multiplicity, generally followed by - Largest quantum yield = brightest emission
vibrational relaxation
Fluorescence lifetime
RADIATIVE - Duration that a molecule spends in an excited
- The molecule in its excited state is transformed
state after being excited → falls back to ground
into lower-energy electronic states emitting
state. When the sample is excited with a very short
electromagnetic radiation
pulse of light, the intensity decreases exponen-
- Fluorescence: “allowed” spontaneous emission of
tially
radiation upon transition between two electronic
I(t) = I0e-t/τ
states with the same spin multiplicity
- Phosphorescence: “forbidden” spontaneous - I(t) is the fluorescence intensity at time t
emission of radiation upon transition between two - I0 is the initial fluorescence intensity at t = 0
electronic states with different spin multiplicity - τ is the fluorescence lifetime.
- When t = 0 → I(t) = I0
OTHER DEACTIVATION PROCESSES - Provides a time window for the detection of
- Photochemical reactions
dynamic processes that occur while the fluoropho-
- Energy transfer
res are in the excited state
- Does not depend on fluorophore concentration
Absorption and emission spectra fluorescence intensity
- Fluorophore spectra exhibit symmetry due to the - Becomes shorter, for instance in case of energy
involvement of the same transitions in both transfer
absorption and emission, and the similarity in
vibrational energy levels of the ground (S0) and
first excited (S1) states.
- In most cases, electronic distributions in S0 and S1
do not significantly alter these energy levels.
- According to the Franck-Condon principle,
Fluorophores and chromophores
electronic transitions are vertical, occurring - Intrinsic chromophores: natural occurring in
without nuclear position changes.
biological systems (aa, nucleotides, proteins)
- The emission spectrum mirrors the vibrational - Extrinsic chromophores: made or created by
energy spacing seen in the absorption spectrum,
humans, can intercalate in biological systems
with the most probable transitions reciprocating
(DAPi in DNA) — selective labeling: where
between the 0th and 1st vibrational levels = mirror
specific molecules bin to and end up ~ use
image rule
different colors of probes → differentiate by using
filters for those specific λ
Advanced Fluorescence and Fluorescence Microscopy
  ↔︎
, Laura van den End
How do we measure abs/ uorescence Lifetime
1. Excitation: pulsed laser emits short pulses
Absorption 2. Pulse Splitting: Light is split into two beams: one
for sample excitation and the other as a reference
3. Sample Interaction: Fluorophores in the sample
absorb light, entering an excited state.
4. Fluorescence Emission: Excited fluorophores
emit fluorescence photons.
5. Start and Stop Signals: Fluorescence triggers a
start signal, and a reference pulse acts as a stop
signal.
6. Time Delay: controlled time delay is introduced.
7. Time Measurement: Time between start and stop
signals is measured for each photon.
- Light source with different λ → detect the light 8. Data Collection: Multiple measurements create a
that was not absorbed fluorescence decay curve.
- Mostly a double team spectrometer because you 9. Analysis: Mathematical modeling analyzes the
can correct for the absorption of the reference and curve to determine fluorescence lifetime.
you don’t want to see the fluctuations in the = Time correlated single proton counting
intensity coming from the light source
Fluorescence anisotropy
Fluorescence - Used polarized light to selectively excite specific
molecules in a sample
- If looking at a dense sample and want a single
molecule excitation → already select a few
molecules → better resolution
Polarized light and photo selection
- Anisotropy measurements rely on selectively
exciting fluorophores with polarized light,
exploiting the alignment of the fluorophore's
transition moment.
- When fluorophores are randomly oriented in an
- Light source → dual grating excitation monochro- isotropic solution, polarized light excitation
mator → filter holder → polarizer → other way selectively energizes those with absorption
around transition dipoles parallel to the electric vector →
- 90º otherwise the detector will be immediately partially oriented fluorophore populations and
saturated partially polarized fluorescence emission. The
emitted light aligns along a fixed axis in the
Emission and excitation fluorophore.
EMMISION DEPOLARIZATION
- Chromophores have transition moments aligned
along specific molecular axis directions.
- Rotational diffusion alters transition moment
directions — influenced by solvent viscosity and
the rotating molecule’s size and shape
- Emission spectrum or fluorescence spectrum: one - In low-viscosity solutions with small fluoro-
excites at one λ and scans the emission phores, rotational diffusion often outpaces
monochromator emission rate, causing depolarization and
- Excitation spectrum: one fixes the emission near-zero anisotropy.
monochromator at one λ and scans the excitation - Anisotropy unveils the avg angular displacement
monochromator of fluorophores between absorption and emission
— depends on the rotational diffusion rate during
the excited state's lifetime.
Advanced Fluorescence and Fluorescence Microscopy
  fl
, Laura van den End
Sequence of events after excitation - Efficiency depends on
- Ground state redox potentials
with polarized light - Excited state energy
- Solvent polarity
- D-A distance
- Inter- and intramolecular
- Only for molecules with closed shell ground states
- E.g. When a chlorophyl molecule at the core of the
photosystem II reaction center obtains sufficient
excitation energy from the adjacent antenna
pigments, an electron is transferred to the primary
electron-acceptor molecule, Pheophytin, through a
process called photoinduced charge separation.
- θ: the rotational correlation time of the sphere is These electrons are shuttled through an electron
transport chain, the so called Z-scheme shown in
dependent on
- η viscosity the diagram, that initially functions to generate a
- T temperature in EK chemiosmotic potential across the membrane.
- κ gas constant
- V volume of rotating unit
Energy and electron transfer
- Radiative: fluorescence from one molecule is
reabsorbed by another (e.g. [ ] is too high)
- Non-radiative: Förster or Dexter type
- Always look at the donor properties because these
change when an acceptor comes by
FRET: FÖRSTER RESONANCE E TRANSFER
- Dipole-dipole interaction: NO electron exchange
process
- Total spin maintained
- Overlap between donor fluorescence and acceptor
absorption required
- FRET between identical chromophores is called
energy hopping
- The critical transfer distance R0 is defined as that
distance at which the transfer rate is equal to the
donor fluorescence decay rate (or the distance at
which half of the excitation energy undergoes
transfer while half is dissipated by all the other
processes including emission).
- Strong distance dependence (molecular ruler)
- One of the moest applied fluorescence methods in
life sciences bv energy transfer can be used as a
convenient spectroscopic ruler to obtain geometric
information
- Can be used to study protein-protein interactions,
conformational changes + proteolytic processing
DEXTER ENERGY TRANSFER
- Electron exchange process
- Distance over 0.5-1 nm
- Spin allowed transition
- Overlap between donor fluorescence and acceptor
absorption required
Advanced Fluorescence and Fluorescence Microscopy
 
