BASIC PHOTOPHYSICS — Visual Study Guide Page 1

BASIC PHOTOPHYSICS

A Complete Visual Study Guide

From Electromagnetic Waves to FRET and Quantum Dots

01 Electromagnetic Radiation & Light

02 Electronic Energy States & Jablonski Diagram
03 Light Absorption — Beer-Lambert Law
04 Fluorescence — Stokes Shift & Franck-Condon
05 Phosphorescence — Triplet States
06 Quantum Yield & Rate Constants
07 Fluorescence Lifetime & Decay Kinetics
08 Fluorescence Anisotropy & Rotational Diffusion
09 Quenching — Static, Dynamic & Stern-Volmer
10 FRET — Molecular Ruler & Distance Measurement
11 Semiconductors, LEDs, Quantum Dots & Solar Cells

Based on: Visser & Rolinski, Basic Photophysics — Comprehensive Student Edition

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CHAPTER 1

Electromagnetic Radiation & Light

Waves, photons, energy, and the electromagnetic spectrum

1.1 What is Electromagnetic Radiation?

Electromagnetic (EM) radiation is a form of energy that travels through space as oscillating electric
and magnetic fields. Unlike sound, it requires no medium — it can travel through a complete vacuum
(like outer space). Light is just one small part of the full electromagnetic spectrum. All EM radiation
travels at the same speed in vacuum: c = 3 × 108 m/s (300,000 km/s — fast enough to circle the
Earth 7.5 times per second!).
Electromagnetic Wave: Oscillating Electric (E) and Magnetic (B) Fields
E field

direction of travel
B field

wavelength λ
Fig 1.1 — An electromagnetic wave consists of an electric field (E, blue) and a magnetic field (B, teal, dashed)
oscillating perpendicular to each other and to the direction of travel.

Key Terms Defined

Wavelength (λ) Distance between two successive peaks of the wave, measured in nanometres (nm)
for visible/UV light. 1 nm = 10■■ m.

Frequency (ν) Number of wave cycles passing a point per second. Unit: Hertz (Hz) = cycles/second.
Higher frequency = more energetic.

Speed of light (c) c = 3 × 10■ m/s in vacuum. Relationship: c = ν × λ (speed = frequency ×

wavelength).

Photon The smallest "packet" (quantum) of electromagnetic energy. Light behaves as both a
wave AND a particle (photon). This is wave–particle duality.

Energy of photon E = hν = hc/λ, where h = Planck's constant = 6.626 × 10■³■ J·s. Higher frequency
(shorter wavelength) = higher energy photon.

Wavenumber (σ) Inverse of wavelength: σ = 1/λ. Unit: cm■¹. Used in spectroscopy. A wavenumber of
40,000 cm■¹ corresponds to 250 nm (UV).

The Electromagnetic Spectrum

The electromagnetic spectrum spans an enormous range of wavelengths, from radio waves
(kilometres long) to gamma rays (smaller than an atomic nucleus). Each region interacts differently
with matter:

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The Electromagnetic Spectrum

400 nm 700 nm

Radio Microwave Infrared Visible UV X-rays Gamma

Low energy / Long wavelength High energy / Short wavelength →

Fig 1.2 — The electromagnetic spectrum. Visible light (400–700 nm) is an extremely thin slice. Photobiology focuses
on the UV (100–400 nm) and visible (400–700 nm) regions.

Wavelength Energy
Spectral Region Common Use in Spectroscopy
Range Range

Radio (VHF) >1m < 10■■ eV NMR (nuclear magnetic resonance)

Microwave (UHF) 1 mm – 1 m ~10■³ eV EPR (electron paramagnetic resonance)

Infrared (IR) 700 nm – 1 mm 0.001–1.7 eV Molecular vibrations, IR spectroscopy

Visible 400–700 nm 1.7–3.1 eV Electronic transitions, colour, vision

Ultraviolet (UV) 100–400 nm 3.1–12.4 eV Aromatic molecule absorption, DNA damage

124–124,000
X-rays 0.01–10 nm Ionisation, crystallography
eV

Gamma (γ) rays < 0.01 nm > 124,000 eV Nuclear transitions

Table 1.1 — Regions of the electromagnetic spectrum and their spectroscopic uses.

Why Does Photobiology Focus on UV and Visible Light?

UV light (100–400 nm) carries enough energy to excite valence electrons in biological molecules (proteins,
DNA, pigments). This can cause photochemical damage (e.g., UV-B causing thymine dimers in DNA) or
useful photoreactions (vitamin D synthesis, photosynthesis).
Visible light (400–700 nm) interacts with chromophores like chlorophylls (photosynthesis), retinal (vision),
and fluorescent proteins (GFP). Infrared is mostly absorbed as heat. X-rays and gamma rays cause
ionisation damage.

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CHAPTER 2

Electronic Energy States & the Jablonski Diagram

Singlet states, triplet states, spin multiplicity, and all possible transitions

2.1 Electronic States of Molecules

Molecules have electrons arranged in orbitals. Each electron has a property called spin — you can
think of it as the electron spinning either "up" (↑) or "down" (↓). The arrangement of spins determines
the quantum state of the molecule. This leads to different types of electronic states: singlet states and
triplet states.

Singlet Ground State (S■) Excited Singlet (S■, S■) Triplet State (T■)

↑ ↓ ↑ ↓ ↑ ↑
Both orbitals filled; electrons have One electron promoted to higher orbital Both excited electrons have PARALLEL
opposite spins. Diamagnetic — not but spin is still paired (opposite). spins. Paramagnetic — interacts with
attracted by magnets. Most stable state. Fluorescence comes from here. Short magnets. Spin-forbidden to return to S■
lifetime (~ns). quickly. Phosphorescence source.

Fig 2.1 — Electron spin configurations for singlet ground state (S■), excited singlet (S■), and triplet state (T■). The
key difference is whether the two relevant electrons have opposite spins (singlet) or parallel spins (triplet).

Singlet state (S) All electron spins are paired (one ↑, one ↓ in the relevant orbitals). Net spin = 0. Spin
multiplicity = 2S+1 = 1. Represented as S■ (ground), S■ (first excited), S■ (second
excited)...

Triplet state (T) Two electrons have parallel spins (both ↑ or both ↑). Net spin ≠ 0. Spin multiplicity =
3. Represented as T■ (lowest triplet), T■, etc. Triplet states have LOWER energy
than the corresponding singlet because electrons avoid each other better (Hund's
rule).

Spin multiplicity Calculated as 2S+1 where S = total spin. For singlet: 2(0)+1=1. For triplet: 2(1)+1=3.
This number determines how many sub-states exist in a magnetic field.

Ground state (S■) The lowest energy state where the molecule "lives" most of the time at room
temperature. All electrons in their lowest-energy orbitals with opposite spins.

Excited state A higher-energy state reached when a molecule absorbs a photon and an electron is
promoted to a higher orbital. Inherently unstable — the molecule quickly returns to
S■.

Vibrational Each electronic state (S■, S■, T■...) contains multiple vibrational energy levels (v=0,
sublevels v=1, v=2...). These arise because bonds in the molecule vibrate like springs, and
quantum mechanics limits the allowed vibration energies.

Boltzmann At room temperature, the thermal energy k_B T is much smaller than vibrational
distribution spacing, so almost ALL molecules sit in v=0 of S■. This is why absorption always
starts from v=0.

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2.2 The Jablonski Diagram

The Jablonski diagram (named after Polish physicist Aleksander Jablonski, 1935) is the central map
of photophysics. It shows all electronic energy levels of a molecule arranged vertically by energy, and
all possible transitions between them as arrows. Every photophysical and photochemical event
begins and ends somewhere on this diagram.

Jablonski Diagram — Energy States and Transitions

S■
2nd exc.
singlet

Internal
conversion

Energy
(~10■¹² s)

Vib. relax.
Absorption
S■ (~10■¹■ s)
1st exc. Intersystem
crossing
LEGEND (ISC)
singlet
Absorption
T■
Fluorescence Triplet
Fluorescence
(~10■■ s) Phosphorescence
Internal conversion Phosphorescence
(>10■³ s)
Intersystem crossing
Vibrational relaxation

S■
Ground
state

Fig 2.2 — Complete Jablonski diagram. Solid arrows = radiative transitions (involve a photon). Dashed arrows =
non-radiative transitions (no photon emitted; energy lost as heat or transferred). The energy axis runs vertically.
Horizontal lines represent vibrational sublevels within each electronic state.

Reading the Jablonski Diagram: Step-by-Step Journey of a Photon

Step 1 — Absorption (10−15 s, femtoseconds): A photon of appropriate energy strikes the molecule.
An electron jumps from S■,■ (ground state, lowest vibration) to a vibrational level of S■ or S■. This
is almost instantaneous — the nuclei don't have time to move during the transition
(Born–Oppenheimer principle).

Step 2 — Vibrational Relaxation (10−12–10−11 s, picoseconds): The molecule vibrates at a high

level within S■ or S■ and quickly loses vibrational energy as heat to the surrounding solvent, settling
to v=0 of the lowest excited singlet (S■,■). This process is called internal conversion between
vibrational levels.

Step 3 — Internal Conversion: S■ → S■ (10−12 s): If the electron went to S■, it crosses
non-radiatively to S■. This is "internal conversion" — a radiationless transition between electronic
states of the same spin multiplicity.

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Step 4 — From S■,■, three pathways compete:

• Fluorescence (~10−9 s, nanoseconds): The electron returns to S■ and emits a photon. Because energy
was lost in steps 2–3, the emitted photon has LESS energy (longer wavelength) than the absorbed
photon. This is the Stokes shift.
• Internal conversion to S■: Non-radiative decay where all remaining energy is lost as heat. Reduces
the quantum yield of fluorescence.
• Intersystem crossing (ISC) to T■: A spin-flip occurs (allowed by spin-orbit coupling) and the molecule
crosses to the triplet state T■. This is slow and normally forbidden by spin selection rules, but heavy
atoms (like bromine) enhance it.

Step 5 — From T■: The molecule is "trapped" in the triplet state for microseconds to seconds
because T■ → S■ is spin-forbidden. Eventually it emits a photon (phosphorescence) or loses energy
non-radiatively.

Kasha's Rule
Regardless of which excited state (S■, S■...) a molecule is excited to, fluorescence and phosphorescence
always originate from the LOWEST excited state of each spin multiplicity (S■ for fluorescence, T■ for
phosphorescence). This is because internal conversion and vibrational relaxation to the lowest level is
always much faster than emission.
Practical consequence: The fluorescence spectrum is the same regardless of excitation wavelength (as
long as you're in the same absorption band). This is used to confirm that emission is truly fluorescence and
not scattered light.

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CHAPTER 3

Light Absorption
Beer-Lambert law, extinction coefficient, oscillator strength, and spectral broadening

3.1 How Molecules Absorb Light

When a photon encounters a molecule, it can only be absorbed if its energy exactly matches the
energy difference between two electronic states of the molecule. It's like a lock and key — only the
right photon energy "fits." The probability of absorption depends on a quantum-mechanical quantity
called the transition dipole moment (µ_eg), which describes how strongly the molecule couples to
the electric field of the incoming light.

Absorption is an almost instantaneous process, occurring in about 10−15 seconds (femtoseconds).

This is so fast that the atomic nuclei — being ~1800 times heavier than electrons — do not move
during the transition. This is the essence of the Born–Oppenheimer approximation and the
Franck–Condon principle: the electronic transition is "vertical" — it happens at fixed nuclear
positions.

3.2 The Beer–Lambert Law

The Beer–Lambert law (also called Lambert–Beer law) is the quantitative relationship between the
amount of light absorbed by a sample and its concentration. It is the foundation of all absorption
spectroscopy. When monochromatic light of intensity I■ passes through a solution of concentration c
and path length d:

A_λ = log■■(I■/I) = ε(λ) × c × d

Absorbance (A) Dimensionless number = log■■(I■/I). If A=1, only 10% of light is transmitted. If A=2,
only 1% is transmitted. A=0 means 100% transmission (no absorption).

ε(λ) Molar extinction coefficient (or molar absorptivity) at wavelength λ. Units: M■¹cm■¹
(litre per mole per cm). It's a property of the molecule, not the sample. Large ε means
strong absorber.

c Molar concentration of the absorbing species (mol/L = M).

d Optical path length through the sample (typically 1 cm in a standard cuvette).

I■ / I I■ = intensity of light entering the sample. I = intensity leaving the sample.

Transmittance (T) T = I/I■ = fraction of light transmitted. T = 10■■. If A=1, T=0.1 (10%).

Practical Limit: A < 0.05 for Fluorescence

When measuring fluorescence excitation spectra, the Beer–Lambert law only applies when absorbance A <
0.05. At higher concentrations, the excitation beam is significantly attenuated as it travels through the
sample (inner filter effect), distorting both excitation and emission spectra. Always dilute fluorescent
samples until A < 0.05 for reliable measurements.

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3.3 Oscillator Strength and Transition Dipole Moment

The oscillator strength (f) is a dimensionless number (0 to ~1) that measures how strongly a
particular electronic transition is "allowed" by quantum mechanics. It is calculated from the area of the
absorption band:

f = 1.44 × 10■¹■ ∫ε(σ)dσ (integral over wavenumber σ)

For a "strongly allowed" transition, f ≈ 1 (e.g., the S■→S■ transition of many aromatic dyes). For a
"forbidden" transition (e.g., S■→T■, which is spin-forbidden), f << 1.

The oscillator strength is directly related to the transition dipole moment (µ_eg), which is the
quantum-mechanical integral that couples the ground-state wavefunction (ψ_g) to the excited-state
wavefunction (ψ_e): µ_eg = ∫ψ_e* · µ · ψ_g dr. This describes the "shift of charge" that occurs when
the electron distribution changes upon excitation. A larger transition dipole means faster absorption
and faster emission (shorter radiative lifetime).

3.4 Why Are Absorption Bands Broad?

In a crystal or gas phase, absorption lines are extremely sharp (narrow). In solution (condensed
phase), they are broad bands. Two mechanisms cause this:

• Homogeneous broadening: Each molecule has many vibrational sublevels superimposed on each
electronic state (S■,■; S■,■; S■,■...). All these vibrational transitions overlap, creating a broad band.
Additionally, rapid collisions with solvent molecules (dephasing) broaden each individual line.
• Inhomogeneous broadening: Different molecules in the solution experience slightly different local
environments (different numbers of solvent molecules, different distances from neighbours). This shifts
each molecule's absorption slightly, and the overall spectrum is the sum of all these slightly shifted
spectra — a broad "inhomogeneous" band.

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CHAPTER 4

Fluorescence
Stokes shift, Franck-Condon principle, solvent effects, and practical measurement

4.1 What is Fluorescence?

Fluorescence is the emission of a photon when an excited molecule returns from the lowest
vibrational level of the first excited singlet state (S■,■) to the ground state (S■). It was named by Sir
George Gabriel Stokes in 1852, who first described this phenomenon in the mineral fluorite. The key
characteristics of fluorescence:

• It always originates from S■,■ (Kasha's rule) — regardless of excitation wavelength.

• It occurs on the nanosecond timescale (1–100 ns, typically). This is much longer than absorption
(~fs) but faster than phosphorescence (~ms to s).
• The emission spectrum is always shifted to longer wavelengths than the absorption spectrum
(Stokes shift).
• The fluorescence spectrum is the mirror image of the absorption spectrum (roughly), because both
report the same set of vibrational modes — absorption gives vibrational structure of the excited state,
fluorescence gives vibrational structure of the ground state.

Absorption and Fluorescence Emission Spectra — Stokes Shift

Absorption Fluorescence (emission)

Intensity

Stokes shift
(emission at longer λ)

300 350 400 450 500

Wavelength (nm)
Fig 4.1 — Absorption (blue) and fluorescence emission (green) spectra. Note the Stokes shift: the emission band is at
longer wavelengths (lower energy) than the absorption band. This energy difference was lost as heat during vibrational
relaxation in the excited state.

4.2 The Stokes Shift — Explained in Detail

After a molecule absorbs a photon and reaches an excited vibrational level of S■ (say S■,■ or
S■,■), it rapidly loses vibrational energy to the surrounding solvent molecules through collisions
(picosecond timescale), settling into S■,■. When fluorescence finally occurs from S■,■, the emitted
photon has less energy than the absorbed photon — hence lower frequency, longer wavelength. This
energy difference (Stokes shift) is the energy lost as heat during vibrational relaxation.

The Stokes shift has an additional contribution from solvent relaxation: the dipole moment of the
fluorophore changes upon excitation, and solvent molecules rearrange around the new dipole (on a
picosecond timescale) to minimize energy. This lowers the excited-state energy further, increasing
the Stokes shift. Polar solvents (like water) cause larger Stokes shifts than non-polar solvents (like

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hexane). This effect is described by Onsager's solvation model.

4.3 The Franck–Condon Principle

The Franck–Condon principle states that the most probable vibronic transition is from the vibrational
state in the ground state to that vibrational state in the excited state that is vertically above it (same
nuclear geometry). This is because the electron moves so fast (~fs) that the nuclei remain stationary
during the transition.

The Franck–Condon factor for a transition is the square of the overlap integral between the
vibrational wavefunctions of the initial and final states: |■ψ_v'|ψ_v■|². This determines the relative
intensities of vibrational bands in absorption and emission spectra. If the excited state geometry is
very different from the ground state (large displacement of equilibrium nuclear positions), high
vibrational levels are favoured, creating a broad spectrum with a large Stokes shift.

4.4 Measuring Fluorescence Spectra

Fluorescence is measured at right angles to the excitation beam. This geometry is used to minimise
detection of scattered excitation light. A monochromator (M1) selects the excitation wavelength from
a light source (xenon arc lamp, LED, or laser). The sample absorbs this light and emits fluorescence
in all directions. A second monochromator (M2) scans through emission wavelengths while a detector
(photomultiplier or CCD) records the intensity.

An excitation spectrum is measured by fixing the emission monochromator at the fluorescence

maximum and scanning the excitation wavelength. Since fluorescence intensity is proportional to ε(λ)
× Φ, an excitation spectrum should match the absorption spectrum (for dilute solutions with A < 0.05).
If it doesn't match, multiple species or artifacts may be present.

4.5 Solvent Effects on Fluorescence

The fluorescence emission maximum of many organic fluorophores shifts dramatically depending on
the polarity of the surrounding solvent. This "solvatochromic" shift occurs because:

• The dipole moment of the fluorophore in the excited state (µ_e) is usually different from the ground state
(µ_g).
• In polar solvents, solvent molecules rearrange after excitation to stabilize the larger excited-state dipole,
lowering the energy of S■ → red-shifted emission.
• In non-polar solvents, this stabilization doesn't occur → emission remains closer to the absorption
wavelength (smaller Stokes shift).
This effect is exploited in environment-sensitive probes like PRODAN, dansyl, or "badan," which
report on the polarity of their immediate environment (e.g., the interior of a protein). In water (very
polar), badan emits at ~520 nm; in toluene (non-polar), it emits at ~450 nm.

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CHAPTER 5

Phosphorescence & the Triplet State

Spin-forbidden transitions, intersystem crossing, singlet oxygen production

5.1 What is Phosphorescence?

Phosphorescence is light emission from the triplet state (T■ → S■). It is often visible after the
excitation source is removed — this is the "glow in the dark" phenomenon seen in toys, watch dials,
and safety signs. The key difference from fluorescence:

Property Fluorescence Phosphorescence

Transition S■ → S■ (singlet to singlet) T■ → S■ (triplet to singlet)

Spin change? No — allowed Yes — formally forbidden

Lifetime 1 ns – 100 ns 1 ms – several seconds (even hours!)

After source off? Stops immediately Continues to glow

Temperature effect Moderate effect Usually stronger in rigid/low-T media

Wavelength vs. At even LONGER wavelength (lower

—
fluorescence energy)

5.2 Why is the Triplet State So Long-Lived?

Quantum mechanics forbids direct transitions between states of different spin multiplicity (selection
rule: ∆S = 0). The T■ → S■ transition requires a spin flip, making it "spin-forbidden." However, this
rule is not absolute — a process called spin–orbit coupling mixes singlet and triplet character into
each state, making the forbidden transition weakly allowed. The rate is very slow (rate constant k_p ~
10²–10³ s−1), giving lifetimes of milliseconds to seconds, or even longer.

Heavy atoms (like bromine, iodine, or heavy-metal ions) increase spin–orbit coupling dramatically.
This is called the heavy-atom effect. Adding a bromine substituent to a fluorophore typically greatly
enhances ISC (populating T■) and phosphorescence rate, at the cost of reduced fluorescence. This
principle is used in photodynamic therapy drugs.

5.3 Intersystem Crossing (ISC)

Intersystem crossing is the non-radiative transition between states of different spin multiplicity: S■
→ T■. It is represented by the dashed orange arrow in the Jablonski diagram. ISC is enhanced by:

• Heavy atoms (Br, I, metals) — increase spin–orbit coupling.

• Oxygen — paramagnetic O■ can induce spin flips through collisional quenching.
• Carbonyl groups (C=O) — the n→π* transition (lone pair to π* orbital) has efficient ISC.
• Rigid environments — in rigid matrices (e.g., frozen solution at 77K), ISC is enhanced because
vibrational dissipation (which competes with ISC) is suppressed.

5.4 Singlet Oxygen — A Dangerous By-Product

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The triplet state has important photochemical consequences. Molecular oxygen (O2) has an unusual
property: its ground state is itself a triplet (³Σ_g−). When a molecule in the excited triplet state T■
collides with ground-state O■ in solution:

M(T■) + O■(³Σ) → M(S■) + O■*(¹∆)

Energy transfer converts the ground-state triplet O■ into highly reactive singlet oxygen (¹∆_g).
Singlet oxygen is extremely reactive and can oxidise (and destroy) proteins, lipids, and DNA. This
process is both harmful (UV-induced skin damage) and medically useful (photodynamic therapy: a
photosensitising drug is administered, activated by light near a tumour, and the resulting singlet
oxygen kills the cancer cells).

Measuring Phosphorescence
Phosphorescence is usually weak in solution at room temperature because oxygen quenches the triplet
state very efficiently. To observe phosphorescence:
1. Remove dissolved oxygen (degas the sample with nitrogen or argon).
2. Use a rigid, viscous, or frozen matrix (e.g., ethanol glass at 77 K) to slow down quenching.
3. Use a time-gated detector: wait a few microseconds after the excitation pulse before opening the
detector — this rejects the short-lived fluorescence and sees only the long-lived phosphorescence.
Edmond Becquerel built the first "phosphoroscope" in 1857 to time how long phosphorescent samples glow
after excitation.

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CHAPTER 6

Quantum Yield of Fluorescence

Efficiency, rate constants, and measurement methods

6.1 Definition and Physical Meaning

The fluorescence quantum yield (Φ) is one of the most important properties of a fluorophore. It tells
you what fraction of absorbed photons are re-emitted as fluorescence photons. Φ ranges from 0
(completely dark — every excited molecule loses energy non-radiatively) to 1 (perfectly efficient —
every absorbed photon produces a fluorescence photon).

Φ = n_E / n_A = k_r / (k_r + k_nr)

Quantum Yield: Competition
Photon absorbed
Between(hν)
Radiative and Non-Radiative Pathways

Fluorescence k_r Excited State S■ k_nr Heat / ISC

photon emitted ✓ (absorbed photon) no photon ✗

Φ = k_r / (k_r + k_nr) = photons emitted / photons absorbed

Φ = 0 means no fluorescence; Φ = 1 means every absorbed photon is re-emitted

Fig 6.1 — Competing pathways from the excited state S■. The rate constant k_r governs the radiative (fluorescence)
pathway. The total non-radiative rate k_nr includes internal conversion and intersystem crossing. Quantum yield =
fraction going through the k_r pathway.

k_r Radiative rate constant. Intrinsic property of the fluorophore. k_r = 1/τ_r (natural
radiative lifetime). Related to the transition dipole moment: k_r ∝ |µ_eg|². Typically
10■–10■ s■¹.

k_nr Total non-radiative rate constant. Sum of all non-radiative pathways: k_nr = k_ic +
k_isc + k_q[Q] + ... (internal conversion + intersystem crossing + quenching...).

Φ vs. temperature Increasing temperature usually increases molecular motion, enhancing non-radiative
decay → lower Φ. Exception: some molecules are more fluorescent at higher T.

Φ vs. rigidity In viscous solvents or proteins, molecular rotation is restricted → non-radiative decay
via rotation is reduced → higher Φ. GFP is bright partly because the chromophore is
rigidly held inside the beta-barrel protein.

6.2 Quantum Yields of Common Fluorophores

Solvent/Conditi Quantum
Fluorophore Notes
ons Yield (Φ)

p-Terphenyl Cyclohexane 0.95 Near-ideal emitter, used as quantum yield standard

Fluorescein 0.1 N NaOH 0.90 Most common fluorescence standard

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Rhodamine 6G Ethanol 0.94 Bright red dye, laser dye

Wild-type GFP Aqueous buffer 0.79 Green fluorescent protein from jellyfish

Enhanced GFP (eGFP) Aqueous buffer 0.64 Mutant GFP, brighter than wt

eYFP (yellow FP) Aqueous buffer 0.61 Common FRET acceptor with CFP

eCFP (cyan FP) Aqueous buffer 0.39 Common FRET donor with YFP

FMN (riboflavin) Water 0.26 Biological cofactor, yellow emission

FAD Water 0.03 Very low Φ; intramolecular quenching

NADH Water 0.03 Biological redox cofactor

eBFP (blue FP) Aqueous buffer 0.24 UV-excitable blue variant

DsRed Aqueous buffer 0.70 Red fluorescent protein from coral

Table 6.1 — Quantum yields of selected biologically important fluorophores. Note the dramatic range from 0.03 (FAD)
to 0.95 (p-terphenyl).

6.3 How to Measure Quantum Yield

Relative method (most common): Compare the integrated fluorescence intensity and absorbance of
your sample with a reference fluorophore of known Φ (e.g., fluorescein in NaOH, Φ = 0.90):

Φ_sample = Φ_ref × (I_sample/I_ref) × (A_ref/A_sample) × (n_sample²/n_ref²)

Where I = integrated fluorescence intensity, A = absorbance at excitation wavelength, n = refractive
index of solvent. Both sample and reference must be measured at low absorbance (A < 0.05) to avoid
inner filter effects.

Absolute method: Use an integrating sphere to collect ALL emitted photons in all directions and
compare with the absorbed photon count. More accurate but requires specialised equipment.

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CHAPTER 7

Fluorescence Lifetime
Exponential decay, TCSPC, phase-modulation, and multi-exponential analysis

7.1 What is Fluorescence Lifetime?

The fluorescence lifetime (τ■) is the average time a molecule remains in the excited state before
emitting a photon. After excitation with an infinitely short (delta-function) pulse, the fluorescence
intensity decays exponentially:

F(t) = F■ × exp(−t/τ■)
The lifetime is the time constant of this decay — the time at which fluorescence has dropped to 1/e (≈
37%) of its initial value F■. It is related to the rate constants by:

τ■ = 1/(k_r + k_nr)
Fluorescence Intensity Decay — Exponential Curve

δ-pulse (excitation)
F■
Fluorescence intensity F(t)

F(t) = F■ × exp(−t/τ■)

τ■ = 5 ns (F drops to 1/e ≈ 37%)

0 5 10 15 20
Time (ns)
Fig 7.1 — Exponential fluorescence decay after a delta-function excitation pulse. The lifetime τ■ = 5 ns is the time for
intensity to drop to 1/e (37%) of its initial value F■. The natural radiative lifetime τ_r = 1/k_r is always longer than the
actual lifetime τ■, which is shortened by non-radiative processes.

τ■ (observed 1/(k_r + k_nr). This is what you actually measure. Depends on both radiative AND
lifetime) non-radiative rate constants. Typically 1–20 ns for common fluorophores.

τ_r 1/k_r. The lifetime in the absence of any non-radiative processes. Always longer than
(natural/radiative τ■. Related to the absorption strength by Einstein's relations. For strongly absorbing
lifetime) dyes, τ_r ~ 5–10 ns.

Relationship to Φ Φ = τ■/τ_r = τ■ × k_r. A bright fluorophore (high Φ) has a shorter radiative lifetime
and/or slower non-radiative decay.

Multi-exponential Real samples often show multi-exponential decays: F(t) = α■exp(−t/τ■) +

decay α■exp(−t/τ■) + ... This occurs when the fluorophore exists in multiple environments,
each with a different lifetime.

Mean lifetime ■τ■ = Σα■τ■/Σα■ (intensity-weighted mean). Used when comparing complex
decays.

7.2 Measurement Technique 1: Time-Correlated Single Photon

Counting (TCSPC)

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TCSPC is the gold standard for fluorescence lifetime measurement. It works as follows:

• Excitation: A pulsed laser (repetition rate ~1–80 MHz) excites the sample with pulses of ~100 fs – 100
ps duration.
• Single photon detection: For each laser pulse, a fast detector (avalanche photodiode or
microchannel-plate PMT) detects the FIRST emitted photon and measures the time elapsed since the
laser pulse (TAC = time-to-amplitude converter, or TDC = time-to-digital converter).
• Histogram building: By repeating this over millions of laser pulses, a histogram of photon arrival times
is built up. This histogram IS the fluorescence decay curve F(t).
• Convolution and fitting: The measured decay is actually a convolution of the true decay with the
Instrument Response Function (IRF). The IRF (the timing jitter of laser + detector) is measured using a
scatterer. Deconvolution (fitting with model × IRF) yields the true lifetime.
Resolution: With mode-locked lasers and microchannel-plate detectors, IRF widths of ~30 ps are
achievable, allowing lifetime measurements down to ~10–20 ps. With cheaper diode lasers and LEDs
(IRF ~200–500 ps), lifetimes down to ~100–200 ps can be resolved.

7.3 Measurement Technique 2: Phase-Modulation (Frequency Domain)

In this technique, the sample is excited with sinusoidally intensity-modulated light at angular
frequency ω. Because of the finite fluorescence lifetime, the emission is delayed in phase and
partially demodulated relative to the excitation. Two observables are measured:

Phase shift: Φ_ω = arctan(ω × τ) Modulation ratio: m_ω = 1/√(1 + (ωτ)²)

By measuring Φ_ω and m_ω at multiple frequencies ω (typically 2 MHz – 2 GHz), and fitting the data
to theoretical expressions, the lifetime (and multi-exponential components) are extracted.
Phase-modulation is complementary to TCSPC: faster data acquisition but less direct visualisation of
the decay.

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CHAPTER 8

Fluorescence Anisotropy & Rotational Diffusion

Photoselection, rotational correlation time, and molecular size measurement

8.1 The Concept of Photoselection

When a sample of randomly oriented fluorophores is excited with linearly polarized light, not all
molecules are equally likely to absorb. Absorption is most probable when the molecule's transition
dipole (a vector fixed in the molecule) is aligned parallel to the electric field of the light. The
probability of absorption is proportional to cos²θ, where θ is the angle between the transition dipole
and the electric field vector.

This selective excitation of aligned molecules is called photoselection. Immediately after a very
short excitation pulse, the excited molecules are anisotropically distributed (preferentially aligned
along the polarisation axis). As time passes, Brownian rotational diffusion randomises the
orientations, and the anisotropy decays.

8.2 Definition of Fluorescence Anisotropy

To measure anisotropy, the sample is excited with vertically polarized light. Fluorescence is collected
at 90°, and a polarizer in the emission path alternately selects the vertical (parallel, I_VV) and
horizontal (perpendicular, I_VH) components. The fluorescence anisotropy r(t) is:

r(t) = [I_VV(t) − I_VH(t)] / [I_VV(t) + 2·I_VH(t)]

The denominator I_VV + 2·I_VH = I_total(t) is the total fluorescence intensity (proportional to the
number of excited molecules), and is independent of rotation. The numerator measures the degree
of polarization of the emission. At t=0 (before any rotation), r■ ≤ 0.4 (limiting anisotropy, maximum
for collinear absorption and emission dipoles). r = 0 means completely isotropic (random
orientations).

8.3 Anisotropy Decay and Rotational Diffusion

For a spherical fluorophore undergoing isotropic rotational diffusion (tumbling freely in all directions),
the anisotropy decays exponentially:

r(t) = r■ × exp(−t/τ_r)
The rotational correlation time (τ_r) is the time constant of this decay. Large molecules rotate
slowly → long τ_r. Small molecules rotate fast → short τ_r. The Stokes–Einstein–Debye equation
relates τ_r to molecular size:

τ_r = (4π r³ η) / (3 k_B T)

r■ Initial (fundamental) anisotropy. Maximum theoretical value = 0.4 for a rigid molecule
with identical absorption and emission dipoles. Less than 0.4 if the absorption and
emission dipoles make an angle (energy transfer between differently oriented
dipoles, or flexible linkers).

τ_r Rotational correlation time. For a protein of molecular weight M (kDa) in water at
20°C: τ_r ≈ 0.6 × M ns (rule of thumb). E.g., a 50 kDa protein has τ_r ≈ 30 ns.

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r Hydrodynamic radius of the rotating sphere. Includes any bound water or associated
molecules.

η Viscosity of the solvent. Increasing viscosity slows rotation → longer τ_r. Used to
study membrane viscosity or cytoplasmic crowding.

k_B T Thermal energy (Boltzmann constant × absolute temperature). Higher temperature =

faster rotation = shorter τ_r.

Steady-state r_ss = r■ / (1 + τ■/τ_r). Measured without time resolution. Perrin equation. Useful for
anisotropy quick estimates of molecular mobility.

8.4 Applications of Fluorescence Anisotropy

• Protein–ligand binding: When a small fluorescent ligand (short τ_r, low r_ss) binds to a large protein,
τ_r increases dramatically and r_ss increases. This is the basis of fluorescence polarisation (FP)
assays widely used in drug discovery (high-throughput screening).
• Membrane fluidity: Fluorescent lipid probes (e.g., DPH) inserted into membranes report on lipid chain
order and membrane viscosity via their anisotropy. More ordered (gel phase) membranes have higher
anisotropy.
• DNA hybridisation detection: A fluorescently labelled oligonucleotide probe tumbles fast when
single-stranded (low r). When it hybridises to its complementary strand, the double helix is larger →
slower rotation → higher r.
• Molecular nanometrology: From τ_r, the hydrodynamic volume (and hence size) of any fluorescently
labelled particle can be determined without physical separation.

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CHAPTER 9

Quenching of Fluorescence
Static quenching, dynamic quenching, Stern-Volmer equation, and common quenchers

9.1 What is Fluorescence Quenching?

Any molecule or process that reduces the fluorescence quantum yield is called a quencher or
quenching process. Quenching always involves the creation of an additional non-radiative pathway
for depopulation of the excited state. Two fundamentally different mechanisms exist: static quenching
and dynamic (collisional) quenching.

Static Quenching Dynamic (Collisional) Quenching

collision!

Q M* Q
M + MQ

quencher quencher
excited
fluorophore dark complex

Result: ↓ Intensity, → Lifetime unchanged Result: ↓ Intensity, ↓ Lifetime

Fig 9.1 — Static quenching (left): the quencher Q forms a dark ground-state complex MQ with the fluorophore M
before excitation. Dynamic quenching (right): the quencher Q collides with the EXCITED fluorophore M* during its
lifetime, quenching it without emission.

9.2 Static Quenching

In static quenching, the quencher Q binds to the fluorophore M in the ground state, forming a
non-fluorescent complex MQ. This complex absorbs light but does not emit (either it has a very fast
non-radiative decay, or the complex chromophore is not fluorescent). Only the unbound fluorophore
M is fluorescent.

The equilibrium is governed by an association constant K_s. The Stern–Volmer equation for static
quenching:

F■/F = 1 + K_S[Q] (where K_S = [MQ]/([M][Q]) is the association constant)

Key diagnostic feature: In static quenching, the fluorescence lifetime τ does NOT change with
quencher concentration. Only the intensity decreases. This is because the remaining unbound
fluorophore M is perfectly normal — it has the same lifetime as without quencher. The overall
intensity decreases simply because fewer free M molecules are present.

9.3 Dynamic (Collisional) Quenching

In dynamic quenching, the quencher Q collides with the excited-state fluorophore M* during its
fluorescence lifetime. Upon collision, energy is transferred to Q (or a chemical reaction occurs), and
M* returns to the ground state without emitting a photon. The quenching rate is limited by the diffusion

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of M* and Q in solution (diffusion-controlled).

F■/F = τ■/τ_q = 1 + K_SV[Q] where K_SV = τ■ × k_q

K_SV Stern-Volmer constant. Units: M■¹. K_SV = τ■ × k_q. Larger K_SV = more efficient
quenching.

k_q Bimolecular quenching rate constant. Units: M■¹s■¹. For diffusion-controlled

quenching in water at 25°C, k_q ≈ 10¹■ M■¹s■¹ (the maximum possible rate).

τ■ Fluorescence lifetime WITHOUT quencher.

τ_q Fluorescence lifetime WITH quencher [Q]. Always shorter than τ■ for dynamic
quenching.

F■/F = τ■/τ_q CRUCIAL DIAGNOSTIC: In dynamic quenching, intensity and lifetime decrease by
the SAME factor. This equality (observed by comparing steady-state and
time-resolved data) is the definitive test for dynamic quenching.

9.4 How to Distinguish Static from Dynamic Quenching

Diagnostic Test Static Quenching Dynamic Quenching

Stern–Volmer plot (F■/F vs

Linear Linear
[Q])

Lifetime vs [Q] τ UNCHANGED τ DECREASES (τ■/τ_q = F■/F)

Temperature effect on K_SV DECREASES with T (binding K_SV INCREASES with T (faster
K_SV weaker at high T) diffusion)

UV absorption of complex Changed (complex absorbs) Unchanged

Upward Stern–Volmer curve Indicates both mechanisms present —

9.5 Common Quenchers and Their Mechanisms

Quencher Type Mechanism Common Use

Triplet energy transfer; charge Dissolved oxygen sensor,

O■ (oxygen) Dynamic
transfer photostability test

Probe accessibility in
I■ (iodide) Dynamic Heavy-atom effect; charge transfer
proteins/membranes

Cs■ Charge transfer from excited Probes on surface (Cs■ can't

Dynamic
(caesium) aromatic cross membrane)

Protein buried vs. exposed

Acrylamide Dynamic Electron transfer from excited state
probes

Br■ (bromide) Dynamic Heavy-atom effect Quenching studies

Nitro-compou Static/Dyn Complex formation + electron

DNA intercalation studies
nds amic transfer

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CHAPTER 10

Förster Resonance Energy Transfer (FRET)

Molecular ruler, distance measurement, orientation factor, and biological applications

10.1 What is FRET?

Förster Resonance Energy Transfer (FRET) is a physical process in which the excitation energy
from an excited donor fluorophore (D*) is transferred non-radiatively to a nearby acceptor molecule
(A) through weak dipole–dipole coupling. No photon is emitted or absorbed during this transfer — it
is a direct quantum mechanical coupling between the transition dipoles of D and A.

The theory was developed by Theodor Förster in 1946–1948. FRET is sometimes called
"fluorescence resonance energy transfer" but this is technically incorrect (no fluorescence takes part
in the energy transfer step itself). FRET is an entirely non-radiative quantum process.

FRET occurring (R < R■) No FRET (R >> R■)

R >> R■ (too far)

FRET

dipole-dipole
coupling
Donor Acceptor Donor Acceptor
(D*) (A*)

excited now excited! emits light! stays dark

Fig 10.1 — FRET occurs efficiently when donor and acceptor are close together (R < R■, left panel): the acceptor
gains energy and emits at its own characteristic wavelength (red-shifted from donor). When they are far apart (R >>
R■, right panel), no energy transfer occurs and the donor emits normally.

10.2 The Förster Equation — Distance Dependence

The rate constant of energy transfer k_T depends on the donor–acceptor distance R with an inverse
6th power dependence:

k_T(R) = (1/τ_D) × (R■/R)■

τ_D Fluorescence lifetime of the DONOR in the ABSENCE of acceptor. The "reference
time."

R Actual distance between donor and acceptor (centre-to-centre). This is what FRET
measures.

R■ Förster critical distance (or Förster radius). The distance at which k_T = 1/τ_D, i.e.,
FRET rate equals the natural donor decay rate. At R = R■, FRET efficiency = 50%.

R■■ dependence Because FRET rate goes as R■■, FRET is exquisitely sensitive to distance. A 10%
change in R changes the FRET rate by ~77%. This makes FRET a precise
"molecular ruler" in the 1–10 nm range.

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10.3 Calculating R■ — The Förster Radius

R■■ = (9000 ln(10) κ² Φ■) / (128π■ N_A n■) × J
κ² Orientation factor. Describes the relative orientation of donor and acceptor dipoles.
Ranges from 0 (perpendicular, no transfer) to 4 (exactly parallel and collinear). For
freely rotating molecules in solution, κ² = 2/3 (dynamic average). This is the main
source of uncertainty in absolute distance measurements.

Φ■ Quantum yield of donor fluorescence in the ABSENCE of acceptor. Higher Φ■ →

more energy available for transfer → larger R■.

N_A Avogadro's number (6.022 × 10²³ mol■¹).

n Refractive index of the intervening medium. Typically n = 1.33–1.40 for biological

samples.

J Spectral overlap integral. Measures the overlap between donor fluorescence

spectrum F_D(λ) (normalised) and acceptor absorption spectrum ε_A(λ) (in
M■¹cm■¹). More overlap → larger J → larger R■. Units: M■¹cm■¹nm■.

Typical R■ values For common FRET pairs: CFP/YFP: R■ ≈ 5.0 nm; Cy3/Cy5: R■ ≈ 6.0 nm;
FITC/TRITC: R■ ≈ 5.5 nm. FRET is useful in the 0.5R■ – 2R■ distance range (≈
2–12 nm).

10.4 FRET Efficiency

E = k_T / (k_T + 1/τ_D) = 1 / [1 + (R/R■)■]
Transfer efficiency E varies from 0 (no transfer, R >> R■) to 1 (complete transfer, R << R■). E can be
measured in several ways:

• Donor lifetime method (best): E = 1 − (τ_DA/τ_D). Compare donor lifetime with acceptor present
(τ_DA) to donor lifetime without acceptor (τ_D). Lifetime is concentration-independent, making this
method reliable.
• Donor intensity method: E = 1 − (F_DA/F_D). Compare donor intensity with and without acceptor.
Requires careful correction for differences in donor labelling efficiency.
• Acceptor sensitised emission: Measure the acceptor emission when the donor is excited. Requires
careful calibration for direct acceptor excitation and donor spectral bleed-through.
• Acceptor photobleaching: Photobleach the acceptor with intense light; donor intensity should increase
(dequenching). E = 1 − (F_D_before/F_D_after). Simple but destructive.

10.5 Requirements for FRET

Requirement Why Needed Typical Value

Donor emission must overlap acceptor absorption — J > 10¹² M■¹cm■¹nm■ for useful
Spectral overlap (J ≠ 0)
"resonance condition" FRET

FRET rate drops as R■■; only works at short

Close proximity R < 2R■ (typically < 10 nm)
distances

Dipole orientation κ² ≠ 0; if perpendicular, no FRET κ² = 2/3 assumed for mobile dyes

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Donor quantum yield >

If Φ■ = 0, R■ = 0 and no FRET occurs Φ■ > 0.1 recommended
0

FRET in Living Cells — A Revolution in Cell Biology

The development of genetically encoded FRET biosensors (using fluorescent proteins like CFP and YFP)
has transformed cell biology. By fusing two fluorescent proteins to different parts of a signalling molecule,
changes in FRET report on protein conformational changes, protein–protein interactions, second
messenger concentrations (Ca²■, cAMP), enzyme activities, and more — all in living cells with nanometre
spatial sensitivity.
Example: FRET-based calcium sensors (cameleons) contain a calmodulin-GFP fusion. When Ca²■ binds,
calmodulin wraps around its target peptide, bringing the two FPs closer together → FRET increases → ratio
of YFP/CFP emission reports on [Ca²■] in real time in living cells.

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CHAPTER 11

Semiconductors, LEDs, Quantum Dots & Solar Cells

Band gaps, p-n junctions, quantum confinement, and photovoltaics

11.1 Conductors, Semiconductors, and Insulators

In a solid material, electrons don't occupy discrete atomic orbital energies but instead fill continuous
"energy bands." The two most important bands are the valence band (completely filled with electrons
at 0 K) and the conduction band (empty at 0 K). The energy gap between them is the band gap
(E_g).

Metal Semiconductor Insulator

Conduction
band (empty)
Conduction
band (empty)
Large gap
Energy

Conduction Band gap > 4 eV

(overlaps!) ~1-3 eV

Valence Valence Valence

band (full) band (full) band (full)

Conducts freely Conducts when excited Does not conduct

Fig 11.1 — Energy band diagrams comparing metals, semiconductors, and insulators. Metals have overlapping bands
(no gap) → always conduct. Semiconductors have a small gap (1–3 eV) → can conduct when electrons are
thermally/optically excited across the gap. Insulators have large gaps (>4 eV) → electrons cannot be excited across
the gap by normal means.

Band gap (E_g) Energy difference between the top of the valence band and the bottom of the
conduction band. For a photon to excite an electron across the gap: hν ≥ E_g. Band
gap determines the minimum wavelength of light that a semiconductor can absorb,
and the maximum wavelength of light it emits.

Conduction band Upper energy band, normally empty. Electrons here are free to move through the
crystal → electrical conduction.

Valence band Lower energy band, normally filled. Electrons here are bound to atoms and cannot
move freely.

Electron Negative charge carrier excited to conduction band by photon absorption.

Hole Positive charge "carrier" left behind in the valence band when an electron is excited.
Holes also move through the crystal (as neighbouring electrons fill in).

Exciton A bound electron-hole pair held together by Coulomb (electrostatic) attraction. Has no
net charge. Can move through the crystal. Recombination of exciton emits a photon
with energy ≈ E_g.

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Doping Intentional addition of impurity atoms to change conductivity. n-type doping (e.g.,
phosphorus in silicon) adds extra electrons. p-type doping (e.g., boron in silicon) adds
extra holes.

11.2 p–n Junctions — The Heart of Semiconductor Devices

When p-type and n-type semiconductors are brought into contact, electrons from the n-side diffuse
into the p-side (and holes do the reverse) until an electric field builds up at the junction (the depletion
region) that prevents further diffusion. This junction diode is the building block of nearly all
semiconductor light-emitting devices.

When forward-biased (positive voltage on p-side), electrons and holes are injected into the depletion
region and recombine, emitting photons with energy ≈ E_g. This is electroluminescence — the
working principle of LEDs and diode lasers.

11.3 Light-Emitting Diodes (LEDs)

An LED is a forward-biased p-n junction. When electrons and holes recombine in the active region, a
photon is emitted with energy equal to the band gap. The emission colour is determined by the choice
of semiconductor material:

Band Gap Emission

Material Application
(eV) Colour

Near-infrared
GaAs 1.43 IR remotes, fibre optics, TCSPC lasers
(~870 nm)

Green (~550
GaP 2.26 Traffic lights, displays
nm)

Blue/UV
GaN 3.4 Blue LEDs, white lighting, fluorescence excitation
(~365–450 nm)

2.0–3.4
InGaN Blue–green White LEDs (with phosphor)
(tunable)

AlGaAs 1.6–2.2 Red–orange Red laser pointers, CD players

NOT efficient LEDs (indirect gap — non-radiative

Si, Ge 1.11, 0.67 IR
recombination dominates)

Table 11.1 — Semiconductor materials and their band gap–determined emission wavelengths. LEDs in fluorescence
spectrometers typically use GaN (blue/UV) or InGaAlP (green-red) LEDs as cheap, compact, long-lived excitation
sources.

11.4 Quantum Dots — Size-Tunable Emitters

Nanometre-sized semiconductor crystals (2–10 nm diameter) exhibit a remarkable phenomenon:
their emission colour depends on their SIZE, not just their composition. This is called quantum
confinement: when the crystal is smaller than the natural exciton Bohr radius, the electron and hole
are spatially confined, increasing their kinetic energy (like particle in a box). This raises the effective
band gap:

∆E ∝ 1/r² (confinement energy scales as 1/radius²)

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Smaller quantum dots → larger effective band gap → higher energy (shorter wavelength, bluer)
emission. For CdSe quantum dots: 2 nm → blue/violet (~450 nm); 7 nm → red (~650 nm).

CdSe/ZnS core–shell quantum dots are used in bioimaging because:

• Broad excitation, narrow emission: All QDs of different colours can be excited with a single UV laser.
Each size emits a narrow band. Ideal for multiplexed imaging.
• Exceptional photostability: Unlike organic dyes that photobleach within minutes, QDs can be imaged
for hours. This enables long-term single-molecule tracking.
• High quantum yield: Up to ~80–90% for optimised core-shell structures (ZnS shell reduces surface
defects).
• Broad application range: From confocal microscopy, tumour targeting, biosensors, to QD-LED
displays (QLED televisions).

11.5 Solar Cells — The Reverse of an LED

A solar cell converts light energy into electrical energy using the photovoltaic effect in a p-n junction.
When a photon is absorbed, an electron–hole pair (exciton) is created. The built-in electric field at the
p-n junction separates the electron (moves to n-side) and hole (moves to p-side) before they can
recombine. This charge separation drives a current through an external circuit.

The analogy with photosynthesis is striking: in both systems, light creates excited states, charge
separation occurs at a special site (p-n junction in solar cells; reaction centre in photosystem II), and
the separated charges drive useful chemistry/electricity. In photosynthesis, positive carriers are
protons (H+), negative carriers are electrons; in solar cells, positive carriers are holes, negative
carriers are electrons.

Analogy: Photosynthesis vs. Solar Cell

Light harvesting antenna (chlorophylls/carotenoids) → equivalent to anti-reflection coating +
broad-spectrum absorber in solar cell.
Energy transfer by FRET (Förster coupling) and exciton coupling to reaction centre → equivalent to charge
carrier diffusion to p-n junction.
Reaction centre: special pair of chlorophylls where charge separation occurs → equivalent to depletion
region of p-n junction.
Positive carrier (proton/hole) and negative carrier (electron) separated → drives ATP synthesis (biology) or
electric current (solar cell).
Both systems operate reversibly and share the same recombination process (back-electron transfer in RCs;
current leakage in solar cells).

11.6 Summary Table: Semiconductor Photonic Devices

Device Input Output Key Physics Biological/Spectroscopy Use

Excitation sources in
Electric e■–hole recombination in
LED Light fluorometers, confocal
ity fwd-biased p-n junction
microscopes

Electric Cohere Stimulated emission + optical TCSPC, confocal microscopy,

Laser diode
ity nt light cavity FRET, FCS

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Electric Photogenerated e■–hole Model system for

Solar cell Light
ity separation at p-n junction photosynthesis studies

Tunabl
Quantum Light e light Quantum confinement shifts Bioimaging, single-molecule
dot (UV) emissio band gap with size tracking, QLED
n

Electric Detectors in
Reverse-biased p-n junction;
Photodiode Light al spectrophotometers,
photocurrent ∝ intensity
current fluorometers

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CHAPTER ★

Quick Reference — All Key Equations & Facts

A one-page summary of every important formula in this guide

Topic Formula / Key Fact What Variables Mean

EM radiation E = hν = hc/λ h=Planck's const, ν=freq, c=light speed, λ=wavelength

EM radiation ν = c/λ frequency × wavelength = speed of light

Beer-Lambert A = ε·c·d ε=extinction coeff, c=concentration, d=path length

Quantum yield Φ = k_r/(k_r + k_nr) k_r=radiative rate, k_nr=non-radiative rate

Fluorescence
τ■ = 1/(k_r + k_nr) Inverse of total depopulation rate
lifetime

Lifetime-QY
Φ = τ■/τ_r = τ■ × k_r τ_r = natural radiative lifetime = 1/k_r
relation

Fluorescence
F(t) = F■ exp(−t/τ■) Exponential decay with time constant τ■
decay

Anisotropy r(t) = [I_VV−I_VH] / [I_VV+2I_VH] Parallel minus perp over total intensity

Anisotropy decay r(t) = r■ exp(−t/τ_r) r■≤0.4, τ_r = rotational correlation time

Rotational time τ_r = 4πr³η / (3k_BT) r=radius, η=viscosity, k_BT=thermal energy

Stern-Volmer F■/F = 1 + K_SV[Q] K_SV=τ■·k_q, k_q=bimolecular quench rate

FRET rate k_T = (1/τ_D) × (R■/R)■ τ_D=donor lifetime, R=distance, R■=Förster radius

E=0 when R much greater than R0; E=1 when R much less
FRET efficiency E = 1/[1 + (R/R0)^6]
than R0

FRET efficiency
E = 1 − τ_DA/τ_D τ_DA=donor lifetime with acceptor
(lifetime)

Förster radius R■■ ∝ κ²·Φ■·J/n■ κ²=orientation, Φ■=donor QY, J=spectral overlap, n=RI

Band gap photon hν ≥ E_g for absorption Only photons with energy ≥ band gap are absorbed

QD confinement ∆E ∝ 1/r² Smaller quantum dot radius r → larger effective band gap

Key Timescales to Remember

10■¹■ s (femtoseconds): Light absorption. The fastest process — nuclei don't move.
10■¹² s (picoseconds): Vibrational relaxation, internal conversion, solvent relaxation.
10■■ s (nanoseconds): Fluorescence emission. FRET if close enough. Rotational diffusion of small
molecules.
10■■ s (microseconds): Intersystem crossing, some phosphorescence, rotational diffusion of large
proteins.
10■³ s (milliseconds) to seconds: Phosphorescence from triplet state.

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Suggested Reading
Lakowicz J.R. — Principles of Fluorescence Spectroscopy, 3rd Ed., Springer (2006). The definitive
reference.
Valeur B. — Molecular Fluorescence: Principles and Applications, Wiley-VCH (2002). Excellent and
readable.
Cantor C.R. & Schimmel P.R. — Biophysical Chemistry Part II: Techniques, Freeman (1980).
Saleh B.E.A. & Teich M.C. — Fundamentals of Photonics, 2nd Ed., Wiley (2007).
Birks J.B. — Photophysics of Aromatic Molecules, Wiley-Interscience (1970). Classic text.

All terms defined • Every diagram explained • Beginner friendly