Figure from W.L. Jorgensen and L.

Salem, The Organic Chemist’s

Book of Orbitals, New York, Academic Press, 1973.
Figure from W.L. Jorgensen and L. Salem, The Organic Chemist’s
Book of Orbitals, New York, Academic Press, 1973.
•  Absorption of electromagnetic radiation is a
fast process

•  typically on the order of 10-15s

•  i.e. the time required for a photon to cross a

distance of 10Å (typical size of a molecular
transition dipole).

Frank Condon Principle Followed:

•  Electronic motions/transitions occur much
faster than nuclear motions
(i.e. 10-16 – 10-14 s versus 10-13 – 10-12 s)
and therefore occur most favourably when
the nuclear structure of the initial and final
states are most similar.
•  i.e. spin quantum numbers don’t change
during electronic transitions.
•  Resonance conditions must exist for
absorption of electromagnetic radiation

•  For Electronic excitation, the resonance

condition is defined as a match between
the energy of the photon (E = hv) and the
energy difference between the S0 and the
excited electronic (S1, S2), vibrational state
(v1, v2, v3,…) and rotational state (not
shown) to which the molecule is excited.

•  The electric field vector of the incident

radiation must be in alignment with the
molecular transition dipole for absorption
to occur.
•  The transition dipole moment is
a vector representing the the
spatial change in charge density
between the ground and excited
state of a molecule.

•  Molecules preferentially absorb

light which is polarized (electric
field) parallel to the direction of
their transition dipole.

•  There is zero probably of

absorbing light polarized
perpendicular to the transition
dipole moment.

Figure from Olympus Microscopy Resource Center: www.olympusmicro.com

Kasha’s Rule: Only the lowest excited singlet state (S1) or
Triplet State (T1) need be considered for most
photochemical reactions owing to rapid radiationless
conversion of higher-order electronic states (S2,T2, etc.) to
the S1 and T1 state.
R The fluorescence lifetime (τ) is the time after which the fluorescence of a
sample population has decayed to 1/e or 37% of its initial intensity:

where I is the intensity and t is the time after excitation.

Some systems have multi-component lifetimes:

n

I(t) = ∑ ai Io exp( − t τ i )
i=1

where ai is the fractional contribution to the total intensity.

The fluorescence lifetime provides information about local environment of the

fluorophore.
€
•  90° Geometry with respect to
excitation and emission path
through the sample
•  Rhodamine B Cell is a Quantum
Counter, used to correct for source
power variability as a function of
wavelength.
•  Quantum counter consist of a highly
concentrated solution of fluorophore
that will absorb any wavelength of
light incident on it with near 100%
efficiency over the wavelength
range of interest and emit light with
100% efficiency (i.e. Φ = 1.0) at a
characteristic emission wavelength
that will be of an intensity directly
proportional to the source intensity
at the excitation wavelength.
•  Corrected spectra achieved by
using the ratio of the Sample PMT
to that of the Ref. PMT
•  Typically, 3g/L Rhodamine B in
ethylene glycol provides for effective
correction over the excitation range
from 220 to 620nm, with emission at
630nm
Figure from “Instrumental Analysis” G.D. Christian, J.E. O’Reilly,
Eds., Second Ed., Allyn and Bacon, Inc., Boston, 1986, p.
 Sources for fluorescence spectroscopy: Arc
Lamps
•  Quartz tubes filled with high-pressure gas
(e.g. mercury or xenon). Operate at several
hundred degrees Celsius.

•  Powered by a DC current (the arc) between

two electrodes. Typically, 50-200 W.

•  High voltage strike needed to ignite.

•  Ionization of gaseous vapour to create

plasma.

•  Free electrons and positive ions stream to

cathode and anode. Collisions excite gaseous
atoms which emit light to relax.

•  Mercury arc lamps contain small amount of

liquid mercury and an inert gas such as argon
or xenon. The arc produces enough heat to
produce mercury vapour. Xenon arc lamps
contain only xenon gas.

Figure from Olympus Microscopy Resource Center: www.olympusmicro.com; photo: R. Algar

 Sources for fluorescence spectroscopy: Arc
Lamps
•  Mercury does not provide
even intensity across the
spectrum. Xenon is better in
this respect, but has only
weak intensity in the UV
region.

•  Mercury and xenon lamps

are good high intensity
polychromatic sources for
fluorescence spectroscopy.

•  When high intensity

monochromatic sources are
required, lasers are used.

Figure from Olympus Microscopy Resource Center: www.olympusmicro.com

 Sources for fluorescence spectroscopy: Arc
Lamps

coherent

incoherent
(different freq.)

incoherent
(different phase)

•  Light Amplification by Stimulated Emission of Radiation

•  High-intensity
•  Coherent (same frequency and same phase)
•  Monochromatic (bandwidth < 0.01 nm)
•  Continuous wave (CW) or pulsed (picosecond and femtosecond pulses are
possible)
 Sources for fluorescence spectroscopy: Arc
Lamps
The laser cavity:

Gain/Lasing Medium
Laser Beam

Fully Reflective Mirror Partially Reflective Mirror

Pump Source

•  Pump source: electrical, radiant (laser, flashlamp), chemical energy

•  Gain or Lasing medium
–  must be pumped for lasing action
–  determines type of laser
–  determines wavelength of laser
–  laser output can be frequency-doubled by certain non-linear crystals
(e.g. BBO = β-barium borate, KDP = potassium dihydrogen phosphate)
Sources for fluorescence spectroscopy: Arc
Lamps
Pumping
•  Supply energy to create excited states
within lasing medium
•  Inverted population is required to sustain
laser operation (excited state is more
populated than ground state)
•  Rapid vibrational relaxation (heat
produced) to metastable excited
electronic state

Spontaneous emission
•  i.e. fluorescence and electronic
relaxation
•  Emission is random in direction and
timing, yielding incoherent light
•  Some photons are directed such that
they traverse the cavity between the
mirrors
Sources for fluorescence spectroscopy: Arc
Lamps
Stimulated emission
•  Excited species are hit by fluorescence
photons traversing the cavity
•  This results in the emission of a second
photon which is precisely in phase and in
the direction of the incident photon
•  Coherent radiation builds up as stimulated
photons traverse the cavity and yield
further stimulated emission
Absorption
•  Ground state species in the lasing medium
can absorb photons to produce the
metastable excited state
•  The number of photons from stimulated
emission must exceed the number of
photons re-absorbed by the lasing medium
for a net gain (i.e. amplification)
•  The necessary population inversion is
achieved/maintained by pumping
•  Four-level systems are more efficient than
three-level systems
 Sources for fluorescence spectroscopy: Arc
Lamps
•  Gas lasers
–  A gaseous atom, ion, molecule, or excimer is the lasing medium
–  e.g. HeNe (632.8 nm), Argon ion (454.6, 488.0, 514.5 nm),
Nitrogen (337.1 nm), XeF (351 nm)

•  Solid-state lasers
–  The lasing medium is often an ion in a host crystal (e.g. Nd3+ in
yittrium aluminum garnet or Ti3+ in sapphire)
–  e.g. Nd:YAG (1064 nm), Ti:sapphire (650-1050 nm),

•  Semiconductor lasers
–  Based on p-n junctions
–  e.g. GaN, GaAs, AlGaAs

•  Dye lasers
–  Organic fluorophores in solution are the active lasing medium
–  Continuously tunable over a certain wavelength range (typically
40-80 nm)
–  Pumped by another laser

•  Also: chemical lasers, metal-vapour lasers, free-electron lasers

 Sources for fluorescence spectroscopy: Arc
Lamps

•  Photoelectric effect to generate a current

•  Amplification through an electron cascade effect at a series of dynodes

•  Current proportional to light intensity

•  Spectral response not uniform; depends on cathode material

Figure from Olympus Microscopy Resource Center: www.olympusmicro.com

Sources for fluorescence spectroscopy: Arc
Lamps
The PIN photodiode is a three layer structure consisting
of: a layer of p-type semiconductor; a layer of intrinsic
(undoped) semiconductor; and a layer of n-type
semiconductor.

In the p-type layer, positive charges (holes) are mobile

and allow electric current to flow.

In the n-type layer, negative charges (electrons) are

mobile and allow electric current to flow.

In the intrinsic layer, there are no charge carries and

thus the layer is an insulator, preventing current from
flowing through the diode.

Absorption of light in the intrinsic layer creates charge

carriers by the excitation of electrons, making the device
conductive and generating an electric current.

The magnitude of the current is proportional to the

amount of incident light.

Semiconductors will be covered in much greater

detail in a later lecture.
 Sources for fluorescence spectroscopy: Arc
Lamps
•  Similar design to PIN photodiode, except
the intrinsic region is thinner.

•  While operating voltages for reversed

biased PIN photodiodes are on the order
of 0-10 V, avalanche photodiodes are
operated at 102-103 V.

•  Electrons generated upon absorption of a

photon are accelerated by the high
potential difference and produce
secondary charge carriers in collisions.

•  The amplification is analogous to dynodes

in a PMT.

•  The gain is typically 102-103, providing

better sensitivity than PIN diodes.
However, dark currents and noise are
higher. The response is also non-linear.
Figure from Olympus Microscopy Resource Center: www.olympusmicro.com
•  Quenching species can include (a) (b) Cl- (c) I- (d) (e) Cl- (f) I-
halide salts (e.g. Cl-, Br-, I-), H+,
oxygen, some metal ions (e.g.
Mn2+, Cu2+), acrylamide, amines,
nitrate, histidine, cysteine,
halogenated hydrocarbons, and
more.

Quinine sulfate: Fluorescein:

•  Heavy atoms often quench by
inducing intersystem crossing (a)  0.1 M H2SO4 d)  0.1 M NaOH
(especially Br - and I-). Metals ions
(b)  0.1 M H2SO4 e)  0.1 M NaOH
can quench by electron transfer. + 0.5 M NaCl + 0.5 M NaCl

(c)  0.1 M H2SO4 f)  0.1 M NaOH

•  Not all species are quenched by
+ 0.5 M KI + 0.5 M KI
the same species, or by the same
mechanism(s).
The orientation factor depends on the
angles and distance between the
transition dipoles for the donor and
acceptor.
Applications of Fluorescence
Quenching and Energy Transfer
Methods:
Wavelength-Shifting Molecular Beacons

From: S. Tyagi, S.A.E. Marras and F. Russell

Kramer “Wavelength-shifting molecular beacons”
Nature Biotechnology,18 (2000) 1191-1196.