Fluorescence spectroscopy analyzes the fluorescent properties of molecules. It works by exciting a molecule to a higher electronic state using a photon, causing it to emit a photon of lower energy as it returns to the ground state. The difference in wavelengths allows detection of emission photons. Key aspects covered include the principles of absorption and emission, instrumentation used, and different types of data that can be recorded such as fluorescence measurements, steady state techniques, and fluorescence anisotropy/polarization.

1. Fluorescence Spectroscopy
Mr.Halavath Ramesh
Department of Chemistry
Loyola College –Chennai
University of Madras

2. Objectives
• 1.What is fluorescence spectroscopy ?
• 2. Brief introduction about fluorescence spectroscopy
• 3. Principle and theory of fluorescence spectroscopy
• 4. Instrumentation
• 5. Different types of data recorded by fluorescence spectroscopy
a) Fluorescence Measurement
b) Steady state fluorescence techniques
c) Fluorescence anisotropy or Fluorescence polarization
d) Luminescence quantum yield
e) Ratio metric fluorescence
f) Excitation Emission Matrix(EEM)

3. a) A-TEEM(Acquire absorbance transmittance and a fluorescence excitation
emission matrix)
b) Singlet oxygen measurement
c) Fluorescence life time techniques
d) Enzymatic reaction rate calculation
e) Spectral measurents
f) Quantitative measurement
g) Fixed wavelength measurents
h) Time course measurents
i) 3-D spectra measurents
j) Absorbance measurents
k) Phosphors measurents
6) How to calculate signal to noise ratio
7) Factors affecting of fluorescence
7) Applications

4. 1. What is fluorescence spectroscopy ?
Fluorescence Spectroscopy analyzes fluorescence from a
molecules based on its fluorescent properties. Fluorescence is a
type of luminescence caused by photons exciting a molecules
raising it to an electronic excited state.
2. Brief introduction about the fluorescence spectroscopy
Fluorescence is the absorption and emission of light of
two different frequencies. Typically, a lower wavelength of
incident light is absorbed from one direction, and a higher
wavelength of light is emitted in all directions. Fluorescent
molecules absorb light at a certain wavelength and emit at
another.

5. With a known incident light wavelength, samples can be
identified by their fluorescent emission spectra. Because
fluorescence occurs on a molecular scale, it is the only
spectroscopic technique capable of identifying single molecules.
Molecules have various states referred to as energy levels.
Fluorescence spectroscopy is primarily concerned with
electronic and vibrational states. Generally, the species being
examined has a ground electronic state (a low energy state) of
interest, and an excited electronic state of higher energy. Within
each of these electronic states there are various vibrational
states

7. 3. Principle and theory of fluorescence spectroscopy
In fluorescence, the species is first excited, by absorbing a photon, from
its ground electronic state to one of the various vibrational states in the excited
electronic state. Collisions with other molecules cause the excited molecule to
lose vibrational energy until it reaches the lowest vibrational state from the
excited electronic state. This process is often visualized with a Jablonski
diagram.
Fluorescence is the result of a three-stage process that occurs in
certain molecules called fluorophores or fluorescent dyes. A fluorescent probe is
a fluorophores designed to localize within a specific region of a biological
specimen or to respond to a specific stimulus. The process responsible for the
fluorescence of fluorescent probes and other fluorophores.
Stage 1.
Excitation. A photon of energy hvEX is supplied by an external source such as an
incandescent lamp or a laser and absorbed by the fluorophores, creating an -

8. Excited electronic singlet state ( S1’) .This process distinguishes fluorescence from
chemiluminescence, in which the excited state is populated by a chemical reaction.
Stage 2. Excited-State Lifetime. The excited state exists for a finite time ( typically 1-10
nanosecond) .During this time, the fluorophores undergoes conformational changes and
is also subject to a multitude of possible interactions with its molecular environment.
These processes have two important consequences. First, the energy of S1’ is partially
dissipated, yielding a relaxed singlet excited state(s1) from which fluorescence emission
originates. Second, not all the molecules initially excited by absorption (stage 1) return
to the ground state (so) by fluorescence emission. Other processes such as collision
quenching, Fluorescence Resonance Energy Transfer(FRET) and intersystem crossing
may also depopulated S1.The fluorescence quantum yield, which is the ratio of the
number of fluorescence photons emitted (stage 3) to the number of photons absorbed
(stage1), is a measure of the relative extent to which these processes occur.

9. Stage 3. Fluorescence Emission. A photon of energy hvEM is emitted ,
returning the fluorophores to its ground state S0. Due to energy dissipation
during the excited-state lifetime, the energy of this photon is lower, and
therefore of longer wavelength, than the excitation photon hvEX.The difference
in energy or wavelength represented by (hvEx-hvEM) is called the stokes shift.

11. The stokes shift is fundamental to the sensitivity of fluorescence
techniques because it allows emission photons to be detected against a low
background isolated from excitation photons. In contrast absorption
spectrometry requires measurement of transmitted light relative to high
incident light levels at the same wavelength.
Fluorescence spectra:
The entire fluorescence process is cyclical. Unless the fluorophores is
irreversibly destroyed in the excited state( an important phenomenon known
as photo bleaching), the same fluorophores can be repeatedly excited and
detected. The fact that a single fluorophores can generate many thousands of
detectable photons is fundamental to the high sensitivity of fluorescence
detection techniques.
Instrumentation:
Four essential elements of fluorescence detection system can be identified
from the preceding discussion:
1. An excitation sources
2. A fluorophores
3. Wavelength filter to isolate emission photons from excitation photons and
4. a detector that registers emission photons and produces a recordable
output.

13. 5. Different types of data recorded by fluorescence spectroscopy.
a) Fluorescence Measurement : 5 different types. Luminescence, emission, excitation,
synchronous and single beam.
Most molecules occupy the lowest energy state at room temperature,
known as the ground state. Within this ground state are vibrational levels.
Before becoming excited, many molecules occupy the lowest vibrational level.
The absorbed photon causes the molecule to adopt a higher vibrational
energy state when a molecule absorbs a certain wavelength of light.
The molecules then collide with other molecules, causing it
to lose its vibrational energy and return to the lowest vibrational level of the
excited state. The molecule can then return to the ground state vibrational
levels.
When the molecule returns to the ground state, it emits a photon of
light at a wavelength different to the wavelength that excited it. This is when
the molecule exhibits fluorescence.

14. Fluorescence is measurable by fluorometers. A fluorometers is an
instrument designed to measure the various parameters of fluorescence,
including its intensity and wavelength distribution of the emission after
excitation. Chemists use this to identify properties and the amount of
specific molecules in a sample.
Most molecules occupy the lowest energy state at room
temperature, known as the ground state. Within this ground state are
vibrational levels. Before becoming excited, many molecules occupy the
lowest vibrational level.
The absorbed photon causes the molecule to adopt a higher
vibrational energy state when a molecule absorbs a certain wavelength of
light. The molecules then collide with other molecules, causing it to lose its
vibrational energy and return to the lowest vibrational level of the excited
state. The molecule can then return to the ground state vibrational levels.
When the molecule returns to the ground state, it emits a photon of light at a
wavelength different to the wavelength that excited it. This is when the
molecule exhibits fluorescence.
Fluorescence is measurable by fluorometers. A fluorometers is an
instrument designed to measure the various parameters of fluorescence,
including its intensity and wavelength distribution of the emission after
excitation. Chemists use this to identify properties and the amount of
specific molecules in a sample.

15. b) Steady state fluorescence techniques
Because fluorescence intensity depends on the concentration of the fluorescent
molecule, standard concentration curves can be generated easily and used to
determine concentrations of the same molecule in unknown samples.
This is useful in quenching experiments, where additives decrease the
intensity of the fluorophores in a systematic way. Concentration curves can also be
created to study how other molecules interact with things like proteins, and can be
used for tracking protein structural changes, folding, unfolding, association and
dissociation systematically.

18. C). Fluorescence anisotropy or Fluorescence polarization
Fluorescence anisotropy or fluorescence polarization is a measurement of the
changing orientation of a molecule in space, with respect to the time between the
absorption and emission events. Absorption and emission indicate the spatial
alignment of the molecule’s dipoles relative to the electric vector of the
electromagnetic wave of excitation and emitted light, respectively. In other words, if
the fluorophores population is excited with vertically polarized light, the emitted light
will retain some of that polarization based on how fast it is rotating in solution. The
faster the orientation motion, the more depolarized the emitted light will be. The
slower the motion, the more the emitted light retains the polarization.
Fluorescence anisotropy the light emitted by chromospheres by light unequal
intensity different access of polarization. Considering partially polarized light
travelling across x- axis's the intensity Iz and Iy can be measured by a detector and
polarizer positioned on x- axis.
For polarization measurement, polarizer are inserted into the excitation and
emission light paths. With the excitation polarizer fixed, the emission polarizater can
be rotated to measure the perpendicular (I) and parallel (I)components of the
fluorescence emission.

21. d) Luminescence quantum yield
Any process that brings about a decrease in the sample emission is referred to as
fluorescence quenching . For proteins the type of quenching we will be primarily
concerned with is that arising from collision between the quencher and the
fluorophores. The fluorescence quantum yield (φ) is the ratio of the number of
photons emitted to the number absorbed. The fluorescence intensity (F) is
proportional to the amount of light absorbed.

23. e) Ratio metric fluorescence:
Ratiometric fluorescence is the method where intensities at two or more
wavelengths of an excitation or emission spectrum are measured to detect changes to
local environment. Typically, a probe is used that is specifically sensitive to an
environmental parameter such as ion concentration, pH, viscosity, or polarity.
The application of ratiometric dyes for finding probe-sensitive
properties such as ion concentration can be used by measuring spectra or kinetics.

24. Excitation Emission Matrix(EEM):
A measurement becoming more widely used in the field of fluorescence
spectroscopy is the excitation emission matrix, or EEM. An EEM is a 3D scan,
resulting in a contour plot of excitation wavelength vs. emission wavelength
vs. fluorescence intensity. EEMs are used for a variety of applications where
multi-component analysis is required and are often referred to as providing a
molecular fingerprint for many different types of samples.
Some of the first published uses of EEM spectroscopy were
in the 1980’s where the technique was used to study tryptophan fluorescence
in low density lipoproteins in human blood serum (Koller, 1986) and to
investigate fluorescent components in human plasma from tumor patients
(Leiner, 1986)

26. A-TEEM(Acquire absorbance transmittance and a fluorescence excitation emission
matrix)
A-TEEM spectroscopy refers to the ability to simultaneously acquire Absorbance,
Transmittance and a fluorescence Excitation Emission Matrix (A-TEEM) of a
particular sample. HORIBA pioneered this technique with the patented Aqualog and
Duetta system, which combines A-TEEM spectroscopy with simultaneous
multichannel CCD detection to provide extremely fast results.
A-TEEM spectrometers can be used for fluorescence EEMs or
for absorbance measurements for multi-component analysis, but its real
power is derived from the fact that the EEMs collected by the instrument are
corrected for inner filter effect. This means they are true and accurate
representations of the molecules of interest over a much broader and more
useable concentration range (typically up to ~2 absorbance units). Therefore,
these EEMs allow for much more precise fingerprinting than is possible with
an EEM collected from a traditional scanning fluorometers.

28. Singlet oxygen measurement
Singlet oxygen is produced from a photosensitizer molecule reacting with ground
state oxygen. Molecular triplet states are chemically reactive due to their long decay
times and the presence of unpaired valence electrons. Reactivity with ground state
oxygen (3O2) will yield singlet oxygen (1O2). Singlet oxygen has an emission spectrum
peak around 1270 nm, which results in photons emitted from a triplet state
(phosphorescence).
The lifetime of singlet oxygen is very long, but quenched
when singlet oxygen reacts or comes in contact with different species.
Instruments used to detect singlet oxygen typically need near-infrared
detectors such as InGaAs detectors (PMT, Analog, or Arrays) and emission
gratings that are blazed for efficiency in the NIR wavelength region.

29. The production of singlet oxygen involves the irradiation
of a photosensitizer molecule in the presence of oxygen and subsequent
reaction of the excited triplet state photosensitizer with the ground state
oxygen resulting in the creation of excited singlet state oxygen. These
include molecules such as Rose Bengal, transition metal complexes (as
shown below), porphyrins, fluorescein, and others. (DeRosa, 2002) Exciting
these molecules at their peak absorbance wavelengths can lead to the
reactivity of these molecules in the excited state with ground state oxygen,
producing singlet oxygen.
Mechanisms that produce singlet oxygen are important
to photodynamic therapeutics, anti-cancer agents and other skin treatments.
The reactivity of singlet oxygen itself can be damaging to organic molecules,
including those in biological systems, but the reactivity, if controlled is also a
potential method of cancer therapy and photodynamic medicine.
In photodynamic therapy, a patient with malignant cancer has a fiber optic light
either inserted into, or placed just outside their body. This light emits visible wavelengths. It
reacts with photosensitizer molecules (photodynamic drugs), provides energy to oxygen in the
microenvironment, which, in turn, generates non-toxic singlet oxygen species to shrink or kill
the tumor.

30. Singlet oxygen species are chemically reactive chemical varieties containing
oxygen.
Also known as light therapy, photodynamic therapy is a treatment for cancers that are
near the surface of a body’s tissue, where the light can act on the chemical substances.
It is not like radiation therapy, which uses radicals and a toxic light source. Nor does it
cause systematic side effects like chemotherapy. The light and molecules
photodynamic therapy uses are non-toxic and benign.

31. Fluorescence Lifetime Techniques
1. What is Time Correlated Single Photon Counting or TCSPC?
TCSPC stands for time-correlated single photon counting. It is a
method of using the timing of a pulsed excitation source, like a laser or
LED, with the timing of the arrival of single photons on a detector to
reconstruct the lifetime decay over many events (repetition of pulses and
photons detected). This technique is based on the fact that the probability of
detecting a single photon at time, t after an exciting pulse is proportional to
the fluorescence intensity at time t.

32. Can I measure kinetic processes with fluorescence lifetimes?
Using the kinetic TCSPC mode, individual measurements in as little
as 1 ms can be made and up to 10,000 measurements can be seamlessly acquired. As
long as a fluorescence lifetime change occurs, then this approach, rather than
intensity, can be used to follow a kinetic process.
Obviously, a sufficient number of photons are needed to be able to
analyze the data. This can be enhanced by using a very high repetition rate, but the
lifetime and time range needs to be considered, so as not to re-excite the sample
before it has completely decayed. The lifetime data can then be used to construct
kinetic traces for the process.

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