FLUORESCENCE
1
SPECTROSCOP
Y
(FLUOROMETR
Y)
BY GROUP 2
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Introduction
Fluorometry is based on a diagram
designed and explained by a
professor called Alexander
Jablonski. And this diagram is
known as the Jablonski diagram
which illustrates the transitions of
electrons between energy states
resulting in the emission of light
after a light source is passed through
the electrons (Lakowicz, 1992;
Albani, 2007).
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Jablonski Diagram
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The Fluorometer
A fluorescent substance (either naturally
fluorescent or labeled with a fluorescent tag)
absorbs light of a specific wavelength called
the excitation wavelength.
The absorbed energy excites the electrons of
the molecule to a higher energy state.
When the electrons return to the ground state,
a part of the energy is released as fluorescent
light at a longer wavelength, known as the
emission wavelength.
The emitted light is measured by a detector,
and the intensity is proportional to the
concentration of the analyte.
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Principle of Fluorometry
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Components of a Fluorometer
Light Source: Xenon lamp, mercury vapour
lamp, LEDs.
Filters/Monochromators: Select excitation
and emission wavelengths.
Sample Holder: Quartz or glass cuvettes.
Detector: Photomultiplier Tubes (PMTs),
highly sensitive.
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Photomultiplier Tube
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Calibration & Standardization
Calibrations
Instrument Preparations
Blank Measurement
Standard Curve Generation
Quality control and verification
Manufacturer specific protocols
Standards
Fluorescein Solution
Specific analyte being measured
Raman band of water
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Analytical Procedure
Load Sample
Measure
Fluorescence
Record Data
Plot Standard Curve
Calculate
Concentration
Background
Subtraction
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Types of Analytes Measured
Biological Analytes eg Retinol
Fluorescent dye analytes eg
fluorescein, rhodamine
Environmental analytes
Industrial analytes eg aromatic
hydrocarbons
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Clinical/Diagnostic Applications
Immunoassays and Serology employs fluorescent
labelled antibodies/antigens to detect analytes.
Molecular Diagnostics employs fluorescently labelled
probes like taqman to quantify viral loads bacteria
pathogens and genetic mutation
Flow Cytometry where cells in suspension are passed
by laser, their light scattering and fluorescent is then
measured
Galamsey
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Quality Control in Fluorometry
Regular calibrations and standardization
Monitor detector and source of light
stability
Control ph and temperature.
Always blank and prepare negative controls
Keep record of all tests
Clean cuvettes, lenses and all equipment
before and after tests.
Ensure proper preparation of sample
concentration and purity.
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Sources of Error
Procedural and
human errors
Instrumental errors
Sample-related errors
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Limitations of Fluorometry
Instrumental costs: Fluorometers are more
expensive, and complex compared to
spectrophotometers.
Matrix effects: Biological samples such as blood, and
serum may contain interfering substances that mask
fluorescence.
Quenching effects: Fluorescence can be reduced by
oxygen, pH changes, or other molecules interfering
with emission.
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Recent Advances: Detectors
Ultra-SensitiveDetectors
(PMTs → APDs → SPADs)
ie Avalanche photodiodes
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Recent Advances: Detectors
Quantum Dots & Nanoparticle Fluorophores is more
sensitive than all other fluorophores. The Nanoparticle
fluorophores allows for multi-color detection in a single
assay assay.
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Recent Advances: Detectors
Smartphone-Based
Fluorometers
Thisis modern smartphones
are combined with LED
excitation modules,
miniaturized detectors, AI
image processing to perform
fluorometric analysis.
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Conclusion
Fluorometry is a
highly sensitive and
specific technique that
relies on the
absorption and
emission of light by
fluorescent molecules.
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References
Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy (3rd ed.). Springer.
[Link]
Valeur, B., & Berberan-Santos, M. N. (2013). Molecular Fluorescence: Principles
and Applications (2nd ed.). Wiley-VCH. ISBN 9783527411357
Albani, J. R. (2007). Principles and Applications of Fluorescence Spectroscopy.
Wiley-Blackwell. ISBN 9781405138918
Guilbault, G. G. (1990). Practical Fluorescence: Theory, Methods, and Techniques
(2nd ed.). Marcel Dekker. ISBN 9780824783150
Thermo Fisher Scientific. (2006). The Molecular Probes Handbook: A Guide to
Fluorescent Probes and Labeling Technologies (11th ed.). Life Technologies
Corporation.
Lakowicz, J. R. (1992). Fundamentals of fluorescence spectroscopy. In Biochemical
and Instrumental Analysis (pp. 1–45). Springer.