Fluorescence Methods
Introduction:
Fluorescence is a phenomenon in which certain substances absorb light of a short
wavelength (usually ultraviolet or blue light) and then emit light of a longer wavelength
(usually visible light). This property is used to study and detect very small quantities of
materials, especially in biological and chemical samples.
Fluorescence methods are powerful techniques used in physics, chemistry, biology, and
material science to study:
• Molecular structure
• Molecular interactions
• Energy transfer
• Dynamics of excited states
• Environmental effects on molecules
Principle of Fluorescence:
When light of a suitable wavelength falls on a fluorescent substance, its molecules absorb
the energy and move to a higher (excited) energy level. This excited state is unstable, so
the molecules quickly return to their normal (ground) state by releasing some of the
absorbed energy as visible light. This emitted light is called fluorescence.
The key idea is that the emitted light always has less energy and hence a longer
wavelength than the absorbed light. This difference is known as the Stokes Shift.
Stokes Shift:
The Stokes shift is the difference between the wavelength (or energy) of absorbed light
and emitted light.
For example, if a molecule absorbs light at 350 nm (ultraviolet) and emits light at 450 nm
(blue light), the difference of 100 nm is the Stokes shift.
This shift occurs because part of the absorbed energy is lost as heat or vibration inside the
molecule before the emission takes place.
A larger Stokes shift means that it is easier to detect fluorescence since the emitted light
is clearly separated from the excitation light.
Types of Fluorescence Methods:
1. Steady-State Fluorescence:
In this method, we measure the intensity of the emitted light when the sample is
continuously illuminated by a light source.
It gives information about the amount of fluorescent material, its environment, and
interactions with other molecules.
This is the most common and simple type of fluorescence measurement.
How it works
• A continuous light beam (UV/Visible) shines on the sample.
• Molecules absorb photons → go to excited state.
• Molecules release energy → fluorescence emission.
• The detector collects this emission as long as the light is ON.
The system is in a steady condition — excitation and emission happen continuously.
2. Time-Resolved Fluorescence:
This method measures how long the fluorescence lasts after the excitation light is turned
off.
The duration for which the molecule stays in the excited state before emitting light is
called its fluorescence lifetime.
Measuring this lifetime helps in understanding molecular behavior, energy transfer, and
environmental effects on fluorescence.
How it works
• A nano-second or pico-second laser pulse excites the sample.
• Molecules go to excited state.
• Fluorescence slowly decays.
• Detector measures the decay curve (Intensity vs Time).
This gives us the fluorescence lifetime.
Fluorescence lifetime
Lifetime is the average time a molecule stays in the excited state before emitting
[Link] lifetimes:
• 1 ns to 10 ns for organic molecules
• Picoseconds for fast processes
Applications:
• Nanoparticle Characterization
Fluorescence is used to study the size, shape, and surface properties of nanoparticles by
observing how they absorb and emit light.
• Energy Transfer Studies in Nanomaterials (FRET)
Time-resolved fluorescence helps measure energy transfer between nanoparticles, useful
in designing solar cells and optical sensors.
• Imaging of Nanostructures
Fluorescent dyes and quantum dots allow high-resolution imaging of nanostructures
inside cells and materials.
• Monitoring Chemical Reactions on Nano Surfaces
Fluorescence helps track fast reactions happening on the surface of nanomaterials, such
as catalysis, drug release, or sensing.