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Spectroscopy Lecture 5 Fluorescence Application

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35 views20 pages

Spectroscopy Lecture 5 Fluorescence Application

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ii.ifc19
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© © All Rights Reserved
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I.

Principles of fluorescence
• Fluorescence energy transfer (FRET)
Molecule 1 Molecule 2
Fluorescence Fluorescence
DONOR ACCEPTOR

Absorbance
Absorbance

Wavelength
Non radiative energy transfer – a quantum mechanical process of
resonance between transition dipoles
Effective between 10-100 Å only
Emission and excitation spectrum must significantly overlap
Donor transfers non-radiatively to the acceptor
II. Quantum yield and lifetime
1a. Quantum yield of fluorescence
• Quantum yield of fluorescence, Ff, is defined as:
number of photons emitted
Ff 
number of photons absorbed
• In practice, is measured by comparative measurements with reference
compound for which has been determined with high degree of accuracy.

• Ideally, reference compound should have


– the same absorbance as the compound of interest at given excitation wavelength
– similar excitation-emission characteristics to compound of interest (otherwise,
instrument wavelength response should be taken into account)
– Same solvent, because intensity of emitted light is dependent on refractive index
(otherwise, apply correction
F uf I uf n 2 (u )
 
F sf I sf n 2 ( s)

– Yields similar fluorescence intensity to ensure measurements are taken within the
range of linear instrument response
II. Quantum yield and life time
1b. Fluorescence lifetime
• Another definition for Ff is kr
Ff 
k
where kr is the radiative rate constant and Sk is the sum of the
rate constants for all processes that depopulate the S1 state.
• In the absence of competing pathways Ff=1
• Radiative lifetime, tr, is related to kr 1
r 
kr
• The observed fluorescence lifetime, is the average time the
molecule spends in the excited state
1
f 
k
II. Quantum Yield and Lifetime
2a. Characteristics of quantum yield
• Quantum yield of fluorescence depends on biological
environment
• Example: Fura 2 excitation spectrum and Indo-1
emission spectrum and quantum yield change when
bound to Ca2+
Fura-2 changes in response to Indo-1 changes in response to
varying [Ca2+] varying [Ca2+]
II. Quantum yield and lifetime
2b. Characteristics of life-time
• Provide an additional dimension of information
missing in time-integrated steady-state spectral
measurements
• Sensitive to biochemical microenvironment,
including local pH, oxygenation and binding
• Lifetimes unaffected by variations in excitation
intensity, concentration or sources of optical loss
• Compatible with clinical measurements in vivo

Courtesy of M.-A. Mycek, U Michigan


II. Quantum Yield and Lifetime
3a. Fluorescence emission distribution
• For a given excitation wavelength, Exc
the emission transition is Emm
distributed among different

Intensity
vibrational energy levels

• For a single excitation wavelength,


can measure a fluorescence Emission Wavelength (nm)
emission spectrum
II. Quantum Yield and Lifetime
3b. Heisenberg’s uncertainty principle
• Values of particular pairs of observables cannot be
determined simultaneously with high precision in
quantum mechanics

• Example of pairs of observables that are restricted in


this way are:

• Momentum and position


• Energy and time
II. Quantum Yield and Lifetime
3c. Heisenberg’s uncertainty principle

h
• Momentum and position : p x x 
2

• Energy and time: h


E t 
2
II. Quantum Yield and Lifetime
3d. Effect on fluorescence emission
• Suppose an excited molecule emits fluorescence in
relaxing back to the ground state

• If the excited state lifetime, t is long, then emission will


be monochromatic (single line)

• If the excited state lifetime, t is short, then emission


will have a wider range of frequencies (multiple lines)
Large t – small E Small t – large E
Exc Emm Exc Emm
Intensity

Emission Wavelength (nm) Intensity Emission Wavelength (nm)


III. Fluorescence Intensity
1. Fluorescence intensity expression
2. Fluorescence spectra
III. Fluorescence Intensities
1a. Fluorescence intensity
The fluorescence intensity (F) at a particular excitation
(lx) and emission wavelength (lm) will depend on the
absorption and the quantum yield:

F x , m   I A x  m 


where,
IA – light absorbed to promote electronic transition
f – quantum yield
III. Fluorescence Intensities
1b. From the Beer-Lambert law, the absorbed intensity
for a dilute solution (very small absorbance)

I A  x   2.303I o  x CL


where, for  CL << 1
x
Io – Initial intensity
e – molar extinction coefficient
C – concentration
L – path length
III. Fluorescence Intensities
1c. Fluorescence intensity expression
The fluorescence intensity (F) at a particular excitation
(lx) and emission wavelength (lm) for a dilute solution
containing a fluorophore is:

Fx , m   I o 2.303 x CL m 


where,
Io – incident light intensity f – quantum yield
C – concentration e – molar extinction
L – path length coefficient
III. Fluorescence Intensities
1d. Measured fluorescence intensity
If we include instrument collection angle:

Fx , m   I o 2.303 x CL m Z


where,
Z – instrumental factor
Io – incident light intensity
e – molar extinction coefficient
C = concentration
L – path length
III. Fluorescence Intensities
2a. Fluorescence spectra
• Emission spectrum
– Hold excitation wavelength fixed, scan emission
– Reports on the fluorescence spectral profile
» reflects fluorescence quantum yield, fk(lm)

Fx , m   I o 2.303 x CL m Z


III. Fluorescence Intensities
2b. Fluorescence spectra
• Excitation spectrum
– Hold emission wavelength fixed, scan excitation
– Reports on absorption structure
» reflects molar extinction coefficient, e(lx)

Fx , m   I o 2.303 x CL m Z


III. Fluorescence Intensities
(a) (b)

Fixed Excitation Wavelength

Fluorescence Intensity
Fixed Emission Wavelength
Fluorescence Intensity

Excitation Wavelength (nm) Emission Wavelength (nm)


III. Fluorescence Intensities
2c. Fluorescence spectra
• Composite: Excitation-Emission Matrix
» Good representation of multi-fluorophore solution
III. Fluorescence Intensities

Excitation Wavelength (nm)


Fixed Excitation Wavelength
Fluorescence Intensity

Emission Wavelength (nm)


Emission Wavelength (nm)

Emission spectrum Excitation-emission matrix

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