Transcript Lecture 7 Part II

Lecture 7
Part II
Tissue optical properties (Absorption)
Acknowledgement
Slides on absorption spectroscopy based on
Lecture prepared by Dr. Nimmi Ramanujam,
University of Wisconsin at Madison
Tissue optical properties
• There are two main tissue optical properties which
characterize light-tissue interaction and determine
therapeutic or diagnostic outcome:
– Absorption coefficient: ma (cm-1)


•
•
•
ma=sa*Na =A/L*ln10
sa=atomic absorption cross section (cm2)
Na=# of absorbing molecules/unit volume (cm-3)
A=Absorbance
L=sample length
Area
sa = Qa Area
Area
ss = Qs Area
– Scattering coefficient: ms (cm-1)
 ms=ss*Ns
 ss=atomic scattering cross section (cm2)
• Ns=# of scattering molecules/unit volume (cm-3)
Qa – absorption efficiency
Qs – scattering efficiency
Population of Energy Levels
•
At any finite T, molecules will be distributed among available E levels due to thermal
agitation
•
The exact distribution among energy levels will depend upon the temperature and
separation between energy levels according to Boltzmann statistics
nupper
nlower

 exp  E

kT
k=1.38*10-23 JK-1 (Boltzmann’s constant)
E = separation in energy level
Energy Levels
Population of energy levels
Absorption Spectroscopy
Population of energy levels
•
Net absorption depends on the difference between the populations of
the energy levels
•
The more populated the ground state, the more intense the net
absorption is
•
Two factors that influence absorption are the energy level spacing and
the temperature
Energy Levels
UV Visible absorption spectroscopy involves transitions between
electronic energy levels
UV ~ 200-400nm
Vis ~ 400-750nm
For  < 200nm (absorption by oxygen in air is significant) – vacuum UV
II. Absorption Spectroscopy
Electron transition rules
•
Energy is absorbed by transitions induced between different electronic
energy states of a molecule
•
Transition occurs only if there is an induced dipole moment
•
Resonance condition; the frequency of radiation must be equal to the
frequency of the dipole
E = hf
where, E = separation of energy states,
h = Planck’s constant, f = frequency
h=6.626x10-34 m2kg/s
II. Absorption Spectroscopy
Absorption strength
• The transition probability from electronic state m to state n is given by:
2
e
d
n

|
m
|
 n
mn
Where, mmn is the transition dipole moment and eu is molar extinction
coefficient at particular frequency, n
II. Absorption Spectroscopy
B. Franck Condon principle
•
The time for an electronic transition is: t=1/n =/c ~ 10-15 s (at 420 nm)
•
Franck Condon principle: electronic transitions occur so rapidly that
during the transition the nuclei are static
•
Thus, all electronic transitions are vertical (internuclear distance
doesn’t change)
II. Absorption Spectroscopy
Energy
B. Franck Condon principle
Internuclear distance
II. Absorption Spectroscopy
B. Spectral line widths
•
Electronic transitions to different vibrational energy levels
•
With enough of these transitions the absorption spectrum looks more
like a smooth curve rather than a line
Absorption spectroscopy
• Lifetime broadening
– A molecule spends only a short amount of time in
its excited state, which defines the lifetime, t, of
the state.
– If a molecule changes states at a rate of 1/t, then
the energy levels become blurred and the
corresponding spread in the energy levels around
E is (Heisenberg uncertainty principle)
E 

t
t = 10-12s
  0.08nm (for  = 400nm)
Absorption spectroscopy
• Doppler broadening
– With the thermal motion of the atoms, those atoms traveling
toward the detector with a velocity v will have transition
frequencies which differ from those of atoms at rest by the
Doppler shift.
wDoppler 
2wo
kT
c 2 ln 2
mo
 wo=frequency for atom at rest
– mo=atomic mass
http://www.walter-fendt.de/ph11e/dopplereff.htm
II. Absorption Spectroscopy
C. Biological chromophores
1.
The peptide bonds and amino acids in proteins
• The p electrons of the peptide group are
delocalized over the carbon, nitrogen, and
oxygen atoms. The n-p* transition is typically
observed at 210-220 nm, while the main p-p*
transition occurs at ~190 nm.
• Aromatic side chains contribute to absorption at
> 230 nm
2. Purine and pyrimidine bases in nucleic acids and
their derivatives
3. Highly conjugated double bond systems
III. Biological Chromophores
1. Amino acids
Molecule
 (nm)
e (x10-3) (cm2.mol-1)
Tryptophan
280, 219
5.6,47
Tyrosine
274,222,193
1.4,8,48
Phenylalanine
257,206,188
0.2,9.3,60
Histidine
211
5.9
Cystine
250
0.3
III. Biological Chromophores
Tryptophan absorption
is used as the basis for
protein concentration
measurements
III. Biological Chromophores
2. Bases and their derivatives
Molecule
 (nm)
e (x10-3) (cm2.mol-1)
Adenine
260.5
13.4
Adenosine
259.5
14.9
NADH
340,259
6.23, 14.4
NAD+
260
5.9, 18
FAD+
450
unknown
III. Biological Chromophores
3. Highly conjugated double bond systems
Spectrum is often in the visible region (electrons less restricted, energy
levels closer)
Metal porphyrin ring system is mainly responsible for the
color in heme proteins
The most intense band is called the Soret band after its
discoverer
3. Hemoglobin Absorption Spectra
• Major absorption peaks around
400 and 540-580nm are due to
pp* transitions of the porphyrin
ring
•The band in HbO2 around 900 nm
arises from charge transfer
between the porphyrin protein and
the Fe(II) atom
Beta carotene absorption spectrum
160000
120000
80000
Series1
Absorption due to pp*
transitions
40000
0
380
480
580
680
Melanin absorption
• eumelanin
A black-to-dark-brown insoluble material found in human
black hair and in the retina of the eye.
• pheomelanin
A yellow-to-reddish-brown alkali-soluble material found
in red hair and red feathers. A variety of low molecular
weight pheomelanins are called "trichromes".
Tissue absorbers
Therapeutic window: 600-900 nm region where not much absorption takes place
II.Absorption Spectroscopy
D. Concentration of molecules
•
Absorption depends on the number of molecules in which transitions
are induced.
•
Absorption spectra can be used quantitatively
•
The effect of sample concentration on the absorption is the basis of
most analytical applications
Molar absorption crosssection=sa*NA (NA is
Avogadro’s number)
If

I0
If
dI
 ln( I f )  ln( I 0 )  ln( )
I
I0
1/log10(e)=2.303
Determining Concentrations (C)
Absorbing sample
of concentration C
Io
I
Path length, L
Io
log 10
 e  CL
If
II. Absorption Spectroscopy
F. Instrumentation and measurement
1.
2.
Typical instrument
Experimental techniques
II. Absorption Spectroscopy
Chopper
BS
S
Monochromator
White
Light
M
R
I
BS
Electronics
Io
Sample
Chamber
Detector
M
Absorption Spectroscopy
2. Experimental technique
a. What can be measured?
b. Modes of measurement
c. Parameters
d. Base line
e. Sample and reference measurements
f. Considerations
II. Absorption Spectroscopy
a. What can be measured?
Absorbing, non-scattering samples
Liquid samples contained in cuvettes
II. Absorption Spectroscopy
b. Modes of measurement
Absorption (A) or optical density (O.D.)
A = log10 (Io/I);
% Transmission (%T)
%T = I/Io; A = log10 (1/ T);
II. Absorption Spectroscopy
c. Setup parameters
A or %T (y-axis) - A
Wavelength range (x-axis) – 350 to
650 nm
Wavelength increment – 5 nm
Averaging time –0.1 s
Spectral band pass – 2 nm
II. Absorption Spectroscopy
d. Record Baseline (for specific setup parameters)
Removes variations in:
• Throughput of reference and sample optics
• Detector sensitivity
Run baseline for setup parameters and no sample inside
spectrophotometer
Store base line file
Run a wavelength scan to make sure that absorbance (A) is zero at
all wavelengths
Absorbance (O.D.)
Baseline Correction
0
Wavelength (nm)
II. Absorption Spectroscopy
e. Sample and reference measurements
Use identical cuvettes in sample and reference arms and ensure
that they have the same path length; cuvette should have low
absorption in region of interest
Fill the sample cuvette with the sample of interest
Fill the reference cuvette with everything in the sample cuvette,
except the absorber
Run a wavelength scan of the sample
Absorbance of Sample
Absorbance (O.D.)
4
0
Wavelength (nm)
II. Absorption Spectroscopy
f. Considerations
Dynamic range (limits on detector sensitivity)
Typical dynamic range – 4 O.D.
If sample has an absorbance of more than dynamic range of
instrument – flat line
Turbid Samples
Light lost to scattering looks like absorption
Need integrating sphere accessory to make accurate absorption
measurements
Absorbance (O.D.)
Dynamic Range
4
Wavelength (nm)