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Lecture 25
Review
Medical Optics and Lasers
• Application of optical methods to medicine
• Why optical methods?
– Non-invasive
– No side-effects
– High resolution
– Functional information
– Real-time information
– Cost effective
– Portable
Medical Optics and Lasers
• Optical methods based on interactions of light with
matter (biological sample)
– Basic Principles
– Absorption
– Scattering
• Multiple scattering/Diffusion
• Single scattering
–
–
–
–
Fluorescence
Microscopy
Optical Coherence Tomography
Photodynamic therapy
Light as a wave
  wavelength (meters)
Period
k
2
 propagation vector(converts distance to angle )

  period (sec onds )

1


c

  2 
time


z, t   o cost  kz
frequency (cycles / s or Hz )
2

 angular frequency (converts time to angle
Phase=f=t-kz
Monochromatic (only one
wavelength/frequency)
waves traveling in phase
Monochromatic (only one
wavelength/frequency)
waves traveling out of phase
Matter: Basic principles
• The basic unit of matter is the atom
• Atoms consist of a nucleus surrounded by
electron(s)
• It is impossible to know exactly both the
location and velocity of a particle at the
same time
• Describe the probability of finding a
particle within a given space in terms of a
wave function, y
Particle in a box
• The particle confined in a one-dimensional box of length
a, represents a simple case, with well-defined
wavefunctions and corresponding energy levels
2
nx
y n ( x) 
sin
a
a
2
2
nh
En 
2
8ma
• n can be any positive integer, 1,2,3…, and represents
the number of nodes (places where the wavefunction is
zero)
• Only discrete energy levels are available to the particle
in a box----energy is quantized
Atomic orbitals: Quantum numbers
• Principal quantum number, n
–Has integral values of 1,2,3…… and is related to size and energy of the
orbital
• Angular quantum number, l
–Can have values of 0 to n-1 for each value of n and relates to the angular
momentum of the electron in an orbital; it defines the shape of the orbital
• Magnetic quantum number, ml
–Can have integral values between l and - l, including zero and relates to
the orientation in space of the angular momentum.
• Electron spin quantum number, ms
–This quantum number only has two values: ½ and –½ and relates to spin
orientation
Molecular orbitals
• Molecular orbitals (chemical bonds) originate from the overlap of
occupied atomic orbitals
• Bonding molecular orbitals
– are lower in energy than corresponding atomic orbitals (stabilizes the
molecule)
• Anti-bonding orbitals
– are higher in energy than corresponding atomic orbitals and destabilizes
the molecule
 s bonds
– involve overlapping s and p orbitals along the line joining the nuclei of
the bond-forming atoms
  bonds
– involve p and d orbitals overlapping above and below the line joining the
nuclei of the bond-forming atoms
Hybrid orbitals and conjugated
bonds
•
The four 2p orbitals can combine to form these  orbitals, arranged according to
energy, with the lowest energy  orbital at the bottom.
•
•
Can you think of a set of wavefunctions that may describe what is going on?
These are similar to the wavefunctions we got for a particle in the box, with the
length of the box corresponding to the length of the carbon chain
Principles of laser operation
• Stimulated emission
• Population inversion
• Laser cavity
– Main components
– Gain and logarithmic losses
– Two vs. three vs. four-level systems
– Properties of laser light
– Homojunction/heterojunction semiconductor
lasers
Cell and Tissue basics
• Basic components of a cell
–
–
–
–
–
Nucleus
Mitochondria
Lysosomes
ER
Golgi
• Basic components of epithelial tissues
– Types of epithelia
– Connective tissue
– Basement membrane
Light-tissue interactions
Optical methods are based on different types of light-matter interactions to
provide structural, biochemical, physiological and morphological
information
• scattering
– elastic scattering
• multiple scattering
• single scattering
Epithelium
• absorption
• fluorescence
Connective Tissue
Tissue optical properties
• There are two main tissue optical properties which
characterize light-tissue interactions 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
– Scattering coefficient: ms (cm-1)
 ms=ss*Ns
 ss=atomic scattering cross section (cm2)
• Ns=# of scattering molecules/unit volume (cm-3)
Tissue absorption
Major tissue absorbers include: Hemoglobin, lipids (beta carotene), melanin, water,
proteins
Oxy and deoxy hemoglobin have distinct spectra. Optical measurements can provide
information on tissue oxygenation, oxygen consumption, blood hemodynamics
Tissue scattering spectra exhibit a
weak wavelength dependence
Structural proteins constitute major tissue scattering centers. Cell nuclei and
membrane rich organelles (e.g. mitochondria) also scatter light
Fluorescence spectra provide a
rich source of information on
tissue state
1.5
450
FAD
Excitation (nm)
1
Protein expression
400
NADH
350
Collagen
300
Trp
350
400
450
0.5
Structural integrity
0
Metabolic activity
-0.5
500
550
600
-1
Emission (nm)
Courtesy of Nimmi Ramanujam, University of Wisconsin, Madison
Which optical method to use?
• Three main questions:
– What is the required depth of penetration?
– What is the acceptable resolution?
– What type of information is needed?
Imaging methods
Resolution (log)
Diffuse optical tomography and
spectroscopy
1 mm
100 mm
10 mm
OCT
Confocal/multi-photon
microscopy
Standard
1 mm
microsc
Penetration depth (log)
100 nm
4-Pi/STED
1 mm
10 mm
100 mm
1 mm
1 cm
10 cm
Spectroscopic methods: Functional information
•
Diffuse reflectance
–
–
–
Penetration depth: microns to centimeters depending on wavelength, souce/detector separation, light
delivery/collection geometry
Resolution not well defined
Absorption
•
•
•
•
–
Scattering
•
•
•
Tissue oxygen saturation
Arterial/venus oxygen saturation
Oxygen consumption
Hemodynamics
Structural changes of the matrix
May be nuclear changes
Light Scattering
–
–
Penetration depth: microns to hundreds of microns depending on how highly scattering is the sample
Inelastic scattering (Raman)
–
Elastic scattering
•
•
Information: biochemical composition
Information
–
–
•
Resolution
–
•
Size distribution of major cell scattering centers (e.g. nuclei, mitochondria)
Cell/tissue organization
Potential to detect size changes on the order of 100 nanometers
Fluorescence
–
–
Penetration depth: microns to centimeters depending on implementation, i.e. wavelength, sample optical
properties, source/detector geometry
Endogenous fluorescence
•
•
–
–
Cell and tissue biochemistry (NADH/FAD, tryptophan, porphyrins, oxidized lipids
Tissue structure (collagen, elastin)
Induced fluorescent protein expression (molecular specificity)
Fluorescent tags
•
•
Antibodies (antigen expression)
Molecular beacons (enzyme activity)
Diffuse optical tomography and
spectroscopy
• Applications
– Breast cancer detection
– Brain function
– Oxygen consumption by muscles
– Arthritis
– atherosclerosis
– Pulse oximeter
– Jauntice (billirubin) test for neonates
Light scattering spectroscopy
• Cancer detection
• Detection of pre-cancerous changes
– Barrett’s esophagus
– Uterine cervix
– Oral cancers
• Biopsy guidance
• Non-invasive patient monitoring
Optical coherence tomography
• Non-invasive detection of morphological
changes
• Applications
– Cancer detection
– Eye diseases
– Atherosclerosis
– Developmental biology
Raman scattering
• Applications
– Atherosclerosis
– Cancer detection
– Blood composition
– Bacterial detection
Tissue fluorescence
• Applications
– Cancer detection
• Pre-cancer detection
• Guide to biopsy
• Patient monitoring
– Atherosclerosis detection
– Bacterial infection (?)
Microscopy
• Cell microscopy
– Understand basic cell functions in healthy and
disease states
– Understand role of specific proteins and cell
component interactions
• Tissue/intravital microscopy
– Understand cell matrix interactions that govern
disease development, progression and regression
• Drug/therapy development and optimization
• Early detection
Multi-modality optical detection
• Goal: Acquire morphological and
biochemical information to achieve more
sensitive/specific detection
• Combined use of fluorescence, diffuse
reflectance and light scattering
• Combined use of Raman and fluorescence
• Combined use of OCT and fluorescence
• Combined use of reflectance and
fluorescence imaging
Photodynamic therapy
• Example of light-based therapeutic
method
• Light used to achieve cytotoxicity
• Optical methods can also be used to tailor
dosimetry to patient and monitor/predict
therapeutic efficacy
• Used for treating a variety of conditions
from cancer to acne to atherosclerosis
Optical methods are a powerful tool for
understanding human health and improving
disease detection and treatment
20
0
18
0.5
mm
1
Enlarged
Nuclei, %
40-50
30-40
20-30
10-20
16
14
0
0.5
1
mm
Adenoma
1.5
Non-dysplastic
mucosa
x (cm)
1.5
12
10
8
6
4
2
0
0
2
4
6
8
y (cm)
10