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2013-02-27
Biophotonics@Lunduniversity 1
Biomedicinsk Optik
STEFAN ANDERSSON-ENGELS
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w w w . a t o m i c . p h y s i c s . l u . s e / b i o p h o t o n i c s
Lund University Medical Laser Centre
► Science and Engineering Faculties– Physics
– Applied Biochemistry
– Analytical Chemistry
– Chemical Physics
– Electrosciences
– Mathematical Statistics
► Medical Faculty– Oncology
– Dermatology and Venereology
– Obstetrics and Gynaecology
– Oto-Rhino-Laryngology
– Surgery
– Neurosurgery
– Ophthalmology
– Urology
– Radiation Physics
– Diagnostic Radiology
– Biomedical Engineering
– Pathology
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Biophotonics@Lunduniversity 2
Biomedicinsk Optik
STEFAN ANDERSSON-ENGELS
BI
OP
HO
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CS
@L
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DU
NI
VE
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IT
Y2012-10-28
3
Biomedicinsk Optik
INNEHÅLL
BI
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HO
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CS
@L
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DU
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Y2012-10-28
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► Introduktion och definitioner
► Ljusutredning i vävnad
► Diagnostiska Tillämpningar
► Behandlingstillämpningar
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Biophotonics@Lunduniversity 3
Biomedicinsk Optik
INTRODUKTION OCH DEFINITIONER
BI
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ina Reistad 2012-10-22
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What is Tissue Optics ?
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Tissue Optics
Investigating PrincipleSubject of Interest
+ = Tissue Optics
• Propagation of Light• Constituents of Tissue• Interaction between Light and Tissue• Diagnostic and Therapeutic Implications
We are interested in …
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MEDLINE Papers with “Tissue Optics” Keywords
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1966-1970 1971-1975 1976-1980 1981-1985 1986-1990 1991-1995 1996-2000 2001-2005 2006-2010
Nu
mb
er o
f P
ub
lica
tio
ns
Medicinska tillämpningar av lasrar
BI
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Medicinska tillämpningar av lasrar
► Parallellt
► Monokromatiskt
► Koherent
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Vad är en laser?
► Monokromatiskt
► Hög fotondensitet
► Riktningsbart
Grundtillstånd
Exciterat tillstånd
λλ
λ
Light Amplification by Stimulated Emission of Radiation
Spegel Spegel
Aktivt material
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Biophotonics@Lunduniversity 6
Medicinska tillämpningar av lasrar
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► Kirurgi
► “Blödningsfri” kniv, bränna, förånga
► Diagnostik
► Pulsoximetri
► Genomlysning, fluorescens, etc
► Fotokemisk behandling
► “Photodynamic therapy” (PDT)
Medicinsk Laserterapi
BI
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► Laserkirurgi
– Ögon (Ar-jon-, Nd:YAG-, Excimer-lasrar)
– Hud (CO2-, Färgämnes-, Rubin-, Ar-jon-lasrar)
– Allmän kirurgi (Nd:YAG-, diod-, CO2-lasrar)
► Hypertermi behandling
► Fotodynamisk tumörterapi
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Biophotonics@Lunduniversity 7
Light – Electromagnetic Radiation
► Soto Thompson, PhD Thesis, 2004
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Ionizing versus optical radiation
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Ionizing Radiation Optical Radiation
h1 h2
Ene
rgy
Ene
rgy
Ene
rgy
h1
h2
Molecular energy level diagram Molecular energy level diagram
Electron shell model Electron shell model
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Light transport in tissueLight transport in tissue
• Scattering, s [m-1]
• Absorption, a [m-1]
s >>a Diffusion
Tissue
Lightsource
• Scattering phasefunction
Refractive index n [-]
Absorptions-koefficient (cm-1)
Wavelength (nm)
0.1
1
10
100
Absorption
500 900 1100 1300
Blod
700
Vatten
Absorption i vävnad
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Absorptions-koefficient (cm-1)
Wavelength (nm)
0.1
1
10
100
Absorption
500 900 1100 1300
Blod
700
Absorption i vävnad
Spridning
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Why do sub-dermal blood vessels look blue? Why do sub-dermal blood vessels look blue?
Tissue optics is complex. One particular observation that is not completely obvious is why sub-dermal blood vessels look blue and not red, as one might have suspected.
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Light‐tissue interactions and photophysical processes
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SpecularReflection
Refraction
DiffuseReflection
Diffuse and specular reflectanceDiffuse and specular reflectance
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Parallel polarisation Perpendicular polarisation
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Light‐tissue interactions and photophysical processes
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Scattering Emission
AbsorptionRefraction
DiffuseReflection
Elastic• Linear• Non‐linearInelastic• Raman• Doppler shifted
Remission photoplethysmography (PPG)
► Courtecy: Prof Janis Spigulis, Riga
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Android: Instant Heart Rate
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p(s,s´) is the probability function for a scattering from direction s to direction s´
It is generally assumed that the scattering probability depends only on (the cosine of) the angle between s and s´:
Scattering phase functionScattering phase function
The g‐factorThe g‐factor
The g-factor is defined as cosg cosg
This is the parameter usually used in tissue optics to describe the anglular distribution of the light scattering
g=0g=0.5
g=0.8
Photon
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Scattering
25
Rayleighscattering
Mie scattering
Raman scattering
Elastic scattering Inelastic scattering
No energy change Energy change
scattered wave ≠ incident wave
Probes vibrational bonds of the molecule
scattered wave = incident wave
Probes static structure of material
Internal energy levels of atoms and molecules are excited
Rayleigh Scattering
264
1I
)cos1(R
N8II 2
24
24
0
LightSource Detector
N = number of scatterers = polarizabilityR = distance from scatterer
Properties of Rayleigh scattering:• Scattering from very small particles ≤ λ/10• Scattering at right angles is half the forward intensity• The strong wavelength dependence enhances the
short wavelengths(Rayleigh scattering is inversely related to the fourth power of the wavelength of the incident light)
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Mie Scattering
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► Size of particles comparable or larger than the wavelength, Mie scattering predominates
► Because of the relative particle size, Mie scattering is not strongly wavelength dependent
► Forward directional scattering
Reduced Scattering Coefficient
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Each step involves isotropic scattering. Such a description is equivalent to description of photon movement using many small steps 1/µs that each involve only a partial deflection angle
Useful for description of photon propagation in diffuse regime
1 iso-scattering step = 1/(1-g) aniso-scattering steps
sss
o
10.0)g1('
2690.0cosg
'
1'mfp
1mfp
ss
Example:
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Elastic Scattering: Biological Scatterers
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Mitochondria
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• 1 mm in size, folded lipid membranes, membranes 9 nm thick
• contains metabolic cofactors NAD, FAD used for proton pump over membrane to generate ATP
• Refractive index mismatch between lipid and water causes scattering
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Collagen fibers and fibrils
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• Cross Links, hydroxylysine pyridinoline and lysyl perydinoline are fluorescent
67 nm
tropocollagen
head groups
Fibers: 2-3 mm in diameter; composed of smaller fibrils (d = 4 nm; l = 300 nm)
Rayleigh scattering
(visible and UV range)
strong Mie scattering
(electron micrograph)
Fibrils: composed of tropocollagen molecules, have banded pattern (67 nm period), optical “crystal” 2nd harmonic generators, periodic structure contributes to...
Rayleigh och Mie spridning
4
1
Wikipedia (2008
Rayleigh – spridare mycket mindre än ljusvåglängden
Mie – sfäriska spridare av godtycklig storlek
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Vad händer med ljuset inne i handen?
• Vilken färg harljuset som kommerut?
• Varför ser man ingaskuggor av benen ihanden?
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Biomedical Optics
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Cuvette filled with water. HeNe laser beam coming in from left
From clear liquid to diffuse media
Increase scattering by adding droplets of milk-like material.
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Light propagation in turbid media
Abs
orpt
ion
ScatteringChromophores (absorbing molecules)
Scattering elements (cells, organellesfibers, etc.)
Incident Light
Tissue
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UV IrradiationUV Irradiation
Visible IrradiationVisible Irradiation
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NIR IrradiationNIR Irradiation
IR IrradiationIR Irradiation
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Light interaction volumeLight interaction volumeVisibleVisibleUVUV
Red - NIRRed - NIR IRIR
Wavelength matters!
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Hand exposed with red and green laser light
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Fluorescencsdiagnostik
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Fluorescensdiagnostik
Vad är fluorescens?
absorption fluorescens
ener
gi
λ
Inte
nsi
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lase
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flu
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What is fluorescence?
Molecularenergy
Absorptionof light
Fluorescence emission
One fluorophore – 109 photons/sec
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Influenced by a varying external EM field
Fluorescence emission
Broad emission
Fluorescence: Tissue autofluorescence Protoporphyrin IX
J. Johansson, Dissertation thesis, LTH (1993)., af Klinteberg et al. (1999)
337 nm excitation
400 500 600 700
Carotene
NADHElastin
Collagen
Wavelength (nm)
Flu
ores
cenc
e in
tens
ity [
a.u.
] 405 nm excitation
500 550 600 650 700 750
Wavelength (nm)
Protoporphyrin IX
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Property Definition SignificanceFluorescenceexcitation spectrum
A plot of excitation wavelength versus fluorescence intensity generated by a fluorophore.
Use excitation light as close to the peak of the excitation spectrum of the fluorophore as possible.
Absorption spectrum
A plot of wavelength versus absorbance of a chromophore or fluorophore.
The absorption spectrum of a fluorophore is usually very similar to the fluorescence excitation spectrum.
Fluorescenceemission spectrum
A plot of emission wavelength versus fluorescence intensity generated by a fluorophore.
Fluorescence emission spectroscopy is the most straightforward basis for distinguishing several fluorophores (incl. tissue autofluorescence).
Extinction coefficient (EC)
Capacity for light absorption at a specific wavelength.
Fluorescence signal (“brightness”) is proportional to the product of the extinction coefficient (at the relevant excitation wavelength) and the fluorescence quantum yield.
Fluorescence quantum yield (QY)
Number of fluorescence photons emitted per excitation photon absorbed.
See “Extinction coefficient”.
Fluorescencelifetime
Time from a short excitation pulse till the fluorescence emission has dropped to 1/e
May be important to distinguish different fluorophores. Is also a parameter that can vary drastically depending on the microenvironment.
Quenching Loss of fluorescence signal due to short-range interactions between the fluorophoreand the local molecular environment
Loss of fluorescence is reversible to the extent that the causative molecular interactions can be controlled. Important in FörsterResonance Energy Transfer (FRET).
Photobleaching Destruction of the excited fluorophore due to photosensitized generation of reactive oxygen species (ROS), particularly singlet oxygen (1O2).
Loss of fluorescence signal is irreversible if the bleached fluorophore population is not replenished (e.g., via diffusion). Extent of photobleaching is dependent on the duration and intensity of exposure to excitation light.
Spectroscopic properties of fluorescent dyes
Fluorescence lifetime
Andersson-Engels et al IEEE JQE (1990)
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Växelverknings-koefficient (cm-1)
Wavelength (nm)
0.1
1
10
100
Absorption
500 900 1100 1300
Blod
700
Viktigaste grafen!!
Spridning
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Vatten
Thanks for the attention, Questions?
