University of Cyprus Biomedical Imaging and Applied Optics Laboratory Light-Tissue Interaction

University of Cyprus Biomedical Imaging and Applied Optics Laboratory Light-Tissue Interaction Costas Pitris, MD, PhD KIOS Research Center Department of Electrical and Computer Engineering University of Cyprus

Introduction Interaction between Light and Tissue Reflection Refraction Absorption Scattering Fluorescence Light Source Tissue Optical Signal Depends on Constituents of tissue Optical properties of tissue Propagation of light 2

Absorption Extraction of energy from light by a molecular species Diagnostic applications: Transitions between two energy levels of a molecule that are well defined at specific wavelengths could serve as spectral fingerprint of the molecule Various types of Chromophores (light absorbers) in Tissue Wavelength-dependent absorption Tumor detection and other physiological assessments (e.g. pulseoximetry) Therapeutic applications: Absorption of energy is the primary mechanism that allows light form a source (laser) to produce physical effects on tissue for treatment purpose Lasik (Laser Assisted in situ Keratomileusis) Eye Surgery Skin defect and tattoo removal Photodynamic Therapy (PDT) 3

Absorption Absorption occurs when the photon frequency matches the frequency associated with the molecule s energy transition Ep=hf = Ε=E2-E1 Electrons absorb the energy of the light and transform it into vibrational motion The absorption of a photon results in: quantized change in charge separation quantized excitation of vibrational modes Electrons interact with neighboring atoms convert vibrational energy into thermal energy E p =hf Heat E 2 E 1 E 2 E 1 4

Absorption Each electronic energy levels is associated with many vibrational energy levels Vibrational Energy Level UV and Visible Light High energy Promotes transition between electronic energy levels Potential Energy ir UV Electronic Energy Level S 1 S 0 Infrared (IR) Lower energy Promotes transitions between vibrational energy levels 5

Absorption Absorption Coefficient, μ a [1/cm] N a a a N a = concentration σ a = absorption crosssectional area Absorption mean free path, l a [cm] l a 1 a Represents the average distance a photon travels before being absorbed 6

Absorption Transmission and Absorbance (macroscopic view) Transmission T I I o Absorbance (attenuation, or optical density) Io A log( T) log I 7

Absorption Lambert Beer Law: The linear relationship between absorbance and concentration of an absorbing species. Relates μ α, transmission, and absorbance I I e o a b I O = The intensity entering the sample at z = 0 [w/cm 2 ] I = The intensity of light leaving the sample [w/cm 2 ] b = pathlength traveled in the sample [cm] 8

Absorption Absorbers in Tissue NIR NIR Hemoglobin Lipids Water VISIBLE * Absorbers that fluoresce when excited in the UV-VIS UV UV-VIS DNA Hemoglobin Lipids Structural protein* Electron carriers* Amino acids* 9

Absorption UV Absorption Protein, amino acid, fatty acid and DNA absorption dominate UV absorption Protein Dominant non-water constituent of all soft tissue, ~ 30% Absorption properties determined by peptide bonds and amino acid residues Peptide excitation about λ = 190 nm Amino acids absorption at λ = 210-220 nm and 260 280 nm DNA Absorbs radiation for λ 320 nm Large water absorption λ< 180 nm Peptide Amino Acid 10

Absorption Infrared Absorption Protein IR absorption peaks at 6.1, 6.45, and 8.3 μm due to amide excitation Absorption depth 10 μm in λ = 6-7 μm region Water absorption peak at 0.96, 1.44, 1.95, 2.94 and 6.1 μm Absorption depth ~ 500 mm at λ = 800 nm <1 μm at λ=2.94 μm 20 μm throughout λ 6 μm 11

Scattering Change of direction of propagation and/or energy of light by a molecular species Diagnostic applications: Scattering depends on the size, morphology, and structure of the components in tissues (e.g. lipid membrane, collagen fibers, nuclei). Variations in these components due to disease would affect scattering properties, thus providing a means for diagnostic purpose Therapeutic applications: Scattering signals can be used to determine optimal light dosimetry and provide useful feedback during therapy 12

Scattering Purely absorbing Photon pathlength = L With Scattering Photon pathlength >> L L 13

Scattering Why is the sky blue, clouds white, and sunsets red? Blue skies are produced due to scattering at shorter wavelengths Visible light (violet & blue) are selectively scattered by O2 and N2 much smaller than wavelengths of the light violet and blue light has been scattered over and over again When light encounters larger particles (cloud, fog), Mie scattering occurs Mie scattering is not wavelength dependent appears white Cigarette smoke, too At sunset The light must travel over a longer path in the atmosphere Blue/green is scattered away and only red/orange (scattered less) reaches your eyes 14

Scattering Mechanism for Light Scattering Light scattering arises from the presence of heterogeneities within a bulk medium Physical inclusions Fluctuations in dielectric constant from random thermal motion Principal parameters that affect scattering Wavelength, λ Relative refractive index of particles Particle radius Shape and orientation Types of scattering: Elasstic vs. Inelastic Rayleigh vs. Mie 15

Scattering Elastic scattering: no energy change Frequency of the scattered wave = frequency of incident wave Probes static structure of material Rayleigh and Mie scattering Inelastic scattering: energy change Frequency of the scattered wave frequency of incident wave Internal energy levels of atoms and molecules are excited Probes vibrational bonds of the molecule Raman scattering (stokes and anti-stokes ) 16

Scattering Rayleigh Scattering Scattering from very small particles λ/10 Rayleigh scattering is inversely related to fourth power of the wavelength of the incident light Light Source Detector I 1 4 λ is the wavelength of the incident light I is the intensity of the scattered light 17

Scattering Mie Scattering For scattering 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 18

Scattering Wavelength Dependence of Mie Scattering As d more rapid oscillations Characteristics spacing of peaks size of scatterer depth of modulation number of such scatterers More often mixture of scatterers Superposition of spectra Normalized Q backsc d=1 μm d=2 μm d=4 μm 400 500 600 700 800 Wavelength (nm) 19

Scattering Scattering Coefficient, μ s [1/cm] μ s =N s σ s, N s = concentration of scatterers σ s = Cross-sectional area for scattering Scattering Mean Free Path, l s [cm] Average distance a photon travels between scattering events l s 1 s 20

Scattering Include the effect of forward scattering events Reduced Scattering Coefficient, μs [1/m] μs incorporates the scattering coefficient, μs and the anisotropy factor, g s ' (1 g) μs can be regarded as an effective isotropic scattering coefficient that represent the cumulative effect of several forwardscattering events s g 1 0 1 totally backward scattering isotropic scattering totally forward scattering Biological Tissues: 0.65 < g <0.95 21

Scattering Scattering in Tissue Tissue is composed of a mixture of Rayleigh and Mie scattering 10 m 1 m Mie Scattering 0.1 m Rayleigh Scattering 0.01 m cells nuclei mitochondria lysosomes, vesicles striations in collagen fibrils macromolecular aggreagates membranes 22

Light Transport in Tissue Scattering and absorption occur simultaneously and are wavelength dependent ' t s a Scattering monotonically decreases with wavelength Absorption is large in UV, near visible, and IR Absorption is low in red and NIR Therapeutic window (600 λ 1000 nm) ' s b '~ s A 0.5 4 23

Light Transport in Tissue Modeling Photon Propagation 24

Measurement of Optical Properties Measurement Strategies Optical Source input Black Box TISSUE H(μa, μs) Detector output H: System Function Goal: To find out H(μa, μs) Requires Non-Static system Perturbations in either optical source or tissue 25

Fluorescence Absorption of light and re-emission at a longer wavelength (loss of energy) Diagnostic applications: Excitation and emission spectra depend on the biochemical constituents of the tissue Variations in these components due to disease affect the fluorescence properties, thus providing a means for diagnostic purpose Therapeutic applications: Provide useful feedback during therapy (especially during PDT) 26

Fluorescence Types of emission Fluorescence return from excited singlet state to ground state; does not require change in spin orientation (more common form of relaxation) Phosphoresence return from a triplet excited state to a ground state; electron requires change in spin orientation Emissive rates of fluorescence are several orders of magnitude faster than that of phosphorescence Energy level diagram (Jablonski diagram) 27

Fluorescence Excitation Light is absorbed For dilute sample, Beer-Lambert law applies Magnitude of ε reflects probability of absorption Wavelength of ε dependence corresponds to absorption spectrum S1 A ( ) Cl So ε = molar absorption coefficient (M -1 cm -1 ) C = concentration, l = pathlength 28

Fluorescence Non-radiative relaxation The electron is promoted to higher vibrational level in S1 state than the vibrational level it was in at the ground state Vibrational deactivation takes place through intermolecular collisions A time scale of10-12 s (faster than that of fluorescence process) S1 So 29

Fluorescence Emission The molecule relaxes from the lowest vibrational energy level of the excited state to a vibrational energy level of the ground state (10-9 s) The energy of the emitted photon is lower than that of the incident photons Emission Spectrum For a given excitation wavelength, the emission transition is distributed among different vibrational energy levels For a single excitation wavelength, can measure a fluorescence emission spectrum S1 So 30

Fluorescence Emission Stokes shift Fluorescence light is red-shifted (longer wavelength than the excitation light) relative to the absorbed light Internal conversion can affect Stokes shift Solvent effects and excited state reactions can also affect the magnitude of the Stokes shift Invariance of emission wavelength with excitation wavelength Emission wavelength only depends on relaxation back to lowest vibrational level of S1 For a molecule, the same fluorescence emission wavelength is observed irrespective of the excitation wavelength S1 So 31

Fluorescence Mirror image rule Vibrational levels in the excited states and ground states are similar An absorption spectrum reflects the vibrational levels of the electronically excited state An emission spectrum reflects the vibrational levels of the electronic ground state Fluorescence emission spectrum is mirror image of absorption spectrum v =5 v =0 v=5 v=0 S 1 S 0 32

Fluorescence Internal conversion vs. fluorescence emission Electronic energy increases the energy levels grow more closely spaced Overlap between the high vibrational energy levels of S n-1 and low vibrational energy levels of S n more likely This overlap makes transition between states highly probable Internal conversion: a transition between states of the same multiplicity Time scale of 10-12 s (faster than that of fluorescence process) Significantly large energy gap between S 1 and S 0 S 1 lifetime is longer radiative emission can compete effectively with nonradiative emission 33

Fluorescence Internal conversion vs. fluorescence emission Mirror-image rule typically applies when only S 0 S 1 excitation takes place Deviations from the mirror-image rule are observed when S 0 S 2 or transitions to even higher excited states also take place 34

Fluorescence Intersystem crossing Intersystem crossing refers to non-radiative transition between states of different multiplicity It occurs via inversion of the spin of the excited electron resulting in two unpaired electrons with the same spin orientation, resulting in a triplet state Transitions between states of different multiplicity are formally forbidden Spin-orbit and vibronic coupling mechanisms decrease the pure character of the initial and final states, making intersystem crossing probable 35

Fluorescence Quantum yield of fluorescence, Φ f, Defined as: f number of photons emitted number of photons absorbed Another definition for f is f k r k where k r is the radiative rate constant and k is the sum of the rate constants for all processes that depopulate the S 1 state. In the absence of competing pathways Φ f =1 Characteristics of quantum yield Quantum yield of fluorescence depends on biological environment Example: Fura 2 excitation spectrum and Indo-1 emission spectrum quantum yield change when bound to Ca 2+ 36

Fluorescence Fluorescence Lifetime Radiative lifetime, τ r, is related to k r r 1 k r The observed fluorescence lifetime, is the average time the molecule spends in the excited state, and it is f 1 k 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 37

Fluorescence Fluorescence life-time methods Short pulse excitation followed by an interval during which the resulting fluorescence is measured as a function of time Provide an additional dimension of information missing in timeintegrated 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 38

Fluorescence Fluorescence Excitation/Emission Emission spectrum Hold excitation wavelength fixed, scan emission Reports on the fluorescence spectral profile reflects fluorescence quantum yield, Φ k (l m ) Excitation spectrum Hold emission wavelength fixed, scan excitation Reports on absorption structure reflects molar extinction coefficient, ε(l x ) Excitation-Emission Matrix (EEM) Composite Good representation of multifluorophore solution Excitation(nm) Fluorescence Ι 550 530 510 490 470 450 430 410 390 370 350 330 310 290 270 250 Fixed Emission λ Excitation λ(nm) NADH Tryp. Fluorescence Ι Fixed Excitation λ Emission λ(nm) FAD 300 350 400 450 500 550 600 650 700 Emission(nm) 39

Fluorescence Quenching, Bleaching & Saturation Quenching Excited molecules relax to ground states via nonradiative pathways avoiding fluorescence emission (vibration, collision, intersystem crossing) Molecular oxygen quenches by increasing the probability of intersystem crossing Polar solvents such as water generally quench fluorescence by orienting around the exited state dipoles 40

Fluorescence Quenching, Bleaching & Saturation Photobleaching Defined as the irreversible destruction of an excited fluorophore Photobleaching is not a big problem as long as the time window for excitation is very short (a few hundred microseconds) Excitation Saturation The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime f) Optical saturation occurs when the rate of excitation exceeds the reciprocal of f Molecules that remain in the excitation beam for extended periods have higher probability of interstate crossings and thus phosphorescence 41

Fluorescence Fluorescence Resonance Energy Transfer (FRET) 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 FRET is very sensitive to the distance between donor an acceptor and is therefore an extremely useful tool for studying molecular dynamics Molecule 1 Molecule 2 Fluorescence DONOR ACCEPTOR Fluorescence Absorbance Absorbance Wavelength 42

Fluorescence Fluorescence Recovery after Photobleaching (FRAP) Useful technique for studying transport properties within a cell, especially transmembrane protein diffusion FRAP can be used to estimate the rate of diffusion, and the fraction of molecules that are mobile/immobile Can also be used to distinguish between active transport and diffusion Procedure Label the molecule with a fluorophore Bleach (destroy) the fluorophore in a well defined area with a high intensity laser Use a weaker beam to examine the recovery of fluorescence as a function of time Nature Cell Biology 3, E145 - E147 (2001) 43

Fluorescence Biological Fluorophores Endogenous Fluorophores amino acids structural proteins enzymes and co-enzymes vitamins lipids porphyrins Exogenous Fluorophores Cyanine dyes Photosensitizers Molecular markers GFP, etc. 44

Applications Optical Biopsy Noninvasive Optical detection Endoscopic compatibility Fiber-optic mediation Diagnosis Optical spectroscopy Various spectroscopies can be used for optical tissue diagnostics Auto-fluorescence Exogenous-drug fluorescence Raman Absorption & FTIR Elastic scattering The internist's dream: smart colonoscope Illumination source (magic laser) Optical biopsy Smart Spectrometer 45

Applications What does a pathologist look for? Biochemical changes Metabolism and gene expression Special stains Architectural changes Shape of cell Shape of nucleus Ratio of nucleus to total cell volume Chromatin distribution Structure of organelles PLEOMORPHISM (variations in nuclear size and DNA density) Cell density and distribution Normal cells Cancer cells 46

Applications Available optical techniques Absorption/Fluorescence (endogenous or exogenous) Biochemical changes or targeted to expressed proteins Scattering Elastic Scattering Spectral and angular patterns of light scattering depend on the size and structure of the scattering particle Inelastic Scattering Raman Spectroscopy (changes in chemical bonds which affect the tissue vibrational levels) Optical Imaging Molecular imaging (biochemistry) Optical Coherence Tomography (microstructure) 47

Applications Light Scattering Spectroscopy Exploit the wavelength dependence of scattering to detect cancer broadband polarized illumination normal colon cells polarization-resolved detection cancerous cells Perelman et al. Phys Rev Let 80: 627-630 (1998); Backman et al. Nature 406: 35-36 (2000) 48

Applications Detection of lung carcinoma in situ with fluorescence using the LIFE imaging system Carcinoma in situ White light bronchoscopy Autofluorescence ratio image Courtesy of Xillix Technologies (www.xillix.com) 49

Applications Detection of lung carcinoma in situ using the LIFE imaging system Autofluorescence enhances ability to localize small neoplastic lesions Severe dysplasia/worse Intraepithelial Neoplasia WLB WLB+LIFE WLB WLB+LIFE Sensitivity 0.25 0.67 0.09 0.56 Positive predictive value 0.39 0.33 0.14 0.23 Negative predictive value 0.83 0.89 0.84 0.89 False positive rate 0.10 0.34 0.10 0.34 Relative sensitivity 2.71 6.3 S Lam et al. Chest 113: 696-702, 1998 50

Applications Fluorescence lifetime measurements Autofluorescence lifetimes measured from colon tissue in vivo Analysis via iterative reconvolution Two component model with double exponential decay M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998 51

Applications Fluorescence lifetime measurements Autofluorescence lifetimes used to distinguish adenomatous from nonadenomatous polyps in vivo M.-A. Mycek et al. GI Endoscopy 48:390-4, 1998 52