Origin of signals in tissue imaging and spectroscopy

Transcription

1 Origin of signals in tissue imaging and spectroscopy Andrew J. Berger The Institute of Optics University of Rochester Rochester, NY 1467 A very brief outline Absorption Emission Scattering 1

2 Who are you? Why are you here? (with apologies to Admiral Stockdale) experienced in some branch of optics biomedical not your main shtick interested in survey of fundamentals want introduction to applications interested in following the later talks want pointers to the literature Fred the photon photons absorption events I I(λ) ( ) 0 λ Absorption = molecular transition between states electronic vibrational rotational (translational)

3 Electronic transitions What's quantized: angular momentum= n h Consequently: 4 me 1 1 E = 8ε 0h n i nf energy ev = 91 nm outer shell: n>1 1 Biologically: typically UV or blue Vibrational transitions energy r r 0 0.5A& U 5 ev r What's quantized: oscillatorlevels: E n n+ 1 = hω Representative values: ( ) U 1 k r r k ~ 6 10 J/m 0 µ = 6amu (carbon nuclei) r 0 r ω= k µ =.5 10 λ = 6µm 14 rad/sec mid-ir 3

4 Rotational transitions What's quantized: angular momentum L ( + ) h = J J 1 Consequently: E J J + h = µ r 1 10 J 3 Representative values: µ = 6amu r =1 A & ev λ mm microwave regime How to talk about absorption I 0 I I ε cl µ a L I 0 molar extinction = 10 concentration = e L µ a ln10 εc "absorption coefficient" [1/length] 4

5 What's absorbing? DNA biological window electronic vibrational rotational courtesy V. Venugopalan, Hemoglobin courtesy V. Venugopalan, 5

6 Typical tissue absorption! adipose tissue ~ 1% blood by volume blood = 45% red blood cells by volume red blood cell = 1/3 hemoglobin by weight Hemoglobin molecular weight = 65,000 mg/mmole Hb concentration = 3 µm Hemoglobin at isosbestic point, -1 µ a = 0.03mM 0.09 mm / mm = Mean free absorption pathlength = 500 mm (!) 0.00mm -1 6

7 single absorber : two absorbers : parameters of interest : Hemodynamics calculations µ a = ln10 εc µ a µ a M 1 = measure the absorption coefficients ln10 ε1 ε M oxygen saturation: total hemoglobin Hb Hb ε ε HbO 1 HbO M c c look up the molar extinction coefficients (e.g. [ HbO ] [ Hb ] + [ HbO ] [ Hb ] + [ HbO ] Hb HbO calculate the concentrations theory works for N> chromophores, too! Further adventures of Fred the photon photons absorption fluorescence 7

8 Fluorescence: level diagram absorption: internal conversion: upper state lifetime: emission: fsec fsec psec-nsec fsec shift is to the RED (Stokes) of the excitation light r 0 r Fluorescence Spectroscopy Major biological fluorophores: structural proteins: collagen and elastin crosslinks coenzymes for cellular energy metabolism (electron acceptors): flavin adenine dinucleotide (FAD) nicotinamide adenine dinucleotide, reduced form (NADH) aromatic amino acids: side groups on proteins porphyrins: precursors to heme Fluorescence Intensity [a.u.] Tryptophan Pyridoxine Collagen Elastin NADH Flavins Porphyrins (Hp) Fluorescence emission wavelength [nm] B Ref. Mycek and Pogue, Handbook of Biomedical Fluorescence courtesy M.-A. Mycek 8

9 A fluorescence scenario cellular epithelium thickening collagen support healthy trending towards cancer increased FAD fluorescence reduced collagen fluorescence (farther from surface) polyp formation neovasculature; increased absorption & decreased fluorescence The time dimension absorption: internal conversion: upper state lifetime: emission: fsec fsec psec-nsec fsec r 0 r radiative decay rate: nonradiative loss rate: k r k nr k nr varies with environment fluorescence decay lifetime varies, too: 1 τ = k not intensity-based! combined spectral and temporal fluorescence measurements: Pitts and Mycek, Rev. Sci. Inst. 7:7, (001). 9

10 More introductions to fluorescence R. Redmond, "Introduction to fluorescence and photophysics," in Handbook of Biomedical Fluorescence (ed. Mycek and Pogue). N. Ramanujam, "Fluorescence spectroscopy of neoplastic and non-neoplastic tissues," Neoplasia, :1, (000). Yet more adventures for Fred photons scattering Stokes Anti-Stokes Raman scattering 10

11 Level diagram for Raman energy incident photon has energy E molecule gains energy E r0 r scattered photon has energy E - E excitation usually in near-ir or <300 nm UV to avoid visible fluorescence Basic mechanism of Raman scattering cosωt cos Ωt induced dipole moment: p = αe α α0 r0 cos t E0 cosωt r = + Ω 0 product term: cos Ωt cosωt = cos ( ω Ω) t + cos( ω + Ω)t STOKES ANTI-STOKES 11

12 Typical spectrum (oral bacteria) intensity (arb. units) phenylalanine guanine adenine 783 cytosine, uracil tyrosine aromatic amino acids 1005 phenylalanine C-N, C-C str amide III C-H def amide I RNA bases Raman shift (cm -1 ) Applications for Raman Chemical analysis of tissue, in vitro or in vivo (breast, artery, blood) Disease classification topical review: Hanlon et al., Prospects for in vivo Raman spectroscopy, Phys. Med. Biol. 45, R1-R59 (000) (or just talk to me!) High-resolution, molecularly specific microscopy go to: FWN4, CARS microscopy: coming of age, Sunney Xie, :45-3:15. FWN5, Interferometric contrast between resonant CARS and nonresonant four-wave mixing, Daniel Marks, 3:15-3:30. 1

13 Fred keeps going, and going, and... photons scattering elastic scattering Elastic scattering caused by variations in refractive index component typical n in the vis/nir extracellular fluid cytoplasm nucleus mitochondria water 1.33 Drezek et al., Appl. Opt. 38:16, (1999). various approaches to modeling: full rigor Maxwell s equations (e.g. Drezek above) Mie theory plane wave on homogeneous sphere (e.g., code at philiplaven.com) van de Hulst three-term approximation to Mie (larger spheres and modest n values) Rayleigh scattering very small particles (compared to ë) 13

14 Wavelength dependence varies w/ scatterer size Polystyrene Spheres of Varying Diameters in Water ) Mie Theory Scattering Coefficient (mm nm 1000 nm 00 nm 100 nm 0 nm λ Wavelength (nm) courtesy Edward Hull, Rochester summer school lecture notes A summary of scattering scales Figure by Steve Jacques, Oregon Medical Laser Center go to: FTuL1, On the microscopic origin of light scattering in tissue, Peter Kaplan, :00-:30. 14

15 Spectral dependence of scattering incident plane wave δ πd ( n n 1 ) n 1 n sphere n n 1 d d/ etalon ( δ λ) sin ( δ λ) sin ~ 1 + δ λ δ λ van de Hulst approximation to Mie theory F sin ~ 1+ F sin ( δ λ) ( δ λ) (F = cavity finesse) Spectral dependence of scattering d=5 microns n 1 = D etalon n /n 1 = D sphere wavelength / nm 15

16 Scattering spectroscopy δ 1 sin sin ~ d 1 + δ λ δ λ ( δ λ) ( δ λ) spacing of peaks: depth of modulation: size of scatterer number of such scatterers δ > δ 1 more rapid oscillations mixture superposition of spectra broadband polarized illumination Scattering spectroscopy polarizationresolved detection normal colon cells cancerous cells Perelman et al., Phys Rev Lett 80:67 (1998) and following. 16

17 Angularly-resolved scattering d n 1 n angular distribution has interferometric (oscillatory) behavior as well go to: FTuR1, Real-time angle-resolved low-coherence interferometry for detecting pre-cancerous cells, Adam Wax, 4:15-4:45. FTuL4, Elastic-scattering spectroscopy for cancer detection: What have we learned from preliminary clinical studies? Irving Bigio, 3:00-3:30. Bulk tissue interrogation reduced scattering coefficient [1/length] ' µ µ s a determine the absorption coefficient (spectroscopy) identify and characterize heterogeneities (functional imaging) note: scattering enables absorption studies in backscattering geometry! 17

18 Absolutely basic photon migration in the limit of: no scattering Detector signal at detector decays according to e µ act absorption no absorption pulse RMS distance from origin ( random walk ) increases according to 1 3 µ ' ( + µ ) s scattering a ct = diffusion coefficient [m /sec] Dct The real deal: diffusion theory pulse scattering and absorption different source-detector separations µ a = mm mm µ s ' = 1 mm -1 n = mm r = 15 mm 18

19 What are the diffusion measurements? source(s) detector(s) time domain: intensity vs. time frequency domain (amplitude-modulation): modulation depth and/or phase vs. distance or frequency steady state: intensity vs. distance go to: FTuK1, Multidimensional diffuse optical imaging in breast cancer detection, Brian Pogue, :00-:30. FTuK5, Functional imaging by optical topography, Randall Barbour, 3:15-3:45. Still hungry? fluorescence: second-harmonic: elastic scattering: polarization: instrumentation: multiphoton-excited microscopy ditto optical coherence tomography, laser scanning confocal microscopy surface-sensitive imaging, intrinsic birefringence Raman fiber probes, fluorescence excitation-emission matrices Thanks to: Mary-Ann Mycek, Vasan Venugopalan, Edward Hull Have a great rest of the conference! 19