Origin of signals in tissue imaging and spectroscopy

1. Origin of signals in tissue imaging and spectroscopy Andrew J. Berger The Institute of Optics University of Rochester Rochester, NY 14627

2. A very brief outline • Absorption • Emission • Scattering

3. 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

4. Fred the photon photons absorption events Absorption = molecular transition between states • electronic • vibrational • rotational • (translational)

5. outer shell: n>1 13.7 eV = 91 nm Electronic transitions What's quantized: energy 4 Consequently: 3 2 1 Biologically: typically UV or blue

6. Representative values: mid-IR Vibrational transitions What's quantized: energy

7. Representative values: microwave regime Rotational transitions What's quantized: Consequently:

8. How to talk about absorption molar extinction "absorption coefficient" [1/length] concentration

9. What's absorbing? DNA biologicalwindow rotational vibrational electronic courtesy V. Venugopalan, http://www.osa.org/meetings/archives/2004/BIOMED/program/#educ

10. Hemoglobin courtesy V. Venugopalan, http://www.osa.org/meetings/archives/2004/BIOMED/program/#educ

11. blood = 45% red blood cells by volume red blood cell = 1/3 hemoglobin by weight Hemoglobin molecular weight = 65,000 mg/mmole Typical tissue absorption! adipose tissue ~ 1% blood by volume Hb concentration = 23 mM

12. Hemoglobin at isosbestic point, Mean free absorption pathlength = 500 mm (!)

13. Hemodynamics calculations single absorber : two absorbers : measure the absorption coefficients look up the molar extinction coefficients (e.g. http:/omlc.ogi.edu) calculate the concentrations oxygen saturation: parameters of interest : theory works for N>2 chromophores, too! total hemoglobin

14. Further adventures of Fred the photon absorption photons fluorescence

15. Fluorescence: level diagram • absorption: fsec • internal conversion: fsec • upper state lifetime:psec-nsec • emission: fsec shift is to the RED (Stokes) of the excitation light

16. 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 courtesy M.-A. Mycek Ref. Mycek and Pogue, Handbook of Biomedical Fluorescence

17. 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

18. The time dimension • absorption: fsec • internal conversion: fsec • upper state lifetime:psec-nsec • emission: fsec • radiative decay rate:kr • nonradiative loss rate:knr • knr varies with environment • fluorescence decay lifetime varies, too: not intensity-based! combined spectral and temporal fluorescence measurements: Pitts and Mycek, Rev. Sci. Inst.72:7, 3061-3072 (2001).

19. 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, 2:1, 89-117 (2000).

20. Yet more adventures for Fred scattering photons Stokes Anti-Stokes Raman scattering

21. incident photon has energy E molecule gains energy DE scattered photon has energy E -E Level diagram for Raman energy excitation usually in near-IR or <300 nm UV to avoid visible fluorescence

22. Basic mechanism of Raman scattering induced dipole moment : product term : STOKES ANTI-STOKES

23. Typical spectrum (oral bacteria) 1005 1092 619 783 667 720 902 1457 853 1340 1127 813 1259 1211 guanine phenylalanine tyrosine adenine intensity (arb. units) C-N, C-C str. 1651 cytosine, uracil phenylalanine amide III C-H 2 def. 1580 amide I aromatic amino acids RNA bases Raman shift (cm-1)

24. Applications for Raman • Chemical analysis of tissue, in vitro or in vivo (breast, artery, blood) • Disease classification • High-resolution, molecularly specific microscopy topical review: Hanlon et al., “Prospects for in vivo Raman spectroscopy,” Phys. Med. Biol. 45, R1-R59 (2000) (or just talk to me!) go to: FWN4, “CARS microscopy: coming of age,”Sunney Xie, 2:45-3:15. FWN5, “Interferometric contrast between resonant CARS and nonresonant four-wave mixing,”Daniel Marks, 3:15-3:30.

25. Fred keeps going, and going, and... scattering photons elastic scattering

26. caused by variations in refractive index Elastic scattering componenttypical n in the vis/NIR extracellular fluid1.35 – 1.36 cytoplasm1.36 – 1.375 nucleus1.38 – 1.41 mitochondria1.38 – 1.41 water1.33 Drezek et al., Appl. Opt. 38:16, 3651-3661 (1999). • various approaches to modeling: full rigorMaxwell’s equations (e.g. Drezek above) Mie theoryplane wave on homogeneous sphere (e.g., code at philiplaven.com) van de Hulstthree-term approximation to Mie (larger spheres and modest n values) Rayleigh scatteringvery small particles (compared to λ)

27. Polystyrene Spheres of Varying Diameters in Water 0 10 ) -1 Mie Theory Scattering Coefficient (mm 2000 nm -1 1000 nm 10 200 nm 100 nm 20 nm -4 l 500 600 700 800 900 1000 1100 Wavelength (nm) Wavelength dependence varies w/ scatterer size courtesy Edward Hull, Rochester summer school lecture notes

28. A summary of scattering scales Figure by Steve Jacques, Oregon Medical Laser Center http://www.omlc.ogi.edu/classroom go to: FTuL1, “On the microscopic origin of light scattering in tissue,”Peter Kaplan, 2:00-2:30.

29. d/2 (F = cavity finesse) etalon Spectral dependence of scattering incident plane wave van de Hulst approximation to Mie theory sphere d

30. Spectral dependence of scattering 1-D etalon • d=5 microns • n1 = 1.36 • n2/n1 = 1.06 3-D sphere wavelength / nm

31. superposition of spectra mixture Scattering spectroscopy more rapid oscillations • spacing of peaks:size of scatterer • depth of modulation:number of such scatterers

32. Scattering spectroscopy broadband polarized illumination polarization-resolved detection normal colon cells cancerous cells Perelman et al., Phys Rev Lett80:627 (1998) and following.

33. Angularly-resolved scattering d 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.

34. Bulk tissue interrogation reduced scattering coefficient [1/length] • determine the absorption coefficient (spectroscopy) • identify and characterize heterogeneities (functional imaging) • note: scattering enables absorption studies in backscattering geometry!

35. absorption RMS distance from origin (“random walk”) increases according to no absorption pulse diffusion coefficient [m2/sec] scattering Absolutely basic photon migration in the limit of: signal at detector decays according to Detector no scattering

36. different source-detector separations ma = 0.001 mm-1 35 mm ms' = 1 mm-1 n = 1.4 25 mm r = 15 mm The real deal: diffusion theory scattering and absorption pulse

37. time domain: intensity vs. time frequency domain (amplitude-modulation): modulation depth and/or phase vs. distance or frequency steady state: intensity vs. distance What are the diffusion measurements? source(s) detector(s) go to: FTuK1, “Multidimensional diffuse optical imaging in breast cancer detection,”Brian Pogue, 2:00-2:30. FTuK5, “Functional imaging by optical topography,”Randall Barbour, 3:15-3:45.

38. Still hungry? • fluorescence:multiphoton-excited microscopy • second-harmonic: ditto • elastic scattering:optical coherence tomography, laser scanning confocal microscopy • polarization:surface-sensitive imaging, intrinsic birefringence • instrumentation: Raman fiber probes, fluorescence excitation-emission matrices Thanks to: Mary-Ann Mycek, Vasan Venugopalan, Edward Hull Have a great rest of the conference!