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? 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 (with apologies to Admiral Stockdale)

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

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

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

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

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

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

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

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

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

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

14. Further adventures of Fred the photon photons fluorescence absorption

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

17. A fluorescence scenario cellular epithelium collagen support healthytrending towards cancer thickening 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:k r nonradiative loss rate:k nr k nr 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 photons Raman scattering scattering Stokes Anti-Stokes

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

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

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

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... photons elastic scattering scattering

26. Elastic scattering caused by variations in refractive index 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 50060070080090010001100 10 10 0 Wavelength (nm) Mie Theory Scattering Coefficient (mm ) 2000nm 1000nm 200nm 100nm 20nm -4 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. Spectral dependence of scattering sphere van de Hulst approximation to Mie theory incident plane wave etalon (F = cavity finesse) d/2 d

30. d=5 microns n 1 = 1.36 n 2 /n 1 = 1.06 1-D etalon 3-D sphere Spectral dependence of scattering wavelength / nm

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

32. Scattering spectroscopy Perelman et al., Phys Rev Lett 80:627 (1998) and following. normal colon cells cancerous cells broadband polarized illumination polarization- resolved detection

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

35. Absolutely basic photon migration Detector signal at detector decays according to no scattering in the limit of: absorption pulse RMS distance from origin (“random walk”) increases according to no absorption scattering diffusion coefficient [m 2 /sec]

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

37. What are the diffusion measurements? 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, 2:00-2:30. FTuK5, “Functional imaging by optical topography,” Randall Barbour, 3:15-3:45. source(s) detector(s)

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 Have a great rest of the conference! Thanks to: Mary-Ann Mycek, Vasan Venugopalan, Edward Hull