Lecture 16 BIOE 498/598 DP 04/02/2014

1. Lecture 16BIOE 498/598 DP04/02/2014

2. Visible & near-UV region wavelength (nm) Microwave & radio wave region frequency (Hz) Infared region wavenumber (cm-1) Far-UV, x-ray, g-ray energy (DE=hn)

3. Absorption & Emission Rapid process(10-15s)

4. UV-Visible Spectroscopy • Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state. • Ultraviolet/Visible light: wavelengths (l) between 190 and 800 nm

5. UV-visible spectrum The two main properties of an absorbance peak are: • Absorption wavelength max • Absorption intensity Amax Housecroft and Sharpe, p. 466

6. Beer-Lambert Law Beer-Lambert Law: log(I0/I) = ebc e = A/cb A = ebc A = ec (when b is 1 cm) I0 = intensity of incident light I = intensity of transmitted light • = molar absoptivity coefficient in cm2 mol-1 c = concentration in mol L-1 b = pathlength of absorbing solution in cm-1 A = absorbance = log(Io/I) ℓ 0.1 cm

7. Beer-Lambert Law • A Absorbance or optical density (OD) • e absorptivity; M-1 cm-1 • c concentration; M • T transmittance

8. Transmittance, Absorbance, and Cell Path Length http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

9. Deviations from the Beer-Lambert Law Low c The Beer-Lambert law assumes that all molecules contribute to the absorption and that no absorbing molecule is in the shadow of another High c http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

10. Sample Concentrations Solution too concentrated Diluted five-fold

11. UV-visible spectrum of 4-nitroaniline Solvent: Ethanol Concentration: 15.4 mg L-1 Path length: 1 cm Molecular mass = 138 Harwood and Claridge, p. 18

12. Molar absorptivities (e) Molar absoptivities are very large for strongly absorbing chromophores (e >10,000) and very small if the absorption is weak (e = 10 to 100). The magnitude of e reflects both the size of the chromophore and the probability that light of a given wavelength will be absorbed when it strikes the chromophore. A general equation stating this relationship may be written as follows: • = 0.87 x 1020P x a where P is the transition probability (0 to 1) a is the chromophore area in cm2 The transition probability depends on a number of factors including where the transition is an “allowed” transition or a “forbidden” transition http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2

13. UV-visible spectroscopy definitions • Chromophore: Any group of atoms that absorbs light whether or not a color is thereby produced. • Auxochrome: A group which extends the conjugation of a chromophore by sharing of nonbonding electrons. • Bathochromic shift: The shift of absorption to a longer wavelength. • Hypsochromic shift: The shift of absorption to a shorter wavelength. • Hyperchromic effect: An increase in absorption intensity. • Hypochromic effect: A decrease in absorption intensity.

14. Absorption and Emission of Photons Absorption Emission Absorption: A transition from a lower level to a higher level with transfer of energy from the radiation field to an absorber, atom, molecule, or solid. Emission: A transition from a higher level to a lower level with transfer of energy from the emitter to the radiation field. If no radiation is emitted, the transition from higher to lower energy levels is called non-radiative decay. http://micro.magnet.fsu.edu/optics/lightandcolor/frequency.html

15. Absorption and emission pathways McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

16. Electronic Transitions: p  p* The pp* transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”. http://www.cem.msu.edu/~reusch/VirtualText /Spectrpy/UV-Vis/uvspec.htm#uv2 McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

17.  * transitions - Triple bonds Organic compounds with -C≡C- or -C≡N groups, or transition metals complexed by C≡N- or C≡O ligands, usually have “low-lying” * orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

18. Electronic Transitions: n  * The n-orbitals do not overlap at all well with the p* orbital, so the probability of this excitation is small. The e of the np* transition is about 103 times smaller than e for the pp* transition as it is “symmetry forbidden”. McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm http://www.cem.msu.edu/~reusch/VirtualText /Spectrpy/UV-Vis/uvspec.htm#uv2

20. Lycopene from Tomatoes http://www.purdue.edu/UNS/html4ever/020617.Handa.lycopene.html

21. Chlorophyll B-carotene hemoglobin

23. UV Absorption of Some Biological Samples UV spectrum of BSA UV spectrum of DNA from E. coli

24. UV Absorption of amino acids

25. Absorption Properties of Nucleotides

27. Optical Imaging Detects Single Stem Engraftment Hematopoesis from a single stem cell Cao et al. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc Nat Acad Sci USA. 2004;101(1):221-226.

28. Optical Imaging Detects Single Stem Cells Optically labeled stem cells can be seen singly in vivo in bone marrow Proc Natl Acad Sci 2009 from University of Tsukuba, Japan and Univ. of Michigan Medical School.

29. Cells can be detected in whole blood with ordinary pathology labels Real-time imaging of labeled probes in 1-10 cc whole blood • 4 billion cells per cc of blood • Large volume cell imaging • 1 min collection, 5 sec imaging time • Useful for: − Circulating rare cell detection − Early sepsis detection (Benaron et al, 2011 Project with Stanford Stem Cell Center, Sloan-Kettering Cancer Center)

30. First clinical translation of fluorescent probes (Top) Clinical IBMI system installed for breast cancer surgery at the University Medical Center, Groningen, Netherlands. (Bottom) Real-time visualization of ovarian cancer surgery on a patient injected with a fluorescent folate-targeting probe (from van Dam G., et. al. Nature Medicine 2011).

31. ICG Intraoperative Coronary Imaging System • A post-CABG intraoperative image shows no flow in graft • (arrow) • Revised, and graft working prior to closure • (from Novadaq, 2008)

32. Three-dimensional rendering of bones, skin and lung based on XCT data and FMT reconstruction of a K-ras mouse with lung tumors. Nature Methods 29(6); 615-620 (2012).

33. Optical imaging geometries for fluorescence detection demonstrating (a) Planar Reflectance, (b) Diffuse Reflectance and (c) Diffuse Transillumination with multiple source (S1-S4) and detector (D1-D4) locations.

34. Planar Reflectance Imaging Example planar reflectance imaging system setup for detection of fluorescence in mouse cancer model. (b) Bright field and (c) fluorescence images of mouse after intravenous administration of tumor-targeted molecular probe in mouse with subcutaneous tumor (arrow).

35. Planar Reflectance Imaging • Camera-based, full-field detection • Good for fast and low-cost screening of PK and bio-d of probes • Simplest and most common geometry for preclinical instrumentation used for fluoresc and biolumin imaging • Can provide the highest acquisition speed and resolution for superficial structures • Spatial resolution quickly diminishes with depth Ntziachristos, Ripoll et al. 2005)

36. Planar Reflectance Imaging (Clinical Use) • Fluorescence endoscopy for urologic surgery(van den Berg, van Leeuwen et al. 2012) • Robot-assisted laparoscopic surgery(Tobis, Knopf et al. 2012) • Fluorescence guided surgery for brain cancer(Roberts, Valdes et al. 2012) • Ovarian cancer(van Dam, Themelis et al. 2011).

37. Planar Reflectance Imaging (Clinical Use)-Obstacles • In the visible wavelength region-background signal from endogenous fluorophores. • Multispectral imaging can be used to separate the signal of interest from these background signals for improved visualization and quantification

38. Carotid endarterectomy specimen in white light (left), near-infrared fluorescence signal before (autofluorescence, middle) and after incubation with MMP-sensitive activatable probe (MMPSense, right) within the IVIS Spectrum.

39. Diffuse reflectance imaging • Utilizes reflectance geometry but with focused excitation and detection of light. • uses the diffuse nature of light propagation in tissues as a means to extend the depth sensitivity. • DRI gave better contrast than planar reflectance systems for imaging at depths greater than 6 mm.(de la Zerda, Bodapati et al. 2010) • The depth sensitivity of DRI is related to the separation of the excitation source from the detector.

40. Approaches of NIR fluorescent imaging probes Isotope and fluorochrome reporters can be used interchangeably for nonspecific and targeted agents; however, fluorochromes can also be used to make activation-sensitive agents for read-out of protein function.

41. How Can These be Administered?

42. Exogenous and Endogenous Contrast Agents http://www.photobiology.info/Photomed.html