INTERNATIONAL SCHOOL OF PHYSICS ENRICO FERMI MICROSCOPY APPLIED TO BIOPHOTONICS Varenna (Lake Como) - July 12th to 22nd 2011

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1 INTERNATIONAL SCHOOL OF PHYSICS ENRICO FERMI MICROSCOPY APPLIED TO BIOPHOTONICS Varenna (Lake Como) - July 12th to 22nd 2011 Basic multiphoton and SHG microscopy Peter T. C. So Department of Mechanical and Biological Engineering Massachusetts Institute of Technology ptso@mit.edu

2 The Need for 3D Imaging Techniques in Thick Tissue Specimens Biological systems are inherently 3D! Biological processes also occur on multiple length scale

3 Histopathology Solution: mechanical sectioning of specimen Comment: (1) Clinical standard (2) Simple technology (3) Sectioning artifacts (4) Not in vivo

4 Total Internal Reflection Microscopy Solution: Evanescence wave at interfaces Objective Lens Comments: (1) only basal surface structure (2) high z resolution, 50 nm Standing wave Sample 1 n Mirror Piezo TIRF Wide Field Sund & Alexrod, Biophys J 2000

5 True 3D Microscopy Techniques Confocal Microscopy: Minsky, US Patent, 1961 Two-Photon Microscopy: Sheppard et al., IEEE J of QE, 1977 Denk et al., Science, 1990

6 The Invention of Confocal Microscopy Confocal microscopy is invented by Prof. Melvin Minsky of MIT in about 1950s.

7 Principle of Confocal Microscopy

8 Early Demonstration of Confocal Microscopy in Biological Imaging White et al., JCB 1987

9 Some Recent Application of Confocal Tissue Imaging

10 Confocal Tissue Imaging Masters, Opt Lett, 1999

11 A Model Tandem Confocal Microscope Utilizing Yokogawa Scan Head C. Elegans Eliminate light throughput Issue by spinning both a plate of lenslets and nother plate of pinholes Calcium events in nerve fiber

12 Two-Photon Excitation Microscopy first electronic excited state vibrational relaxation excitation photon emission photon excitation photons emission photon one-photon excitation fluorescence emission two-photon excitation fluorescence emission electronic ground state vibrational states (a) (b)

13 An abbreviated history of two-photon microscopy (1930) Maria Goeppert-Mayer predicted the existence of two-photon effect (1961) Franken et al. demonstrated second harmonic generation in ruby (1961) Kaiser et al. showed two-photon fluorescence in a solid state material (1964) Singh and Bradley reports three-photon fluorescence (1970s-1980s) Two-photon effect has been used in biological spectroscopy by researchers such as Birge, Fredrich, and McCain (1970s-1980s) Microanalysis based on non-linear 2nd harmonic generation was developed by researchers such as Freund and Hellwarth ( s) A number of researchers such as Sheppard, Kompfner, Gannaway and Wilson suggested the possibility of incorporating non-linear excitation \into scanning microscopy (1989)Denk, Webb and coworkers definitively demonstrated two-photon scanning microscopy and a number of its unique properties such as the triggering of localized chemical reaction

14 A comparison of two-photon and confocal microscopes (1) Confocal microscopes have better resolution than two-photon microscopes without confocal detection. (2) Two-photon microscope results in less photodamage in biological specimens. The seminal work by the White group in U. Wisconsin on the development of c. elegans and hamsters provides some of the best demonstration. After embryos have been continuously imaged for over hours, live specimens are born after implantation.

15 (3) Two-photon microscope provides better penetration into highly scattering tissue specimen. Infrared light has lower absorption and lower scattering in turbid media.

16 Point Spread Function of Two-Photon Microscopy Lateral Dimension: Airy function PSF confocal k is the wave number 2J ( kr) ( kr) [ kr Axial Dimension : Sinz function 1 ] 4 PSF confocal ( kz) sin( kz) [ ] ( kz) 4 Resolution: Lateral NA Axial 2 NA

17 Depth discrimination of Two-Photon Microscopy For a uniform specimen, we can ask how much fluorescence is generated at each z-section above and below the focal plane assuming that negligible amount of light is absorbed throughout. u (z-axis) v (r-axis) F z sec u) 2 F( u, v) 0 ( vdv

18 Depth discrimination One-Photon Tw o-photon

19 Two-photon excitation cross section Since two-photon absorption is a second-order process involving the almost simultaneous interaction of two photons with one fluorophore, this process has a small cross-section, d, on the order of cm 4 s (defined as 1 GM, Goppert-Mayer). The instantaneous intensity of the fluorescence signal: F (t) (photons/sec/molecule) The incident photon flux: I (t) (photon/sec/m 2 )

20 One-photon excitation fluorescence: 1 F P ( t) I( t) where is the one-photon cross section with unit of cm 2 : Two-photon excitation fluorescence: 2 F P 2 F P ( t) ( t) di( t) d 2 2 P( t) 2 2 NA 2c P(t) is the laser instantaneous power is the wavelength of light NA is the numerical aperture of the focusing objective and c are Planck s constant and the speed of light

21 Comparing CW and pulse laser excitation efficiency *For CW laser, T is an arbitrary time interval *For pulse laser, T is the inverse of the pulse repetition rate, f P T 1 f P For CW lasers, where P( t) P the average power 0 2 Fcw P 2 dp NA 2 c

22 For pulsed lasers with pulse width,, repetition rate, f P and averaged power, P 0, and assuming the following pulse profile: P P ( t) 0 f P for 0 t P( t) 0 for t 1 f P 2P Fp 2 P f P dt d c T f 0 P c 2 NA 1 P NA d For CW and pulse lasers to have equivalent excitation efficiency, the average power of the CW laser has to be higher by a factor of 1 f P

23 Assumptions: Calculation of Two-Photon Emission Rate Laser parameter: wavelength : 780 nm, repetition rate f P : 80 MHz pulse width : 100 fs Probe parameter: two-photon cross section d: 40 GM = cm 4 s Microscope parameter: numerical aperture NA = 1.0 Fundamental constants: speed of light c: m/s, Planck s constant : Js Fluorescence (photon/sec) 1.00E E E+08 y = 2E+11x E E E E E E E E E E+00 Average Power (W)

24 Saturation Effect We can also calculate the number of photon pairs absorbed per laser pulse per molecule n 2P P d 2 P 0 2 f P 2 NA 2 c 2 n (photon pair abs/mol/pulse) 1.00E E E E E E E E E E E E E E E E E E+00 Power (mw) We should not be saturated otherwise PSF will broaden

25 The design of a two-photon microscope Two-photon microscope design is actually significantly simpler than that of confocal microscope and has much in common.

26 Fluorophores used in two-photon microscopy Most conventional fluorophores can be used in two-photon microscopes. As a guideline, fluorophores can be excited in twophoton mode at twice their one-photon absorption wavelength. However, it should be recognize that this twice wavelength rule is only a rough approximation because one- and two-photon absorption processes have different quantum mechanical selection rules. Today, the two-photon excitation spectra of many fluorophores have been measured. In general, a fluorophore s twophoton excitation spectrum, scaled to half the wavelength, can be very different from its one-photon counterpart (See Xu et al, 1995, PNAS). Equally important, fluorophores designed for one-photon excitation is not optimized for two-photon excitation. Therefore, effort involved in the design of fluorophores with significantly high two-photon cross section is essential (Albota et al., 1998, Science).

27 Two-photon cross sections of some common fluorophores, is the fluorescence quantum efficiency under two-photon excitation (Xu, 1996; Xu, 1997; Chen, 1997) Fluorophores Excitation Wavelength (nm) d(10-50 cm 4 s /photon) d(10-50 cm 4 s /photon) 1. Extrinsic Fluorophores Rhodamine B Fluorescein (ph~11) Fura-2 (free) Fura-2 (with Ca 2+) Indo-1 (free) Indo-1 (high Ca) Bis-MSB Dansyl Dansyl Hydrazine DiI Couramin Cascade Blue Lucifer Yellow DAPI BODIPY Intrinsic Fluorophores GFP wild type GFP S65T NADH FMN Phycoerythrin

28 In collaboration with I. Kochevar, Wellman Labs, MGH and B. Masters

29 Application of Multiphoton Microscopy in Neurobiology Structural Basis of Memory Plasticity Z-Stack, Individual Slices Computational Model of Dendrite Branches Lee et al., PloS Biology 2006, Lee PNAS 2009 Reconstructed 3-D View

30 Discovery of neuron remodel on the dendrite level As potential of memory plasticity Lee et al., PloS Biology 2006 Lee et al., PNAS 2009, Chen et al. Nat. Neuro 2011

31 SECOND HARMONIC GENERATION (SHG) Virtual state w 2w Coherent process resulting from induced nonlinear polarization: (1) (2) P 0 E EE... 2 nd order susceptibility vanishes in centrosymmetric media No transition to higher electronic state Phase of SH wave is deterministic with respect to the incident field Complementary to multiphoton excitation

32 SHG IN BIOLOGY Early demonstrations: Fine (1971), Freund (1986) Subcellular and membrane imaging: Campagnola (1999), Moreaux (2000) Imaging of bulk connective tissue: Freund and Deutsch (1986) Guo (1997), Campagnola (2002), Brown (2003), Sun (2003) Quantitative SHG measurement: Kim (1999,2000), Stoller (2003)

33 SHG MICROSCOPY Intrinsic 3D sectioning capability due to nonlinear excitation process Sensitive to noncentrosymmetric media such as lipid bilayers, structural proteins Reduced photodamage, photobleaching Complementary to multiphoton fluorescence microscopy

34 Review of Second Harmonic Generation (Materials follow discussion of Jerome Mertz web site) Two useful concepts of light and electromagnetic field (1) Accelerating charges produce light Antenna Light emisson from oscillating dipole E + + E k oscillation oscillation k - -

35 + + + (2) Dielectric materials are made up of molecules; some of this molecules are polarizable E

36 Molecular Emission Under Strong EM Field Hyper-Rayleigh Scattering Symmetric molecule w w Excitation Emission w Asymmetric molecule 2w w

37 Light Emission From Two Molecules Constructive Interference Destructive Interference Scattered light is coherent Emission intensity depends on molecular orientation and interference

38 Light Emission From a Collection of Molecules Incoherent Coherent Coherent sum of hyper Rayleigh scattered light is second harmonic generation Second harmonic emission is DIRECTIONAL because of phase matching condition

39 Tissue Imaging Based on SHG of collagen SHG From Fish Scale Muscle and ECM Structure Of Mouse Lower Leg Data are either extracted from: Campagnola, Biophys J, 2002 Millard, Biophys J, 2004

40 SHG Imaging of C. Elegans Embryo

41 SHG and Fluorescence (GFP) Imaging of Microtubule

42 Excitation Polarization Dependence of SHG SHG Does Not Generate Photobleaching

43 SHG Spectra of Specimen Studied and Its Excitation Wavelength Dependences

44 Apply TP & SHG Microscopy to study regeneration scar regeneration Regeneration efficacy is dependent on optical scaffold degradation rate Scaffold degradation and new collagen synthesis can be monitored spectroscopically Good Poor week1 week9 green (collagen fluorescence) Red (collagen SHG)

45 Other 3D microscopy based on non-fluorescent contrast Going beyond multiphoton, multiple harmonics, & CARS: Many new modalities are developed leading by the Warren and Xie groups. Stimulated Raman (Freudiger, 2008, Fu, 2008, Volkmer, 2009) Transient absorption (Fu, 2007) Stimulated emission (MIN, 2009) The list goes on Cheng, CARS + 2P, demyelination diseases Matthews, Warren, Absorption, melanoma