Multiphoton microscopy Joonas Holmi ELEC October 6, 2016
Multiphoton microscopy 1. 2. 3. 4. Multiphoton microscopy 2/14
Intro: Multiphoton microscopy Nonlinear optical characterization method Pulsed laser scanning microscopy technique Excels at depth profiling and sectioning Used in ie. tissue and nano engineering History 1931: Concept introduced (by Maria Goeppert-Mayer) 1960: First laser (by Theodore Haiman) 1961: First 2-photon absorption (by Wolfgang Kaiser) 1990: 2-photon microscopy (by Winfried Denk et al.) Multiphoton microscopy 3/14
Intro: Widefield Confocal Multiphoton Figure : Microscopy techniques. Source: candle.am/microscopy Multiphoton microscopy 4/14
Intro: Key advantages over other techniques Advantages Less Mie scattering due to larger λ More penetration Scattered excitation photons too dilute Localization Localized excitation All photons useful if detected All photons useful No pinhole Higher useful signal Localized high signal High 3-D contrast + resolution Requirements Focused narrow pulse IR laser with low average power Low magnification lens with high numerical aperture (NA = n sin θ max ) Increased penetration depth Multiphoton microscopy 5/14
: Linear vs. nonlinear excitation Figure : Jablonski Energy Diagram. 2PE is rare to occur because an absorption event has very small cross-section and short duration ( 10 16 s). Source: www.microscopyu.com Multiphoton microscopy 6/14
: Linear vs. nonlinear excitation Taylor series expansion of polarization density, P and electric displacement field, D: P = P 0 + ɛ 0 χ (1) E + ɛ 0 χ (2) E 2 + ɛ 0 χ (3) E 3 +..., where χ (1) = ɛ r 1 = n 2 1 D = ɛ 0 E + P linear ɛ 0 (1 + χ (1) )E = ɛ 0 ɛ r E = ɛ 0 n 2 E Focused Gaussian TEM 00 -beam with waist, ω 0 : I(r, z) parallax ( ) 2 ( ) I ω0 0 ω(z) exp 2r 2 Im{n} 0 Re{n} ω(z) 2 2µ 0 c 0 E 2, where intensity I 0 = 2P 0, power P πω0 2 0, waist (at z) ω(z) = ω 0 1 + z2 z 2 R and Rayleigh length z R = πω2 0 λ. Multiphoton microscopy 7/14
: Linear vs. nonlinear excitation Figure : Fluorescence by 488 nm (left) and IR (right) lasers. 2PE does not generate out-of-focus light like 1PE does. Source: Image by Steve Ruzin and Holly Aaron, UC Berkeley Multiphoton microscopy 8/14
: Optical sectioning Figure : Depth scan for fluorescent pollen grain using widefield, confocal and 2-photon microscopy techniques. Source: candle.am/microscopy Multiphoton microscopy 9/14
: In vivo 3-D imaging Figure : Cortical layer V pyramidal neurons in living young adult mouse brain using 1064 nm gain-switched semiconductor laser diode and 7.5 ps pulse width. Source: Kawakami, R., et al. Biomed. Opt. Express 6 (3), 891-901. Video: youtu.be/ot9tbashgoo Multiphoton microscopy 10/14
: 2-D crystal optical characterization Figure : 2PE and 3PE for exfoliated GaSe. Source: Karvonen, L. et al. Sci. Rep. 5, 10334 Multiphoton microscopy 11/14
: Coherent anti-stokes Raman scattering Figure : Two pulsed lasers: pump (=probe) and Stokes. Source: leica-microsystems.com and www.physik.uni-bielefeld.de Figure : A larvae of a fruit fly. Overlay of CARS (red) and autofluorescence (green) images. Fat cells shown in red. Source: leica-microsystems.com Multiphoton microscopy 12/14
Advantages of multiphoton microscopy Increased penetration depth, contrast and resolution Excitation only at focal point due to E n dependence Less scattering and damage (ie. phototoxicity) At best like ideal confocal microscopy Faster scanning speed Requirements of multiphoton microscopy Extremely high power and narrow laser pulses Best with low magnifying but high NA objective lens Fully open pinhole if operated in confocal mode Multiphoton microscopy 13/14
(CLARIFIED 10.10.2016) The sample is excited using 1064 nm pulsed laser beam with peak power 2.5 kw. The beam is focused through a 20 (NA = 0.90) objective lens so that 3 ω(z ) portion of the beam will fit in. You can assume that the laser beam is an ideal Gaussian TEM00-beam. Suppose that the sample is a bottle of liquid, in which the objective lens is immersed. Liquid electric susceptibilities are χ (1) = 0.20, χ (2) = 0.05 nm V. a) 2-D plot the electric field amplitude, E and that amplitude squared, E 2, where both r and z range from -10 µm to +10 µm. b) Calculate at which distance (along the symmetry axis, z) the 2 nd order term of electric polarizability will become a dominating term? For simplicity, treat P and E as scalars. Multiphoton microscopy 14/14