Advanced Microscopy. Wintersemester 2012/13 Alexander Heisterkamp Nonlinear Microscopy
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1 Advanced Microscopy Wintersemester 2012/13 Alexander Heisterkamp Nonlinear Microscopy
2 Contact Prof.&Dr.&Alexander&Heisterkamp& Friedrich9Schiller9Universität&Jena& D907743&Jena,&Germany& phone:&+49&3641&947&660& fax:&&&&&&&+49&3641&947&652& Internet:&hXp://
3 last lecture:
4 Fluorescence excitation by nonlinear absorption sample& fluorescence&
5 Fluorescence excitation by nonlinear absorption sample& two9photon& fluorescence&
6 Cross sections for 2-photon-fluorescence in dyes σ TPA 1 10 cm s = 1GM GM& & Goeppert9Mayer & & bright&sunlight:& & 19photon&every&second!& &29photon&absorp@on&every&10&million&years!!!& &
7 Cross sections for 2-photon-fluorescence in tissue Zipfel&2003&
8 Cross sections for 2-photon-fluorescence in dyes Maria&Göppert9Mayer&
9 Sources for nonlinear excitation average power = very low peak power = kw-mw!!! focused by high NA: GW/cm x sec. = 100 fs pulsed laser
10 Sources for nonlinear excitation Denk&et&al,&1990:&CPM&Laser,&colliding&pulse&modelocked&laser& & & & today:&ti:sa9laser,&tuneable&between& nm,&pulse&width&approx.&120fs.& & &several&w&output&power& & & problem:& & & & & & &
11 fs-oscillators Ti:sapphire& slit& Lyot&filter& fiber&coupler& diode& fiber& beam& splixer& isolator& output&pulse&
12 Propagation of short pulses & red &wavelength&are&faster&compared& & to& blue &wavelengths.& wavelength& t_in& t_out& GDD&[s²]& GDD&[fs²]& 720& 1,52E913& 2,08E913& 7,80071E927& 7800,71332& 760& 1,46E913& 2,21E913& 8,71501E927& 8715,010082& 800& 1,63E913& 2,44E913& 1,06502E926& 10650,2068& 840& 1,31E913& 2,09E913& 7,69799E927& 7697,991824& 880& 1,25E913& 2,03E913& 7,21462E927& 7214,622738& 920& 1,20E913& 1,61E913& 4,64778E927& 4647,781729&
13 dispersion of ultrashort laser pulses n! 0! (! w! )! w! because of normal dispersion, the red part of the spectrum will be faster, the blue parts are delayed (GVD group velocity dispersion) chirp blue& red& n! 2! (! I! )! Ι# the pulse broadens due to n self phase modulation (SPM) 2
14 Excitation of multiple dyes multicolor imaging 1.0 DAPI DAC FITC R110 Spctrm O.Texas Red Cy5 Cy Fluoreszenz λ(nm) 29photon9& 750!%!950!nm!
15 Multicolor-Imaging Wokosin,&Centonze,&CriXenden,&and&White&(1996),&Bioimaging&
16 Comparison linear/nonlinear excitation W.!Webb,!Nature!Biotechnology!2003!
17 Comparison linear/nonlinear excitation Olympusmicro!
18 Setup of a nonlinear microscope descanned!deteccon! detector& z! fs9laser& scan%mirror! UV9lampe& detector& whole!area!deteccon!!!!
19 fs-clsm descanned scattering signal at 800nm Epithelium&
20 fs-clsm descanned scattering signal at 800nm Epithelium&
21 fs-clsm descanned scattering signal at 800nm Nerve&plexus&
22 fs-clsm descanned scattering signal at 800nm Stroma&
23 fs-clsm descanned scattering signal at 800nm Stroma&
24 fs-clsm descanned scattering signal at 800nm Stroma&
25 Nonlinear absorption leads to confocal like imaging sample& two9photonen& fluorescence&
26 granulosa&cells& (staining:& MitoTracker&Orange,& exc.:&554&nm,&em.:& 576&nm)& 5&µm&
27 granulosa&cells& (staining:& MitoTracker&Orange,& exc.:&554&nm,&em.:& 576&nm)& 5&µm&
28 MPM of drosophila larves (fruit fly) dye:&gfp& SupaXo&et&al.,&2005,&PNAS&
29 Absorption 1 absorption [µm -1 ] hemo- globine proteins melanin water nm 1µm wavelength 10µm
30 Low penetration depth of visible light z = 100µm z Tibor Veres, Fraunhofer Institut für Toxikologie Hannover
31 High penetration depth due to long wavelength Olympus, Scale-View Objectives (optical clearing agent used to reduce scattering)
32
33 for&example:&
34 Gerthsen,&Wöhlecke&
35 Bradley,&Cracknell,&Oxford,&1972&
36
37
38 Second harmonic generation - SHG SHG&is&sensi@ve&for&local&anisotropies& NL9polarisa@on:& P( ω SHG ) χ (2) ( ω SHG ) E( ω L ) E( ω L ) scales&with&intensity:& P (2) 2 2 SHG χ I Laser
39 Cells under a MPM/SHG SHG& MPM& &symmetric&molecule&distribu@on¬&visible&within&the&shg&image& L.&Moreaux&et&al.,&Opt.&LeX.&25,&320&(2000)&
40 MPM- & SHG-microscopy L.!Moreaux!et!al.,!Opt.!LeH.!25,!320!(2000)!
41 Second-Harmonic-Generation (SHG) P (1) = ε 0 χ E + ε E (2) (3) 0 χ E E + ε 0χ E E +... non-isotropic media, for example uni-axial crystals, frequency doubling by P (2) IVA, Ulm,
42 Second-Harmonic-Generation (SHG) collagen:& & in&mammals)& molecule&is&1.5nm&width&and& 300nm&in&length& highly&organized& IVA, Ulm,
43 MPM image of collagen matrix
44 SHG signal depends on alignment of structure
45 example: heart muscle tissue lightµscopy/histology& MPM/SHG& University!of!Western!Australia & & W.W.!Webb!! HE&staining& intrinsic&nad(p)h&(red)&& &&SHG&(green)&&
46
47 Label free-imaging of corneal tissue f9shg& & & & b9shg& & & & NLO& & & & f9clsm&
48 Investigation of corneal cross-linking by nonlinear microscopy 3&days& 6&days& M.&Hovakimyan,&R.&Guthoff,&S.&Knappe,&A.&Zhivov,&A.&Wree,&A.&Krueger,&A.&Heisterkamo,&O.&Stachs& Short&Term*Corneal*Response*to*Cross&Linking*in*Rabbit*Eyes*Assessed*by*In*Vivo*Confocal*Laser*Scanning* Microscopy*and*Histology,&Cornea,&2011&
49 SHG9&and&THG9microscopy& P (1) (2) 2 (3) 3 = ε 0 ( χ E + χ E + χ E + ) SHG!microscopy! THG!microscopy! non9centro& symmetric&media& needed& & for&example&collagen& E& THG&is&generated&wll& at&interfaces,& between&water&and& absorbing&layers&like& fat& E&
50 Cell und a multiphoton microscope SHG& MPM& &symmetric&molecule&distribu@on&within&the&adhesion&site,&thus&no&signal&visible& L.&Moreaux&et&al.,&Opt.&LeX.&25,&320&(2000)&
51 MPM- & SHG-Mikroskopie L.!Moreaux!et!al.,!Opt.!LeH.!25,!320!(2000)!
52 Example&for&SHG9&and&THGµscopy:&Cornea& z1& z2& z3& z4& Plamann,!Schanne%Klein,!Beaurepaire!2011!
53 MPM of a drosophila larvae SupaHo!et!al.,!2005,!PNAS!
54 MPM ex vivo, fluorescence + SHG + THG Aptel!et!al.,!IOVS,!2011!
55 Nonlinear imaging in the eye diagnosis of Diabetes Multimodal Nonlinear Imaging of the Human Cornea Florent Aptel et al, (group of Carsten Plamann, LAO, ENSTA Paris, France)
56 Nonlinear imaging in the eye diagnosis of Diabetes
57 MPM in vivo, Fluorescence + THG Schaffer!et!al.,!2011,!Biophys.!J.!
58 Label-free imaging autofluorescence & CLSM Cross&linked& control& 20 µm 20 µm 100 µm 100 µm nuclei& no&nuclei&
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