Nonlinear Microscopy and Temporal Focusing Microscopy Y Silberberg

Nonlinear Microscopy and Temporal Focusing Microscopy Y. Silberberg Physics of Complex Systems Weizmann Institute of Science Rehovot, Israel CREOL April 2008

Nonlinear Microscopy 1. Nonlinear Microscopy 2. Pulse Shaping and Microscopy 3. Temporal Focusing Microscopy

Nonlinear Laser Scanning Microscopy computer Photomultiplier current amplifier tube filter condenser z • Optical Sectioning • Deep Penetration • Contrast mechanism sample y x Optical scanners microscope objective Short-pulse Laser

Two-Photon Microscopy Reduced photo-bleaching Denk &. Webb, 1990 Cornell

Two-Photon Microscopy Hayashi Lab, MIT www. bris. ac. uk • Natural extension of standard fluorescence microscopy • Long wavelength excitation: reduced scattering, deep penetration • Reduced photobleaching

SHG Microscopy New Contrast Mechanisms Collagen, Skin tissue Neural imaging, Webb’s lab

THG images of biological specimen Third-Harmonic Generation Universal process General structural imaging Coherent process c(3)

THGTHG Microscopy images of biological specimen Mouse bone fossil Xenopus embryo Drosophila ovary Yelin & Silberberg, Opt. Express 5, 169 (1999) Yeast cell

THGOptical biological specimen images of Sectioning Optical sections of a live neuron by THG Sections separated by 1 m Yelin et al. , Appl. Phys. B 74, S 97 (2002)

Metal nanoparticles as markers for THG Nuclear membrane labeling by 10 nm particles and silver enhancement Control

THG images of biological specimen Multi-Modal Nonlinear Microscopy Optical section of a seed by TPFE, SHG and THG Debarre et al, Nature Method 4, 47 (2006)

THGCARS Microscopy images of biological specimen CARS Image tuned to DNA backbone vibration at 1090 cm-1 in mitosis CARS image of fibroblast cells that are stimulated to synthesize lipids. The lipid droplets are visualized with CARS tuned to the C-H vibration at 2845 cm-1. Xie’s group, Harvard

THGSTEDof biological specimen images Microscopy Nonlinear Saturation for Enhanced Resolution Stefan Hell, MPI Goettingen

Why Nonlinear Microscopy 1. Optical sectioning (all) 2. Reduced photobleaching (TPFE) 3. New contrast mechanisms, no labeling, live specimens (SHG, THG, CARS. . ) 4. Reduced scattering, deep imaging (TPFE, SHG, THG) 5. Molecular imaging (CARS) 6. Enhanced resolution (STED)

Nonlinear Microscopy 1. Nonlinear Microscopy 2. Pulse Shaping and Microscopy 3. Temporal Focusing Microscopy

Short Pulse = Broad Band I(t) E(w) Broad, COHERENT Bandwidth 10 fs pulses @ 800 nm ~130 nm FWHM

THG images biological specimen Femtosecondof. Pulse Shaping Weiner & Heritage pulse shaper: Phase, amplitude and polarization synthesizer Spectral SLM plane 10 fs pulses @ 800 nm ~130 nm FWHM

Control of TPA in Cesium f f computer Lock-in amplifier SLM input pulse PMT output pulse Cs cell 8 S 1/2 7 P =822 nm Dw flr=460 nm Meshulach & Silberberg, Nature, 396, 239 (1998) =822 nm 6 S 1/2

Control of TPA by a narrow atomic transition scan of a periodic phase mask Sinusoidal phase Cosinusoidal phase 2 p 1 p I 0 Meshulach & Silberberg, Nature, 396, 239 (1998) w 0 F

Control of TPA Atomic two-photon transitions can be controlled with excellent contrast Can this concept be used for controlling organic chromophores with broad absorption bands?

Coherent control for selective two-photon fluorescence microscopy of live organisms Microscope Shaper J. P. Ogilvie, D. Débarre, X. Solinas, J. -L. Martin, E. Beaurepaire, M. Joffre Opt. Express 14, 759 (2006)

Linear combinations yield two selective images of Drosophila embryo Blue pulse Yolk emission 25 µm Red pulse GFP emission J. P. Ogilvie, D. Débarre, X. Solinas, J. -L. Martin, E. Beaurepaire, M. Joffre Use of coherent control for selective two-photon fluorescence microscopy of live organisms Opt. Express 14, 759 (2006)

THGCARS Microscopy images of biological specimen CARS Image tuned to DNA backbone vibration at 1090 cm-1 in mitosis CARS image of fibroblast cells that are stimulated to synthesize lipids. The lipid droplets are visualized with CARS tuned to the C-H vibration at 2845 cm-1. Xie’s group, Harvard

THG images of biological specimen Single-Pulse CARS spectroscopy A single ultrashort, broadband pulse (shorter than the vibrational period) to provide all 3 frequencies High frequencies blocked to detect CARS signal |v> |g> Issues: Resolution Nonresonant Background

CARS control schemes • Goal: to achieve high-resolution (ps) CARS spectroscopy using a single broadband source through coherent control • Methods: – Selective excitation Use quantum control to excite just a single Raman level – Multiplexed CARS Excite with wide band, read with an effective narrow probe to resolve spectrum

Impulsive excitation

Selective excitation Weiner et al. , Science 273, 1317 (1990)

Single-pulse CARS microscopy Pulse bandwidth 1500 cm-1 SLM 15 fs input pulse output pulse blocker filtered signal Dudovich et al. , Nature 418, 512 (2002)

Single-pulse CARS microscopy Maximal resonant contribution Minimal resonant contribution Nonresonant Resonant + nonresonant Dudovich et al. , Nature 418, 512 (2002) Resonant contribution Pulses are shaped to maximize CARS signals extracted fromexclusively molecules specific New fast pulse-shape modulation techniques are useful for Lock-in detectioncapillary plate with 10 m holes filled with CH Br The sample: glass on pulse shapes Maximal-minimal difference Transform limited 2 2

Nonlinear Microscopy 1. Nonlinear Microscopy 2. Quantum Control and Microscopy 3. Temporal Focusing Microscopy

THG images of biological Temporal Focusing specimen Microscopy

THG images of biological specimen Temporal Focusing Microscopy

THG images of in a 4 -f shaper Pulse evolutionbiological specimen Short pulse at grating surface Longest pulse at Fourier plane Pulse short Focus Temporal again at second grating

THG images of biological specimen Temporal Focusing Microscopy

Geometries for temporal focusing Head-on (diffuser) 10 fs pulse Tilted (grating) NA 1. 4 X 100 objective 300 l/mm grating 20 cm achromat

Time domain picture of temporal focusing By Fermat’s principle, moving line focus is generated in sample Oron and Silberberg, JOSA B 22, 2660 (2005)

THG images of biological specimen Temporal Focusing Microscopy CCD Grating 300 l/mm Lens 20 cm Objective X 100 1. 4 f 2 f 1 10 fs pulse in

Scanningless imaging with temporally focused pulses 4. 5 m Depth resolution equivalent to line-scanning Drosophila egg-chamber stained with DNA binding dye, sections separated by 5 m Oron et al. , Opt. Express 13, 1468 (2005) Image obtained with regular mirror, eliminating temporal focusing

THG images of biological specimen Temporal Focusing Microscopy • Full field image is obtained simultaneously • Beam power is distributed among all pixels • With appropriately designed amplified pulses image may be obtained in a few s • Useful for time-resolved microscopy, FLIM • Depth resolution is reduced (1 dimensional shortening)

THG images of biological specimen Z-scan through temporal focus Z-resolution is limited by lens NA, and is equivalent to that achieved with line-scan microscopy 4. 5 m

Depth resolution enhancement in line-scanning multiphoton microscopy Combining temporal focusing with spatial focusing along one axis A line is formed on grating normal to groves Tal et al. , Opt. Lett 30, 1686 (2005)

Depth resolution enhancement in line-scanning multiphoton microscopy 1. 7 m Depth resolution equivalent to point-scanning sections separated by 2 m

THG images of biological Focusing Video-Rate line scanning Temporal specimen Microscopy Z-scan through temporal focus

THG images biological specimen Nonlinearof. Microscopy Quantum Coherent Control via femtosecond pulse shaping offers new functionalities in nonlinear microscopy, including high-resolution single-pulse CARS microscopy and scanningless microscopy by temporal focusing www. weizmann. ac. il/~feyaron

Thanks… Coherent Control: Nonclassical Light: Microscopy: Solitons: Doron Meshulach Nirit Dudovich Dan Oron Thomas Polack Evgeny Frumker Adi Natan Barry Bruner Barak Dayan Avi Pe’er Itay Afek Yaron Bromberg Hagai Eisenberg Yaniv Barad Roberto Morandotti Daniel Mandelik Yoav Lahini Asaf Avidan Dvir Yelin Eran Tal Navit Dori www. weizmann. ac. il/~feyaron