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

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

Nonlinear Laser Scanning Microscopy Optical Sectioning Deep Penetration Contrast mechanism

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

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

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

THG images of biological specimen Third-Harmonic Generation Universal process General structural imaging Coherent process

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

Yelin et al., Appl. Phys. B 74, S97 (2002) Optical sections of a live neuron by THG Sections separated by 1  m THG images of biological specimen Optical Sectioning

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

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

CARS Image tuned to DNA backbone vibration at 1090 cm -1 in mitosis THG images of biological specimen CARS Microscopy 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 STED Microscopy Nonlinear Saturation for Enhanced Resolution Stefan Hell, MPI Goettingen

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) Why Nonlinear Microscopy

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

Short Pulse = Broad Band   Broad, COHERENT Bandwidth 10 fs 800 nm ~130 nm FWHM

THG images of biological specimen Femtosecond Pulse Shaping SLM Weiner & Heritage pulse shaper: Phase, amplitude and polarization synthesizer Spectral plane

Control of TPA in Cesium ffff PMT Lock-in amplifier computer input pulse output pulse SLM Cs cell 6S 1/2 8S 1/2 7P7P  =822nm flr =460nm =822nm Meshulach & Silberberg, Nature, 396, 239 (1998)

Control of TPA by a narrow atomic transition scan of a periodic phase mask Control of TPA by a narrow atomic transition scan of a periodic phase mask  I   0 1   Meshulach & Silberberg, Nature, 396, 239 (1998) Sinusoidal phaseCosinusoidal phase

Control of TPA 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?

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

Linear combinations yield two selective images of Drosophila embryo 25 µm Blue pulse Red pulse Yolk emission 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)

CARS Image tuned to DNA backbone vibration at 1090 cm -1 in mitosis THG images of biological specimen CARS Microscopy 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

A single ultrashort, broadband pulse (shorter than the vibrational period) to provide all 3 frequencies |v> |g>  High frequencies blocked to detect CARS signal THG images of biological specimen Single-Pulse CARS spectroscopy 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 15 fs input pulse output pulse SLM blocker filtered signal Dudovich et al., Nature 418, 512 (2002) Pulse bandwidth 1500cm -1

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

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

THG images of biological specimen Temporal Focusing Microscopy

THG images of biological specimen Temporal Focusing Microscopy

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

THG images of biological specimen Temporal Focusing Microscopy

Geometries for temporal focusing Head-on (diffuser) Tilted (grating) 10fs pulse 300 l/mm grating 20cm achromat NA 1.4 X100 objective

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)

f1f1 f2f2 Grating 300 l/mm Lens 20 cm Objective X THG images of biological specimen Temporal Focusing Microscopy 10 fs pulse in CCD

Scanningless imaging with temporally focused pulses 4.5  m Drosophila egg-chamber stained with DNA binding dye, sections separated by 5  m Image obtained with regular mirror, eliminating temporal focusing Depth resolution equivalent to line-scanning Oron et al., Opt. Express 13, 1468 (2005)

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 4.5  m Z-resolution is limited by lens NA, and is equivalent to that achieved with line-scan microscopy

Depth resolution enhancement in line-scanning multiphoton microscopy A line is formed on grating normal to groves Combining temporal focusing with spatial focusing along one axis 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

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

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