Nonlinear Microscopy and Temporal Focusing Microscopy Y. Silberberg Physics of Complex Systems...

Nonlinear MicroscopyNonlinear Microscopy

and Temporal Focusing Microscopyand Temporal Focusing Microscopy

Y. Silberberg

Physics of Complex Systems

Weizmann Institute of ScienceRehovot, Israel

CREOL April 2008

1. Nonlinear Microscopy

2. Pulse Shaping and Microscopy

3. Temporal Focusing Microscopy

Nonlinear MicroscopyNonlinear Microscopy

z

x

y

Short-pulse Laser

computer current amplifier

Photomultiplier tube

filtercondenser

sample

microscope objective

Optical scanners

Nonlinear Laser ScanningNonlinear Laser Scanning MicroscopyMicroscopy

• Optical Sectioning• Deep Penetration• Contrast mechanism

Two-PhotonTwo-Photon MicroscopyMicroscopy

Denk &. Webb, 1990 CornellReduced photo-bleaching

Two-PhotonTwo-Photon MicroscopyMicroscopy

• Natural extension of standard fluorescence microscopy

• Long wavelength excitation: reduced scattering, deep penetration

• Reduced photobleaching

Hayashi Lab, MIT www.bris.ac.uk

SHGSHG MicroscopyMicroscopy

Collagen, Skin tissue Neural imaging, Webb’s lab

New Contrast Mechanisms

THG images of biological specimenTHG images of biological specimenThird-Harmonic GenerationThird-Harmonic Generation

Universal processGeneral structural imagingCoherent process

THG images of biological specimenTHG images of biological specimen

Yelin & Silberberg, Opt. Express 5, 169 (1999)

Mouse bone

Xenopus embryo

fossil Yeast cell

Drosophila ovary

THG MicroscopyTHG Microscopy

Yelin et al., Appl. Phys. B 74, S97 (2002)

Optical sections of a live neuron by THG

Sections separated by 1m

THG images of biological specimenTHG images of biological specimenOptical SectioningOptical Sectioning

Nuclear membrane labeling by 10nm particles and silver enhancement

Control

Metal nanoparticles as markers for THGMetal 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 specimenTHG images of biological specimenMulti-Modal Nonlinear MicroscopyMulti-Modal Nonlinear Microscopy

CARS Image tuned to DNA backbone vibration at 1090 cm-1 in mitosis

THG images of biological specimenTHG images of biological specimenCARS MicroscopyCARS 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 specimenTHG images of biological specimenSTED MicroscopySTED 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 MicroscopyWhy Nonlinear Microscopy

1. Nonlinear Microscopy

2. Pulse Shaping and Microscopy

3. Temporal Focusing Microscopy

Nonlinear MicroscopyNonlinear Microscopy

Short Pulse = Broad BandShort Pulse = Broad Band

Broad, COHERENT Bandwidth

10 fs pulses @ 800 nm ~130 nm FWHM

10 fs pulses @ 800 nm ~130 nm FWHM

THG images of biological specimenTHG images of biological specimenFemtosecond Pulse ShapingFemtosecond Pulse Shaping

SLM

Weiner & Heritage pulse shaper:

Phase, amplitude and polarization synthesizer

Spectral plane

Control of TPA in CesiumControl of TPA in Cesium

f f f f

PMT

Lock-in amplifier

computer

input pulseoutputpulse

SLM

Cs cell

6S1/2

8S1/2

7P

=822nm

flr=460nm

=822nm

Meshulach & Silberberg, Nature, 396, 239 (1998)

Control of TPA by a narrow atomic transitionControl of TPA by a narrow atomic transition

scan of a periodic phase maskscan of a periodic phase mask

I

0

1

Meshulach & Silberberg, Nature, 396, 239 (1998)

Sinusoidal phase Cosinusoidal phase

g

f

Control of TPA Control of TPA

g

f

Atomic two-photon transitions can be controlledwith excellent contrast

Can this concept be used for controllingorganic chromophores with broad absorptionbands?

g

f

Microscope

Shaper

J.P. Ogilvie, D. Débarre, X. Solinas, J.-L. Martin, E. Beaurepaire, M. JoffreOpt. Express 14, 759 (2006)

Coherent control for selective two-photonfluorescence microscopy of live organisms

Linear combinations yieldtwo 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. JoffreUse of coherent control for selective two-photon fluorescence microscopy of live organismsOpt. Express 14, 759 (2006)

CARS Image tuned to DNA backbone vibration at 1090 cm-1 in mitosis

THG images of biological specimenTHG images of biological specimenCARS MicroscopyCARS 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 thevibrational period) to provide all 3 frequencies

|v>|g>

p

pr

s

CARS

High frequencies blocked to detect CARS signal

THG images of biological specimenTHG images of biological specimenSingle-Pulse CARS spectroscopySingle-Pulse CARS spectroscopy

Issues: Resolution Nonresonant Background

CARS control schemesCARS control schemes

• Goal: to achieve high-resolution (ps) CARS spectroscopy using a single broadband source through coherent control

• Methods:

– Selective excitationUse quantum control to excite just a single Raman level

– Multiplexed CARSExcite with wide band, read with an effective narrow probe to resolve spectrum

Impulsive excitationImpulsive excitation

Selective excitationSelective excitation

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

Single-pulse CARS microscopySingle-pulse CARS microscopy

15 fs input pulse

outputpulse

SLM

blocker

filtered signal

Dudovich et al., Nature 418, 512 (2002)

Pulse bandwidth 1500cm-1

Single-pulse CARS microscopySingle-pulse CARS microscopy

Transform limited

Maximal resonant contribution Minimal resonant contribution

Maximal-minimal difference

Resonant + nonresonant

Nonresonant

The sample: glass capillary plate with 10 m holes filled with CH2Br2

Resonant contribution extracted exclusively

Pulses are shaped to maximize CARS signalsfrom 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 MicroscopyNonlinear Microscopy

THG images of biological specimenTHG images of biological specimenTemporal Focusing MicroscopyTemporal Focusing Microscopy

THG images of biological specimenTHG images of biological specimenTemporal Focusing MicroscopyTemporal Focusing Microscopy

THG images of biological specimenTHG images of biological specimenPulse evolution in a 4-f shaperPulse evolution in a 4-f shaper

Short pulse at grating surface

Longest pulseat Fourier plane

Pulse short againat second grating

Temporal Focus

THG images of biological specimenTHG images of biological specimenTemporal Focusing MicroscopyTemporal Focusing Microscopy

Geometries for temporal focusingGeometries 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 focusingTime domain picture of temporal focusing

By Fermat’s principle, moving line focus is generated in sample

Oron and Silberberg, JOSA B 22, 2660 (2005)

f1f2

Grating300 l/mm

Lens 20 cm

ObjectiveX100 1.4

THG images of biological specimenTHG images of biological specimenTemporal Focusing MicroscopyTemporal Focusing Microscopy

10 fspulse in

CCD

Scanningless imaging with temporally focused pulsesScanningless imaging with temporally focused pulses

4.5m

Drosophila egg-chamber stained with DNA binding dye, sections separated by 5m

Image obtainedwith regular mirror,eliminating temporal focusing

Depth resolution equivalent to line-scanning

Oron et al., Opt. Express 13, 1468 (2005)

THG images of biological specimenTHG images of biological specimenTemporal Focusing MicroscopyTemporal 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 specimenTHG images of biological specimenZ-scan through temporal focusZ-scan through temporal focus

4.5m

Z-resolution is limited by lens NA, and is equivalentto that achieved with line-scan microscopy

Depth resolution enhancement in line-scanning Depth resolution enhancement in line-scanning multiphoton microscopymultiphoton microscopy

A line is formedon grating normalto groves

Combining temporal focusing with spatial focusing along one axis

Tal et al., Opt. Lett 30, 1686 (2005)

Depth resolution enhancement in line-scanning Depth resolution enhancement in line-scanning multiphoton microscopymultiphoton microscopy

1.7m

Depth resolution equivalent to point-scanning

sections separated by 2m

Z-scan through temporal focus Z-scan through temporal focus

THG images of biological specimenTHG images of biological specimenVideo-Rate line scanning Temporal Focusing MicroscopyVideo-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

www.weizmann.ac.il/~feyaron

THG images of biological specimenTHG images of biological specimenNonlinear MicroscopyNonlinear Microscopy

Thanks…Thanks…

www.weizmann.ac.il/~feyaron

Coherent Control:

Doron Meshulach

Nirit Dudovich

Dan Oron

Thomas Polack

Evgeny Frumker

Adi Natan

Barry Bruner

Nonclassical Light:

Barak DayanAvi Pe’erItay AfekYaron Bromberg

Microscopy:

Dvir YelinEran TalNavit Dori

Solitons:

Hagai EisenbergYaniv BaradRoberto MorandottiDaniel MandelikYoav LahiniAsaf Avidan