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