Transcript Nonlinear Microscopy with Shaped Femtosecond Pulses

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
• Optical Sectioning
• Deep Penetration
• Contrast mechanism
z
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
images Microscopy
of biological specimen
Mouse bone
fossil
Xenopus embryo
Drosophila ovary
Yelin & Silberberg, Opt. Express 5, 169 (1999)
Yeast
cell
THGOptical
images of biological
specimen
Sectioning
Optical sections of a live neuron by THG
Sections separated by 1mm
Yelin et al., Appl. Phys. B 74, S97 (2002)
Metal nanoparticles as markers for THG
Nuclear membrane labeling by 10nm
particles and silver enhancement
Control
THG imagesNonlinear
of biological specimen
Multi-Modal
Microscopy
Optical section of a seed by TPFE, SHG and THG
Debarre et al, Nature Method 4, 47 (2006)
THGCARS
images ofMicroscopy
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
THGSTED
images ofMicroscopy
biological specimen
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 ofPulse
biologicalShaping
specimen
Femtosecond
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
f
f
computer
Lock-in
amplifier
SLM
input pulse
PMT
output
pulse
Cs cell
8S1/2
7
P
l=822nm
Dw
lflr=460nm
Meshulach & Silberberg, Nature, 396, 239 (1998)
l=822nm
6S1/2
Control of TPA by a narrow atomic transition
scan of a periodic phase mask
Sinusoidal phase
f
Cosinusoidal phase
g
2p
1
p
I
0
Meshulach & Silberberg, Nature, 396, 239 (1998)
w
0
F
Control of TPA
f
Atomic two-photon transitions can be controlled
with excellent contrast
g
Can this concept be used for controlling
organic chromophores with broad absorption
bands?
f
g
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
images ofMicroscopy
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 CARS
of biological
specimen
Single-Pulse
spectroscopy
A single ultrashort, broadband pulse (shorter than the
vibrational period) to provide all 3 frequencies
High frequencies
blocked to detect
CARS signal
Dw
ws
w pr
w
p
w
CARS
|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
1500cm-1
SLM
15 fs input
pulse
output
pulse
blocker
filtered signal
Dudovich et al., Nature 418, 512 (2002)
l
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
specific molecules
New fast pulse-shape modulation techniques are useful for
Lock-in
detection
on pulse
The sample:
glass capillary
plateshapes
with 10 mm holes filled with CH Br
Maximal-minimal difference
Transform
2 limited
2
Nonlinear Microscopy
1. Nonlinear Microscopy
2. Quantum Control and Microscopy
3. Temporal Focusing Microscopy
THG images of biological specimen
Temporal
Focusing Microscopy
THG images of biological specimen
Temporal
Focusing Microscopy
images of biological
specimen
PulseTHG
evolution
in a 4-f
shaper
Short pulse at
grating surface
Longest pulse
at Fourier plane
Pulse
short Focus
again
Temporal
at second grating
THG images
of biological
specimen
Temporal
Focusing
Microscopy
Geometries for temporal focusing
Head-on
(diffuser)
10fs
pulse
NA 1.4 X100
objective
Tilted (grating)
300 l/mm
grating
20cm
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
X100 1.4
f2
f1
10 fs
pulse in
Scanningless imaging with temporally focused pulses
4.5mm
Depth resolution equivalent
to line-scanning
Drosophila egg-chamber
stained with DNA
binding dye, sections
separated by 5mm
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 ms
•
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
4.5mm
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
1.7mm
Depth resolution equivalent
to point-scanning
sections separated by 2mm
THG imagesTemporal
of biological specimen
Video-Rate line scanning
Focusing Microscopy
Z-scan through temporal focus
THG images of biological specimen
Nonlinear
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