Medical Imaging with Ultrashort Laser Pulses

1
Medical Imaging with Ultrashort Laser Pulses
Multi-Photon Imaging Optical Coherence
Tomography Ballistic Photon Imaging Stimulated
-Emission Depletion Imaging
2
The goal of medical imaging
3
Problems with standard microscopy
Many objects are clear and require generally
toxic stain to be seen. Objects must usually be
sectioned (sliced) to be observed. Some objects
are inaccessible to microscopes, such as the
inside of a blood vessel or the retina of the
eye. Most objects are turbid, that is, they
scatter a lot. This is the big challenge in
medical imaging inside the human body.
Optical microscopy has limited spatial
resolution (l/2), and a lot of stuff is smaller.
Ultrashort-pulse imaging techniques address these
problems.
4
Multi-photon imaging
In multi-photon imaging, we focus an ultrashort
pulse tightly into an object and observe the
multi-photon signal light.
F I2
F Two-photon Fluorescence energy
5
Whats wrong with single-photon imaging?
Focusing a laser beam to a spot and
raster-scanning across the medium has poor
longitudinal (z) resolution
Sample
w(z)
z
0
If we look at a linear process, such as
fluorescence, as much signal is created in the
unfocused regions of the beam as at the focus.
The w- and z-dependences cancel out!
Scanning the focus through a medium and measuring
any linear signal light yields an image, but
which averages over z. Not so good.
6
Nonlinear-optical processes emphasize a focus.
In nonlinear processes, in which the signal
scales nonlinearly with the intensity (e.g.,
I2), much more signal is created at the focus.
NLO signal
w(z)
z
0
Using an effect that scales as I3 yields signal
even more concentrated at the focus.
7
Two-photon fluorescence emission from a focused
pulse
One-photon fluorescence from a beam entering from
the right
Two-photon fluorescence from an identical beam
entering from the left
8
NLO processes used in imaging
In third-order, one must use a longer wavelength
laser (gt1.0 µm) in order to avoid direct
absorption of the third harmonic by the specimen.
9
Multi-photon imaging
We raster-scan the focus throughout the medium.
We can scan out a 2D or 3D image by using an xy
or xyz stage.
Images due to Chris Schaffer, UCSD
10
Making a 3D image involves raster-scanning the
focus throughout the sample.
11
Two-photon fluorescence imaging
Pollen grain (Clivia Miniata)
Conventional image (using fluorescence)
14 µm
46 sections separated by 0.5 µm in the axial
dimension. 2 seconds/image
1.5 µm axial resolution 200 mW in 16 beamlets
Images due to Jeff Squier, Colorado School of
Mines
12
Two-photon images of a rat brain
Blood vessels
Fast imaging allows red-blood-cell motion to be
discerned.
Images due to Chris Schaffer, UCSD
13
Two-photon fluorescence of brain tissue
Living rat hippocampal neurons were stained with
DiO, and imaged using pulsed illumination at 900
nm. The image is a projection through 50 sections
of 0.3 mm each. No dye bleaching was observed
during scanning. The imaging had no adverse
effect on the health of the cells.
Steve Potter, Georgia Tech
14
Why no third harmonic occurs in a uniform medium
Dc(3)
The tight focus provides high intensities,
allowing non-linear optical effects to occur.
Breaking the symmetry of the focus
prevents totally destructive interference and
some of the third harmonic light is emitted.
Third harmonic light is produced on one side of
the focus...
... and the other...
but they interfere destructively.
Guoy Phase Shift
So third-harmonic imaging is sensitive to
interfacesa good thing.
Images due to Jeff Squier, Colorado School of
Mines
15
Third-harmonic generation is much stronger at
interfaces.
Note the increase in signal at the interfaces. So
THG imaging yields an image of the interfaces of
the specimen.
Images due to Jeff Squier, Colorado School of
Mines
16
Third-harmonic microscopy
The third harmonic of the incident light is
produced when an interface breaks the symmetry of
the focus, providing inherent optical
sectioning. Normal optical microscope objectives
are used to focus the input light and collect the
TH signal light. The sample can be scanned in
x and y (and maybe z) directions.
This work has been pioneered by Squier and
Muller, UCSD
17
Characteristics of THG imaging

• Background-free imaging technique, requiring no
  additional staining.
• Provides inherent optical sectioning.
• Non-fading in nature (stains fade with time).
• Performed under same excitation intensities as
  2-photon microscopy.
• Performed in transmission.
• Coherent imaging technique.
• Uses IR, rather than visible or UV, so is less
  damaging to the specimen.
• Is less bothered by phase distortions in the
  medium than conventional microscopy.

18
Detection of low contrast interfaces
Demonstration using an optical fiber in
index-matching fluid.
The third harmonic signal is generated at the
interface of jacket and cladding no image
processing or background subtraction was used
here. (100 fs pulses at 1.2 µm, 1 kHz repetition
rate.)
Images due to Jeff Squier, Colorado School of
Mines
19
Orientation dependence of THG imaging
The different images show the fiber along
different paths.
Images due to Jeff Squier, Colorado School of
Mines
20
Sectional THG images of spiral algae formation
Excitation pulse 100 fs, at 1.2 µm, at 250 kHz
repetition rate. 1.2 mW average power at
sample. Excitation objective 20x, 0.6 NA Zeiss
Plan-Apochromat Collection objective 20x, 0.4 NA
Olympus Cursor single point, rastered in a
traveling Lissajou pattern. Dispersion 500 fs2,
which results in 30 pulse broadening.
21
3D reconstruction of spiral algae
The THG images of the previous slide allow this
reconstruction.
Images due to Jeff Squier, Colorado School of
Mines
22
Third harmonic real-time imaging of living root
1.5 sec per frame
THG cross sectional images showing motion of
statoliths (Barium Titanite crystals) in a living
plant root (Chara rhizoids). Motion is due to
turbulence.
Images due to Jeff Squier, Colorado School of
Mines
23
More real-time THG images
Artificial blood vessel (two cover slips) with
real red blood cells flowing in it. Scanning
scheme used a Lissajou pattern.
Images due to Jeff Squier, Colorado School of
Mines
24
Still more real-time THG images
Anonymous microbes in Amsterdam canal water
Images due to Jeff Squier, Colorado School of
Mines
25
SHG and THG imaging simultaneously
Images of a Zebrafish larva
20 mm
20 mm
THG (blue) shows edges the larva skin and
boundary of somite and notochord.
Muscle fibers exhibit strong SHG (green) due to
crystalline nano-structure.
Sun and coworkers, Opt. Expr. Nov. 2003
26
A home-built THG microscope
Feature camera detection, or PMT detection for
scattering media Lock-in detection
capability Line scan rates of 1 kHz Multiple
channels for physiology Arbitrary area scan
control About 18 inches high Uses two input
beams to cause fringes to enhance the spatial
resolution
Images due to Jeff Squier, Colorado School of
Mines
27
CARS Imaging
Coherent Anti-Stokes Raman Scattering (CARS) is
another nonlinear process used for biomedical
imaging.
CARS can preferentially image molecules with
certain vibrations.
Ji-xin Cheng, Andreas Volkmer, X. Sunney Xie,
Theoretical and experimental characterization of
coherent anti-Stokes Raman scattering
microscopy, J. Op. Soc. Am. B, 19, 1363-1375
(2002).
28
CARS imaging of cells undergoing apoptosis
(programmed cell death)
Early stage
Late stage
15 ?m
CARS images of of NIH3T3 cells undergoing
apoptosis. The pump and Stokes frequencies were
tuned to the CH2 symmetric vibration at 2845
cm1. Each image was acquired in 8.5 seconds.
29
CARS images of chromosomes
10 ?m
CARS Images of Chromosome distribution at
different depths in a Mitotic NIH3T3 Cell. The
pump and Stoke frequency difference is tuned to
the DNA PO2 stretching at 1090 cm1.
30
Optical Coherence Tomography
Optical ranging in biological tissue can also
produce an image.
This work has been pioneered by Jim Fujimoto and
coworkers of MIT, who have kindly provided their
slides for this section.
31
Optical coherence distance ranging
Just time-resolve the back-scattered light, and
then scan transversely.
32
Cross-sectional imaging
Transverse Scanning
Backscatter Intensity
Axial Scanning (Depth)
Tissue Specimen
Huang, et al., Science, 254 (1991)
33
Low-coherence interferometry
Reference
Michelson Interferometer
Sample
When the interferometer paths are equal, the
intensity fringes are the strongest. The accuracy
is the coherence length.
Detector
34
Fiber-optic interferometer for OCT
An all-fiber system can be constructed.
The two detectors see opposite-phase fringes, so
subtracting their signals doubles the signal and
subtracts off noise.
35
Retinal imaging normal human subject
At the moment, ophthalmologists essentially use
flashlights and look for shadows. Or they take
low-contrast photographs.
Nerve fiber layer
Hee, et al., Ophthalmology 102 (1995)
36
Measurements of a live tadpole
Dorsal
Ventral
Boppart, et al., Dev. Biology 177 (1996)
37
Cardiovascular Imaging unstable plaque in vitro
Comparison of OCT and ultrasound
Unstable plaque has a thin wall of less than 100
microns and is prone to rupture. Standard OCT
with its resolution of 15 microns provides a
significant improvement over conventional
ultrasound with a 100 micron resolution.
Brezinski, et al, Circulation 93 (1996)
38
Ex vivo OCT imaging of cervical cancer
Invasive Carcinoma
Carcinoma In Situ
Normal Cervix
e
e
Ultrasound
500 mm
500 mm
500 mm
OCT
Pitris, et al, Obstetrics and Gynocology 93 (1999)
39
Real-time OCT imaging
(African frog heart in vivo )
Log Reflectivity
500 µm
Images of 512 pixels square can be acquired 4 to
8 frames per second.
Tearney, et al., Opt. Lett. 21 (1996)
40
In vivo ultrahigh resolution OCT
Xenopus laevis (African frog tadpole)
100µm
1 x 5 µm (long. x trans.) 0.54 x 2 mm 1200 x
1000 pixels
Drexler, et al., Opt. Lett. 24, (1999)
41
Inside a blood vessel (in vitro)
The OCT images have significantly higher
resolution than intravascular ultrasound (IVUS).
IVUS
OCT
Brezinski, et al., Am. J. Cardiology 77 (1996)
42
Features of optical coherence tomography
Cross-sectional micron-scale imaging In situ and
in real time, without the need for tissue
excision and processing Catheter / endoscope
imaging of internal organs Consistent with
Minimally Invasive Surgery Electronic information
enables processing, storage, transmission Commerci
al device available for ophthalmologists
43
Ultrafast ballistic-photon imaging
All imaging techniques are intended for nearly
transparent media. Unfortunately, most tissue is
opaque due to massive amounts of
scattering. Its easy to image when light rays
travel straight through a medium. But when they
scatter randomly, how do you image anything?
Most medical imaging problems (e.g., mammography)
are plagued with scattering. Even x-rays are
scattered significantly. This is a major
unsolved problem.
44
Ultrafast ballistic-photon imaging
Since scattering is probabilistic, there will
usually be some photons that experience no
scattering and pass straight through the medium.
Note that rays that travel straight through a
medium take the least time. A tortuous path with
many scatterings takes much longer. So
illuminate the medium with an ultrashort pulse
and time-gate the transmitted beam, detecting
only the photons that arrive earliest (i.e., that
pass straight through).
45
Ultrafast ballistic-photon imaging
The transmitted light will have a fast
ballistic component of unscattered photons,
followed by a slower diffuse scattered component.
Using ultrafast time-gating to detect only the
ballistic component will yield an image of
absorption vs. transverse position.
46
Results using ballistic photon imaging
Alfano and coworkers 24 May 1999 / Vol. 4, No. 11
/ OPTICS EXPRESS
47
Ultrafast Ballistic-Photon Imaging
Unfortunately, the ballistic component is weak,
as most light is scattered away. If Ls is the
mean free path for photons, the energy of the
ballistic photon pulse, Uballistic, is
Uballistic Uincident exp(L / Ls) where L
is the sample length and Uincident is the
incident pulse energy. Example Suppose we wish
to perform mammography. Human breast tissue has
Ls 0.5 mm. If the breast is only 25 mm thick,
then exp(L / Ls) exp(50)
1022 Using 1 J 1019 photons, the ballistic
component has less than one photon!
Ballistic-photon imaging has achieved only
limited success.
48
Imaging with resolution better than a wavelength
Near-field Scanning Optical Microscopy (NSOM)
Light can only image structure as small as l/2 in
size. Wed like to do better. In NSOM, light is
transmitted through a small aperture next to the
object. But this requires a very sharp fiber tip
to be placed on the object, often damaging it.
This method doesnt require short pulses, but
they can be used if ultrafast temporal resolution
is desired.
This method is often used to study inanimate
surfaces.
49
Stimulated-Emission Depletion (STED) Microscopy
Excite with an ultrashort pulse. Probe with a
STED beam pair.
Intense STED beams deplete fluorescence except
where their intensity is zero.
Fluorescing regions as small as 28 nm (l/25) has
been achieved!
x
50
Stimulated-Emission Depletion Microscopy
Laser beams
Experimental STED image of individual molecules
(compared to a conventional image)
Improved resolution is only achieved in one
direction. But a donut-shaped beam could do so
in both directions.
V. Westphal, L. Kastrup, and S.W. Hell, Appl.
Phys. B 77, 377380 (2003).