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O.SolgaardStanford
Fiber optics: Window on human biology
H. Ra, W. Piyawattanametha, E. Gonzalez-Gonzalez, J.-W. Jeung, Y. Taguchi, D. Lee, U. Krishnamoorthy, I.W. Jung, M. Mandela, J. Liu, K.
Loewke, T. Wang G.S. Kino, C. Contag, O. SolgaardStanford University
Support: CIS, NIH, NSF
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O.SolgaardStanford
MiniaturizedMicroscope
TabletopMicroscope
10 cm
In-vivo MicroscopyCombined with image-guided,
endoscopic surgery
Tools for continuous observations of biological systems Fundamental biology Optical microscopy is non-invasive with sub-cellular resolution
How do we see through tissue with a miniaturized optical microscope?
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O.SolgaardStanford
Use lasers, detectors, and lenses!
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O.SolgaardStanford
Optical Fiber
core (n1)
cladding (n2)
~10um 125um
Optical fibers are glass cylinders Highways of the internet
Transmission band: 1,280 to 1,610 nm => 50 THz! Transmission band of coaxial cable <10GHz
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O.SolgaardStanford
My Favorite Optical Device!
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O.SolgaardStanford
Camera Obscura – Pin Hole Cam
The pinhole projects a scene on the camera screen Object at any distance are imaged with high fidelity
(but upside down) The pinhole must be small to give a sharp image
=> low light efficiency
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O.SolgaardStanford
The LENS enabled Telescopes and Microscopes!
The lens projects a scene on the camera screen Objects in focus (1/a+1/b=1/f) are imaged with high
fidelity The lens can be large and still give a sharp image
=> high light efficiency
a b
f
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O.SolgaardStanford
Why combine a Pinhole Camera with a lens microscope?
The lens projects a single volumetric pixel (voxel) on the pinhole
Only light from the single voxel in focus is registered on the detector
We scan the voxel around to get a 3-D image
Detector
Pinhole
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O.SolgaardStanford
We get 3-D AND we can see through scattering media!
We still see the voxel even if it is embedded in a scattering medium, e.g. tissue! We get a less bright voxel, but it is not obscured by light from
other voxels
We can see into the body! (Camera not-obscura?)
Detector
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O.SolgaardStanford
AcceptRejectImag
M. Minsky, Memoir on inventing the confocal microscope, Scanning, Vol. 10, Issue 4, 1988.
Point SourceIllumination Beamsplitter
Detector
Pinhole orSMF
Sample
RejectedLight
AcceptedLight
ImagePlane
RejectedPlane
Confocal MicroscopyConfocal Microscopy
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O.SolgaardStanford
MicroElectroMechanical System (MEMS) Mirror
SubstrateThermal Oxide
Top Device Layer Bottom Device Layer
substrate 1mm
substrate 1mm
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O.SolgaardStanford
Operation of 2-D Scanner
V1
V2
V3
V4
GND
GND Outer axis rotation = V1 and V2 Inner axis rotation = V3 and V4
substrate 1mm
substrate 1mm
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O.SolgaardStanford
2-D Scanner Characterization
0 20 40 60 80 100 120 140 160 180 200-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
Opt
ical
def
lect
ion
angl
e (d
egre
e)
DC voltage (V)
V2 V1 V3 V4
102 10310-2
10-1
100
101
Opt
ical
def
lect
ion
angl
e (d
egre
e)
Driving frequency (Hz)
Outer axis Inner axis
− Outer axis: ±5.5°− Inner axis: ±3.8°
− Outer axis: ±11.8°@1.18kHz− Inner axis: ±8.8°@2.76kHz
V1 and V2 = Outer-axis rotationV3 and V4 = Inner-axis rotation
Static mode Dynamic mode
13
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O.SolgaardStanford
DAC Design
Schematic of the dual-axis confocal (DAC) microscope HL: hemispherical lens MEMS: microelectromechanical systems scanning mirror PMT: photomultiplier tube
The laser, PMT, and transimpedance amplifier setting and gains are constant within and across 3-D datasets for quantification.
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O.SolgaardStanford
Dual Axes Confocal Microscope
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O.SolgaardStanford
3-D MEMS Scanning
Two MEMS mirrors are used to enable 3-D scanning
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O.SolgaardStanford
Dual Axis Confocal Microscope
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O.SolgaardStanford
143um
27.5um
Z-scanning by 1-D depth scanner
2.7º
170um
X-Y scanning by 2-D lateral scanner
FOVz (z axis=+/-27.5um) = 286um
FOVx(q=+/- 2.7deg) = 340um / FOVy (q=+/- 1.9deg) = 236um
Total scanning volume = 340um × 236um × 286um
Raytracing of 3-D Scanning
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O.SolgaardStanford
Dual Axis Confocal microscopes
10 mm with alignment optics Skin
Miniaturized 5 mm for endoscopy GI tract
10 mm with GRIN extender Brain imaging
Implantable DAC ….
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O.SolgaardStanford
Multimodality Package 1Wide-Field Fluorescence + DAC Microscope
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O.SolgaardStanford
70 deg. FOV (wide-field)
300 micron FOV (confocal)
Multimodality: DAC + Wide-field
21
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O.SolgaardStanford
Multimodality Package 2Ultrasound + DAC Microscope
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O.SolgaardStanford
DAC Applications: Cancer Screening
The first in vivo imaging in the GI tract of a patient using a MEMS-based confocal microscope has been demonstrated with the 785 nm 5-mm-diamter DAC microscope Images are taken at 5 Hz with 2 frame averaging, yielding FWHM
transverse and axial resolutions of 4 um and 7 um The DAC endomicroscope was loaded in the instrument channel of a
therapeutic upper GI endoscope Topical application of ICG (25 mg of medical grade ICG diluted in 4 ml of
aqueous solvent) ICG is a chromophore as well as a fluorophore, so we identify areas
where ICG is binding well with a wide-field CCD camera, and then bring the DAC into contact with the tissue of interest.
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O.SolgaardStanford
Visualizing the Vasculature
Normal Tumor
Mouse Ear Tumor ModelJonathan Liu
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O.SolgaardStanford
Imaging of GFP in a Reporter Mouse of Medulloblastoma
Medulloblastoma Normal brain
In vivo tumor: through the skull Ventral side of the brain
C.
Jonathan Liu
Maestro Image
IVIS200
DAC DAC
B.
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O.SolgaardStanford
Experimental methods Silencing the GFP reporter gene in the epidermis by intradermal
injection of siRNA Intradermally inject irrelevant control siRNA and specific siRNA
(targeting GFP mRNA) in each footpad for 14 days siRNA potently and specifically inhibits GFP expression in the
epidermis, control siRNA has no effect
20 µm 20 µm
Ex vivoskin
sections
Irrelevant control siRNA Specific siRNA
Footpad skinGreen – GFP
Stratum corneumGranulosum
gene silencing
In vivo sequential imaging: siRNA silencing
Standard fluorescence microscope
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O.SolgaardStanford
Clinical test Topical application of IC-GREEN cream formulation Excess cream removed with cotton pads after 15 - 30
mins Gel used as an optical coupling agent
Volunteer
PC patient Prior treatment Intradermal injection of TD101 siRNA (right) and
vehicle control solution (left) in symmetric plantar calluses Twice weekly for 17 weeks
Imaged 48 days after last siRNA treatmentLeachman, et al., Mol Ther, 2010
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O.SolgaardStanford
Lieb
erm
an e
t al.,
Cel
l(20
06)
siRNA as a Therapeutic
Short, 19-23 nucleotides long, double stranded RNA
Any gene can be theoretically be silenced
Easy to synthesize
Can target multiple genes
Highly specific and efficient (in cell culture)
Delivery is the rate limiting step to translation
More than $4 billion worth of deals struck since 2000.Yet, no effective delivery tools described to date.
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O.SolgaardStanford
Evolution of the DAC Microscope
at Stanford
29
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O.SolgaardStanford
System ConceptSpatial Light
Modulator (SLM)
Multimode Fiber (MMF)
Focused Light in 3D
A cylindrical, step-index waveguide can support propagating modes
NA = 1.33, a = 50 µm, λ = 550 nm => N ~ 175,000 = 4202
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O.SolgaardStanford
Impact Studies of mammalian gene function and
regulation Models of human diseases Molecular reporters
Fluorescent markers
Continuous intravital optical microscopy will lead to new understanding of fundamental biological processes Investigations of Biological processes over
extended time Cancer progression and metastasis Stem cell regeneration and differentiation Neurology Optogenetics