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

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?

O.SolgaardStanford

Use lasers, detectors, and lenses!

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

O.SolgaardStanford

My Favorite Optical Device!

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

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

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

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

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

O.SolgaardStanford

MicroElectroMechanical System (MEMS) Mirror

SubstrateThermal Oxide

Top Device Layer Bottom Device Layer

substrate 1mm

substrate 1mm

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

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

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.

O.SolgaardStanford

Dual Axes Confocal Microscope

O.SolgaardStanford

3-D MEMS Scanning

Two MEMS mirrors are used to enable 3-D scanning

O.SolgaardStanford

Dual Axis Confocal Microscope

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

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 ….

O.SolgaardStanford

Multimodality Package 1Wide-Field Fluorescence + DAC Microscope

O.SolgaardStanford

70 deg. FOV (wide-field)

300 micron FOV (confocal)

Multimodality: DAC + Wide-field

21

O.SolgaardStanford

Multimodality Package 2Ultrasound + DAC Microscope

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.

O.SolgaardStanford

Visualizing the Vasculature

Normal Tumor

Mouse Ear Tumor ModelJonathan Liu

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.

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

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

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.

O.SolgaardStanford

Evolution of the DAC Microscope

at Stanford

29

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

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