1. 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. Solgaard
Stanford University
Support: CIS, NIH, NSF O.Solgaard
Stanford

2. In-vivo Microscopy
Tabletop Miniaturized
Combined with image-guided,
Microscope Microscope
endoscopic surgery
 Tools for continuous observations of biological systems
 Fundamental biology
10 cm Optical microscopy is non-invasive with sub-cellular resolution

 How do we see through tissue with a miniaturized
optical microscope?
O.Solgaard
Stanford

3. Use lasers, detectors, and lenses!
O.Solgaard
Stanford

4. Optical Fiber
cladding (n2)
~10um core (n1) 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.Solgaard
Stanford

5. My Favorite Optical Device!
O.Solgaard
Stanford

6. 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.Solgaard
Stanford

7. The LENS enabled Telescopes
and Microscopes!
f
a b
 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
O.Solgaard
Stanford

8. Why combine a Pinhole Camera
with a lens microscope?
Detector
Pinhole
 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
O.Solgaard
Stanford

9. We get 3-D AND we can see
through scattering media!
Detector
 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?)
O.Solgaard
Stanford

10. Confocal Microscopy
Confocal Microscopy
Sample
Point Source
Illumination Beamsplitter
Rejected
Light
Pinhole or Imag Rejected
Image Reject
Plane Plane
SMF Accepted
Accept
Light
Detector
M. Minsky, Memoir on inventing the confocal microscope, Scanning, Vol. 10, Issue 4, 1988. O.Solgaard
Stanford

11. MicroElectroMechanical System
(MEMS) Mirror
substrate 1mm
Top Device Layer
Bottom Device Layer
Substrate
Thermal Oxide
1mm
substrate
O.Solgaard
Stanford

12. Operation of 2-D Scanner
GND  Outer axis rotation = V1 and V2
V3  Inner axis rotation = V3 and V4
V2
substrate 1mm
V1
GND
V4 1mm
substrate
O.Solgaard
Stanford

13. 2-D Scanner Characterization
V1 and V2 = Outer-axis rotation
V3 and V4 = Inner-axis rotation
Static mode Dynamic mode
6
V2 Outer axis
5 10
1
V1 Inner axis
4
Optical deflection angle (degree)
Optical deflection angle (degree)
V3
3 V4
2
0
1 10
0
-1
-2
-1
10
-3
-4
-5
-6 10
-2
0 20 40 60 80 100 120 140 160 180 200 2 3
10 10
DC voltage (V) Driving frequency (Hz)
− Outer axis: ±5.5° − Outer axis: ±11.8°@1.18kHz
− Inner axis: ±3.8° − Inner axis: ±8.8°@2.76kHz
13
O.Solgaard
Stanford

14. 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.Solgaard
Stanford

15. Dual Axes Confocal Microscope
O.Solgaard
Stanford

16. 3-D MEMS Scanning
 Two MEMS mirrors are used to enable 3-D
scanning
O.Solgaard
Stanford

17. Dual Axis Confocal Microscope
O.Solgaard
Stanford

18. Raytracing of 3-D Scanning
Total scanning volume = 340um × 236um × 286um
Z-scanning by 1-D depth scanner
27.5um
143um
FOVz (z axis=+/-27.5um) = 286um
X-Y scanning by 2-D lateral scanner
2.7º
170um
FOVx(q=+/- 2.7deg) = 340um / FOVy (q=+/- 1.9deg) = 236um
O.Solgaard
Stanford

19. 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.Solgaard
Stanford

20. Multimodality Package 1
Wide-Field Fluorescence + DAC Microscope
O.Solgaard
Stanford

21. Multimodality: DAC + Wide-field
300 micron FOV (confocal)
70 deg. FOV (wide-field)
O.Solgaard 21
Stanford

22. Multimodality Package 2
Ultrasound + DAC Microscope
O.Solgaard
Stanford

23. 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.Solgaard
Stanford

24. Visualizing the Vasculature
Normal Tumor
Mouse Ear Tumor Model
Jonathan Liu
O.Solgaard
Stanford

25. Imaging of GFP in a Reporter Mouse of Medulloblastoma
Jonathan Liu
In vivo tumor: through the skull Ventral side of the brain
IVIS200 B.
Maestro Image
C. DAC DAC
Medulloblastoma Normal brain
O.Solgaard
Stanford

26. In vivo sequential imaging: siRNA silencing
 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
Standard fluorescence microscope
Stratum corneum
Granulosum
Ex vivo
Footpad skin
skin
gene silencing Green – GFP
sections
20 µm 20 µm
Irrelevant control siRNA Specific siRNA
O.Solgaard
Stanford

27. 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 treatment
Leachman, et al., Mol Ther, 2010
O.Solgaard
Stanford

28. siRNA as a
Therapeutic
Lieberman et al., Cell (2006)
 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.Solgaard
Stanford

29. Evolution of the
DAC Microscope
at Stanford
O.Solgaard 29
Stanford

30. System Concept
Spatial Light Multimode Fiber Focused Light in
Modulator (MMF) 3D
(SLM)
 A cylindrical, step-index waveguide can support
propagating modes
 NA = 1.33, a = 50 µm, λ = 550 nm => N ~ 175,000 = 4202
O.Solgaard
Stanford

31. 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
O.Solgaard
Stanford