Outline - Abbe School of Photonics · Cellular-Resolution Optical Coherence Tomography Sheng-Lung...
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Cellular-Resolution Optical Coherence Tomography
Sheng-Lung Huang
Institute of Photonics and Optoelectronics
National Taiwan University
5/6/2014@Abbe Center of Photonics
Outline
- OCT introduction
- In vitro single cell analysis
- Ex vivo tissue imaging
- In vivo human skin diagnosis
Optical Coherence Tomography (OCT)-
a low coherence interferometry
Specimen
Scattered
light
)()()( tEtEE
Axia
l po
sitio
n (D
ep
th)
1 D 2 D 3 D
High-Dynamic-Range Imaging
3
- Phase sensitive (i.e. optical path sensitive)
- At least 3-dB advantage on S/N compared with direct PD detection
- Insensitive to unwanted background
- Why Interference? Homodyne detection
- Broadband light source while narrow band detection
Broadband interference
22ln2 o
Axial resolution
Noise:
Shot noise: Due to random arrival of photons
Excess intensity noise: Due to different wavelength
Thermal noise: Electronic noise
L
Bre
TBk
42
BI dc
ex
22
4
S/N Ratio in OCT System
BqI dcsh 22
Signal: )]cos(2[ 0 lkPPPPI srsr
)cos(2 0 lkPPI srs
22 I
)/4(]/)1()4/(2[)4/(8 200
02 2
LBrr
r
TkVRPqRP
RP
BSNR
Ref: A. F. Fercher, W. Drexler, C. K. Hitzenberger, and T. Lasser, “Optical coherence
tomography-principles and applications,” Rep. Prog. Phys., 66, 239 (2003).
5
S/N Ratio Simulation
-4 -3 -2 -1 0
80
90
100
110
120
130
SN
R (
dB
)
log Rr
SNR
SNsh
R
SNex
R
SNthR
Light
source
P0 1.5 mW
0 830 nm
25 nm
V 1
Photon
detector
NEP 6.25×10-13 W
B 100 kHz
0.8 A/W
Beam splitter ratio is 0.5.
If system is shot-noise-limited:
Parameter
qB
PSNR
4
0
Speckle
6
Definition: Speckle appears in a signal when that signal is imposed of a
multitude of independently phased additive complex components.
Largely constructive addition Largely destructive addition
i.e. random walks
Ref: J. W. Goodman, Speckle phenomena in optics- theory and applications, 2007.
7
Spatial Resolution
Rayleigh
criterion
Sparrow
criterion
Axial resolution Lateral resolution
)()()( tEtEE
Axial
resolution
22ln2
2
oCl
NAr o
46.0NA
r o61.0
)(: ECl FWHM of
High axial resolution needed
High lateral resolution needed
Isotropic resolution needed
8
Isotropic Resolution
0
5
10
15
0 50 100 150 200 250
0.0
0.1
0.2
0.3
0.4
0.5
Reso
luti
on
(m
icro
n)
Bandwidth (nm)
Re
qu
ired
NA
fo
r is
otr
op
ic
res
olu
tio
n
20x
10x
Objective
Required NA for isotropic resolution
9
OCT vs. Confocal Microscopy
OCM can image with high transverse resolution at much lower numerical
aperture than confocal microscopy because it does not depend on high axial
resolution for optical sectioning. Ref. J. Fujimoto 2005 report
22ln2 c
Cl
10
Penetration Depths and Ranges of Lateral Dimensions
AFM: atomic-force
microscopy
AOH: all-optical histology
BL: bioluminescence
FL: fluorescence
microscopy at visible
wavelengths
MEG: magnetoencephalography
MRI: functional magnetic
resonance imaging
OCT: optical coherence
tomography
PET: positron emission
Tomography
TIR-FM: total internal
reflection fluorescence
microscopy
US: ultrasound.
Imagining imaging’s future, Nature Reviews of Molecular Cell Biology, 2003.
11
Light Sources Used in OCT
Light source c (nm) c (mm) Coherence power
SLD
(super luminescent light diode)
675 nm 10 20 40 mW
820 nm 20 15 50 mW
820 nm 50 6 6 mW
930 nm 70 6 30 mW
1300 nm 35 21 10 mW
1550 nm 70 15 5 mW
Ti:sapphire femtosecond laser 810 nm 260 1.5 400 mW
Cr:forsterite femtosecond laser 1280 nm 120 6 100 mW
Yb-doped fiber 1064 nm 30 17 40 mW
Er-doped fiber 1550 nm 80 16 100 mW
Tm-doped fiber 1800 nm 80 18 7 mW
fs laser + Photonic crystal fiber 1300 nm 370 2.5 6 mW
725 nm 370 0.75
Thermal tungsten halogen 880 nm 320 1.1 1 mW
CW and Bright Broadband Sources
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
0 200 400 600 800 1000 1200 1400 1600 1800
No
rm. sp
ectr
al
den
sit
y (
a.u
.)
Wavelength (nm)
Ce3+:YAG
Ti3+:sapphire
Cr4+:YAG
- LCD devices
- Ocular applications
- OCT+confocal
- Semiconductor devices (IC, solar cell)
- Moisture sensing
- Ophthalmology
- Dermatology
- Other biomedical organs
DCF
cross section
SAED of
DCF core
22ln2
2
oCl
13
Glass-Clad Crystal Fibers base OCT
0.00
2.00
4.00
6.00
8.00
10.00
0 100 200 300 400
Bandwidth (nm)
Ax
ial
res
olu
tio
n (
mm
)
Wavelength= 0.56 micron
Wavelength= 0.84 micron
Wavelength= 1.15 micron
Wavelength= 1.31 micron
Wavelength= 1.38 micron
Ultra-broad band Gaussian-like spectrum
in air
Point spread
function
- Higher resolution
- Less speckle
- Less image-pixel cross talk
- Power independent resolution
- CW and cost effective
- Satisfy FDA regulation: 20 mJ/cm2
-10 -8 -6 -4 -2 0 2 4 6 8 10
0.0
0.5
1.0
1.5
Inte
nsit
y (
a.u
.)Path length (mm)
14
A. C. Akcay, J. P. Rolland, and J. M. Eichenholz,
“Spectral shaping to improve the point spread
function in optical coherence tomography,” Opt.
Lett. vol. 28, p. 1921, 2003.
Side Lobe Influence on Image Quality
1 2
1 (large
side lobe)
2 (small
side lobe) Onion measured using CF light source
Single layer
Multi-layer
OCT Schemes
15
3D tomography Source arm Reference arm Detection
Scan PD/CCD/CMOS
Time-domain OCT Δω τ(t) 2 D 0 D
Spectral-domain OCT Δω τo 2 D 1 D
Sweep source OCT ω(t) τo 2 D 0 D
Spatial-domain OCT Δω τo 2 D 1 D
Full-field OCT Δω τ(t) 0 D 2 D
OCT = Broadband light source
+ Michelson interferometer
+ scanning
τ
ω
DUT Source
arm
Reference
arm
16
CW Crystal Fiber Light Source
Ce:YAG crystal fiber emission spectrum
Theoretical analysis
Out of a 70-μm crystal fiber
Out of a 200-μm MMF
Power and luminous flux
Point spread function
Pixel number AIC (dB)
1st adjacent -20
2nd adjacent -46
3rd adjacent -61
Axial image crosstalk
- In vitro single cell analysis
- Ex vivo tissue imaging
- In vivo human skin diagnosis
Outline
18
Google on “Cell Image”
19
Cell Tomography Technologies
Frozen cell by X-ray In vitro with tissue staining
C. A. Larabell and M. A. Le Gros, “X-ray tomography generates
3-D reconstructions of the yeast, Saccharomyces cerevisiae, at
60-nm resolution,” Molecular Biology of the Cell, 15, pp. 957-
962, 2004.
Yeast cell: 5-μm diameter
20-μm
https://www.invitrogen.com.tw/shop/index.asp
Skin Cells Tomography using Full-Field OCT
20
Skin cells
Keratinocyte
Mature keratinocyte
Melanocyte
En face Cross section
Intra-cellular Organelle Imaging of BCC
Microscope 2D image (50x objective )
J. W. Tjiu, NTU Hospital
Basal cell carcinoma cell line
– BCC number: 15000
– Matrigel dilution: 5X
– Matrigel volume: 50 μL
– 24hrs after seeding BCCs
Feature of the Week, 2012.
Single Cell 3D Tomography Analysis
22
Cell boundary
Normal BCC Apoptotic BCC Sum
SMF 11 6 17
Free space 15 3 18
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
Normal Apoptotic
Sig
na
l a
ve
rag
e (
a.u
.)
P < 0.001
Normal group, 0.01506 ± 0.00065
Apoptotic group, 0.01262 ± 0.00111
The measured signal average & cell density of single
cells show statistically significant difference between
normal & apoptotic BCC cells.
Raw image w/ Gaussian filter w/ bilateral filter
Cell Segmentation
3D segmented cell
4
HaCaT Tomography
Axial step per frame= 0.84 µm
10 µm
Axial resolution= 0.9 µm
Lateral resolution= 0.9 µm
Frame rate: 10 fps
Skin Cell Identification
25
Signal
average
Cell volume
Fibroblast (Hs68) Melanoblast
(M5)
Keratinocyte
(HaCaT)
Fibroblast
(Hs68) P=0.0007 P=0.0018
Melanoblast
(M5) P=0.0034 P=0.0052
Keratinocyte
(HaCaT) P=0.0097 P=0.0292
Parameters been considered:
Signal average, signal dynamic range, signal variance, cell density, cell volume
Ca2+ Induced Keratinocyte Differentiation
Br. J. Dermatol. 1994; 130: 139-47
Increased [Ca2+] results
• Rapid redistribution of molecules from
the cytosol to the membrane in the
formation of intercellular contacts
• Formation of cornified envelope by
crosslinking loricine, involucrine and
other proteins (Mol Cell Endocrinol 2001; 177:
161-71)
• These subcellular changes results in
alteration in back-scatting signals and
might be detected by ultrahigh resolution
OCT.
Formation of desmosome 24 hours
after increase Ca2+ level to 1.2 mM
(1550x) (Cell 1980; 19: 245-54)
Cell Discrimination:
Keratinocyte in Differentiation States
Epidermis structure
Keratinocytes begin in the basal layer as
undifferentiated cells, the daughter cells lose
proliferative ability and give rise to differentiated
cells comprising the spinous, granular, and
cornified layers.
Control
Treated
Epidermis cells from
newborn BALB/c mice
(in normal keratinocyte-SFM for 6 days)
(1.2mM CaCl2 in keratinocyte-SFM for 6 days)
6
Features Extraction/Reduction
Components
1 2 3 4 Skew .577 .620 .513 .034
Kurt .482 .534 .651 .058
SA_dB .808 -.535 -.003 -.010
SV_dB .749 .312 -.305 .127
Skew_dB .185 .660 .222 -.069
Kurt_dB .495 .331 .611 -.071
ADR .793 .113 -.451 -.103
VDR .628 .628 -.144 .201
Skew-DR .105 -.007 .721 -.630
Kurt-DR -.322 .135 .358 .774
TDR .763 .548 .048 .096
CD .800 .198 -.266 -.066
Cell_pixel -.100 .686 -.552 -.153
CV -.100 .686 -.552 -.153
SA_mask .797 -.556 -.001 .059
SV_mask .984 .023 .010 .034
SA .853 -.485 -.039 .011
SV .973 -.151 -.062 .060
H3 .492 -.816 .131 .053
L3 .890 -.224 -.168 -.047
Features and components
60%
70%
80%
90%
100%
SVM analysis
5
A-scan of Primary Melanocyte
T-mode en-face image
Point A
Point B Point C
Point D
Melanocyte
Immature keratinocyte
Point A
Point B
Point C
Point D
Water Distribution in Cell: Simulation Model
Collaboration with Prof. Snow Tseng
A-scan
B-scan
Impact of Rough Boundary
31
𝑎 =2 ∗ 𝑛2𝑛1 + 𝑛2
𝑏 =2 ∗ 𝑛1𝑛1 + 𝑛2
𝑛3 =𝑎 ∗ 𝑏 ∗ 𝑛2 ∗ 𝐸𝑖 − 𝑛2 ∗ 𝐸𝑏𝑎𝑐𝑘
𝐸𝑏𝑎𝑐𝑘 + 𝑎 ∗ 𝑏 ∗ 𝐸𝑖
𝑛3 = 1.3400
Melanoma Cell Line
32 Cell size: ~ 30 μm
2D
microscope
3D OCT cross sections
- In vitro single cell analysis
- Ex vivo tissue imaging
- In vivo human skin diagnosis
Outline
Ex Vivo Anatomic Imaging of Skin
34
Stratum corneum
Stratum spinosum
Stratum granulosum
Stratum basale
Stratum lucidum
Epidermis
Dermis
20 μm
FF-OCT cross section Skin schematic
Incident power: 3 mW
En face frame rate: > 10 frame/s
http://en.wikipedia.org/
wiki/Epidermis_(skin)
Female 1963 / inner arm Female 1923 / inner arm
Aging Evaluation
20 mm 20 mm
Male 1945 / face
30 mm 30 mm
SC
SS
36
Skin Quality Evaluation
OCT cross-sectional image Layer number and total thickness
Total thickness: 33.8 mm
Layer number: 12
Average layer thickness: 2.82 mm
Expansion of hydration: 40%
With hydration
Total
thickness
(μm)
# of layers Average layer
thickness (μm)
Maximum 41.42 17 3.65
Minimum 24.50 8 2.04
Average 32.94 13.05 2.55
SD 4.48 2.09 0.31
20 μm
37
Skin histology
Optical Biopsy?
In vivo Mirau based OCT
OCT Endface and Biopsy Comparison
38
Stratum corneum
Epidermis
Dermis
Hair follicle
Nude mouse biopsy Stained
biopsy OCT
endface
Fat cell Hair follicle
In-Vivo Fish Cornea Stroma Observation
Ce:YAG DCF based OCT Conventional OCT
Clinical
significance ?
Normal
Fuchs’
Keratoconus
: Feature of the Week, 2010.
Time Evolution of Fish Cornea
Time= 0 minute Time= 30 minutes
Time= 60 minutes Time= 90 minutes
Average thickness: 2.69 mm
Standard deviation: 1.49 mm
- In vitro single cell analysis
- Ex vivo tissue imaging
- In vivo human skin diagnosis
Outline
42
In Vivo Skin Diagnosis at Forearm
Melanin Cap
In vivo, forearm, 35-year-old male
z=192
z=209
z=176
145 x100 μm2
En face Cross sections (max projection)
x-z plane y-z plane
110 x100 μm2
Refractive Indices of Bio Specimen
1.3
1.35
1.4
1.45
1.5
0.3 0.4 0.5 0.6 0.7 0.8
Wate
r re
fracti
ve i
nd
ex
Wavelength (µm)
Tissue Index
Epidermis 1.34
Dermis 1.41
Organelle ---
Mitochondria 1.42
Nucleus 1.39
Ingredient ---
Collagen 1.43
Keratin 1.51
Melanin 1.70
Protein 1.353
DNA 1.345
Extracellular fluid 1.35
cytoplasm 1.37
Lymphocyte
1. Plasma membrane (lipid): n=1.48, < 1% volume
2. Cytoplasm (protein & water): n=1.35, 49% volume
3. Nucleus (protein & water): n=1.39, 48% volume
4. Average: n=1.37
1. Normal cell: water content: 20 – 60 %, average n=1.385
2. Cancer cell: water content 80 %, average n=1.399
Breast cell
- V. A. Loiko, et al, Conference on Light Scattering by Non-spherical
Particles,105 (2007).
- http://web.mit.edu/newsoffice/2001/fenn-0110.html
OCT on Deterministic Biometry?
45
Melanin cap in epidermis
Dispersion and Extinction Coeff.
Measured by OCT Validation
T. S. Ho, P. Yeh, C. C. Tsai, K. Y. Hsu, and S. L. Huang, “Spectroscopic measurement of absorptive thin films
by spectral-domain optical coherence tomography,” Optics Express, 22, No. 5, pp. 5675–5683, 2014.
From Epidermis to Dermis
En face Cross section
In vivo flow cytometry?
Toward In Vivo Flow Cytometry: RBC Detection
48
30 mm
30
mm
30 mm
- Typical dimension
Diameter: 6.2~8.2 mm
Thickness: 2~2.5 mm
- Measured dimension
Diameter: 7.2 mm
Thickness: 1.8 mm
Red blood cell
Toward In Vivo Flow Cytometry: White Blood Cell
49
30 mm
30 mm
30
mm
- Typical dimension (lymphocyte)
Diameter: 7~15 mm
- Measured dimension
Diameter: 10.0 mm
White blood cell
50
Blood Flow Measurement at Forearm
Real-time blood flow monitoring
Orthogonal polarization imaging
OCT cross section
52
From Cellular Resolution Tomography
to Early Disease Diagnosis
Structure
Function
Severity of disease
Ch
an
ge
Disconnect between structure and function in bio system.
Beyond anatomy
- Necrosis/ apoptosis
- Cellular interaction
- Activation/ pathway
- System coordination
- Disease diagnosis
High resolution
OCT
Broadband
spectroscopic
OCT, Raman,..
Cellular
signaling
path
Take home message…
53
- Full-field OCT with high resolution, high speed, and
low image pixel cross talk could enable real-time
cellular diagnosis of skin during clinics or in surgical
operation.
- It is crucial to link OCT tomogram features with
clinical meaning, and OCT validation is ongoing in
worldwide laboratories.
- The integration of OCT (cellular-level resolution) with
molecular-level spectroscopy could be extremely
powerful in clinics for early disease and cancer
diagnosis.
54
Acknowledgment
NTU Hospital:
MD Jeng-Wei Tjiu
MD Chang-Hao Yang
MD Chia-Tung Shun
MD Yu-I Li
Taipei Veterans General Hospital:
MD Chang-Youh Tsai
MD Yen-Bo Tsao
Dr. C. K. Wei
Dr. R. T. Ueng
Dr. William Wang
Dr. C. P. Chuang
Dr. Mason Yen
NTU GIPO:
Prof. Snow Tseng
Vielen Dank für Ihre Aufmerksamkeit.