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

51

On-Line Database

http://cellmu.com/

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.