integrated lasers for biophotonics

Integrated Lasers for Biophotonics

Peking University Summer SchoolBeijing, ChinaJuly 19, 2013

James S. HarrisStanford University

Peking University Summer School, July 19, 2013

STANFORD

JSH 2

Outline

Motivation and background Implantable sensor design and fabrication In vivo monitoring of a molecular probe in

a tumor Blood Coagulation Sensor Neural Activity Sensor Summary


STANFORD

JSH 3

The NANO-BIO-TECH Revolution

Integrated Circuit-1961 STM-1981 AFM-1986

Vo-Dinh, Nanobiotech 1, 3 (2005)


STANFORD

JSH 4

Medical imaging

MRI

From structural/anatomical……to functional imaging

PETPET

MRI

Bioluminescence

Fluorescence


STANFORD

JSH 5

Multimodality Imaging Strategies


STANFORD

JSH 6

Light-tissue interactions

Incident Light

DiffuseReflectance

SpecularReflectance

Chromophore in ground state

Photon at incident wavelength

Chromophore in excited state

Fluorescent photon

Raman-shifted photon

TissueAbsorption

Multiple elasticScattering

Single Backscattering

Fluorescence

RamanScattering

Fluorescence

Raman Scattered

Provides molecular functional information


STANFORD

JSH 7

Advantages of near IR Window of operation: 650–900 nm

Integrated semiconductor sensors

Low intrinsic absorption & scattering

Shah. Et al, NeuroRx Review 2, 215 (2005)Day vs night star viewing

mabs = 0.04 cm-1 mscattering = 10 cm-

1No tissue autofluorescence


STANFORD

JSH 8

• Integration of laser-induced fluorescence (LIF) detection

Example Integrated Sensor

Photonic systems integration

LIF Detection System

– Expensive, bulky and non-portable

• Discrete components • Integrated system– Cheap, portable and

parallel

1 meter 1 mm – 100 m


STANFORD

JSH 9

Vertical Cavity Surface Emitting Lasers: Miniature + can be integrated Low-cost manufacturing/packaging Arrayable

Images: Logitech, M-ComImages: Molec. Devices ,D. Armani et al, Nature 2003;

B. Cunningham et al, Sens Act B 2002; Biacore

Bulky, expensive external light sources and delicate alignment

not realistic for point-of-care/ bedside

2 ft long, 30 lbs

VCSELs are key for low-cost, compact biosensors


STANFORD

JSH 10

Materials challenges for biosensors

InAs

AlSb

GaSbGeBULKSLE

(Ge)

a-SiC

GaSbInSb

AlSb CdTe

InAs

InN

GaN

AlN

ZnS MgSe

CdSAlP

CdSe

ZnTe

ZnSe

GaAs

GaPAlAs

InPSi

Lattice Constant (Å)

(.41 mm)

(.62 mm)

6.0

4.0

3.0

2.0

1.0

3.0 3.2 3.4 5.4 5.6 5.8 6.0 6.4

Ban

dgap

ene

rgy

(eV

)

(1.24 mm)

Visible Spectrum

(.31 mm)

(.21 mm)

.31

.41

.62

1.2

Wavelength (µ

m)

Telecom & IT Revolution

Fluorescent Proteins


STANFORD

JSH 11

Outline

Motivation and background Implantable sensor design and fabrication In vivo monitoring of a molecular probe in a

tumor Blood Coagulation Sensor Neural Activity Sensor Summary


STANFORD

JSH 12

n-DBR

Laser Cavity

675nm VCSEL Detector

Absorption region (i-GaAs)

p-DBR

GaAs Substrate

Fluorescent Molecules

Excitation

EmissionBack-scattered excitation light

Emission Filter

Detector

VCSEL

Detector

VCSEL

Detector

VCSEL

Top view

Side view

Integrated sensing geometry

Sponta

neo

us

em

issi

on


STANFORD

JSH 13

Implantable Fluorescence Sensor


STANFORD

JSH 14

Detector

Laser (VCSEL)High quality optical filter blocks fluorescence

excitation from reaching detector (Desire >OD6 99.9999% isolation)Requires combination of spatial and spectral

isolation

Optical Filter (above detector)

Gallium Arsenide (GaAs)-based Integrated Optical Sensor


STANFORD

JSH 15

What are we sensing?

• Cyanine 5.5 (Cy5.5) dye – Many existing preclinical probes in use

• Near-Infrared (NIR) imaging– Favorable optical properties in

tissue – Low tissue autofluorescence– Increased availability of near-

IR molecular probes – Emerging NIR fluorescent

proteins*

*X. Shu, et al, Science 324, 804 (2009)

650 700 750 800 850 900 950 10000.0

0.2

0.4

0.6

0.8

1.0

Abs

orpt

ion

(mm

-1m

M-1)

Wavelength(nm)

Tissue NIR absorbers

HHb

O2Hb

BULK LIPID

H2O


STANFORD

JSH 16

What are we sensing?

• Cyanine 5.5 (Cy5.5) dye – Many existing preclinical probes in use

600 650 700 750

0

20

40

60

80

100

Inte

nsi

ty (

a.u

.)

Wavelength (nm)

Excitation Emission

Cy5.5 Absorption/Emission


STANFORD

JSH 17

Sensor components: Laser

• VCSEL Characteristics– Optical output power (12µm oxide aperture)– 1-2mW at room temperature– Lasing up to 50°C– Wavelength: 675nm (+/- 1nm)– Multimode linewidth: <0.2nm (FWHM)

673.5 674.0 674.5 675.0 675.5 676.0

0.0

0.2

0.4

0.6

0.8

1.0

Integrated VCSEL Spectra

Inte

nsi

ty (

a.u

.)

Wavelength (nm)

5.0mA 6.0mA 7.0mA 8.0mA 9.0mA 10.0mA 11.0mA 12.0mA

0 1 2 3 4 5 6 7

0.0

0.5

1.0

1.5

2.0

1

2

3

4

Vo

ltag

e (V

)

Lig

ht

Ou

tpu

t (m

W)

Current (mA)

20C 25C 30C 35C 40C 45C 50C

Integrated VCSEL IV Characteristics


STANFORD

JSH 18

Sensor components: Detector

Detector characteristics– Area: 0.75mm2

– Internal quantum efficiency: >75%– Dark current: <5pA/mm2 (0.1V)

0.0 0.2 0.4 0.6 0.8 1.01E-14

1E-12

1E-10

1E-8

1E-6

Da

rk C

urr

en

t (A

)

Reverse Bias (V)

No Passivation SiNx Passivation without wet etch SiNx Passivation with wet etch

Reduction of Dark Current

-1.0 -0.5 0.0 0.5 1.0 1.5

0

1

3

4

Fo

rwa

rd C

urr

en

t (m

A/m

m2)

Detector Bias (V)

Integrated Detector Dark Current

-0.4 -0.2 0.0 0.2-30

-20

-10

0

10

20

pa

/mm

2

V

T. O’Sullivan et al, Proc. SPIE 7173, (2009)


STANFORD

JSH 19

Sensor specifications:emission filtering

600 650 700 750 800

0.0

0.2

0.4

0.6

0.8

1.0In

ten

sity

(a

.u.)

Wavelength (nm)

Cy5.5 Excitation Cy5.5 Emission Detector Response VCSEL Excitation

Overlap betweendetector response and Cy5.5 emission

VCSEL Excitation

• Consists of thin-film interference filter and thick hybrid (absorption) filter element

• 15-20% overlap with fluorescence emission

Emission Filter Spectral Response


STANFORD

JSH 20

Summary: Sensor design

• Designed for Cy5.5 sensing• Vertical-cavity surface-emitting laser

(VCSEL), emitting at 675nm • Large-area, low dark current, uncooled

GaAs photodiode • Integrated excitation blocking elements

– Thin-film fluorescence emission combined with miniature optical filter

– Metal blocking layers

Packaged sensor with collimation lens

5x10 array of sensors Excitation blocking elements


STANFORD

JSH 21

Outline

Motivation and background Implantable sensor design and fabrication In vivo monitoring of a molecular probe

in a tumor Blood Coagulation Sensor Neural Activity Sensor Summary


STANFORD

JSH 22

How to monitor a cancerous tumor

Molecule of interest

Cy5.5 dye

Cancer

● Tumors start as a single cell, and divide/grow to a mass of cells

● At some point their growth is limited because of a lack of nutrients/oxygen recruits new blood vessels (angiogenesis)

● New blood vessels (neovasculature) have an up-regulated integrin receptor (αVβ3)

Fluorescent probe

?


STANFORD

JSH 23

Sensor performance:In vivo sensitivity

• Injected live anesthetized mouse subcutaneously on dorsum with 50µL dilutions Cy5.5

• Sensed Cy5.5 concentration down to 50nM• Correlates with CCD-based fluorescence imager

10 100 1000

1E9

1E10

N=2 mice each concentration

Correlation of Sensor and CCD Imager

CC

D M

ax R

adia

nce

(p/s

ec/c

m2 /s

r)

Sensor Photocurrent (pA)

50nM

100nM

250nM

500nM

2.5M

5M

10M

100 1000 10000

10

100

1000

Variation due to background

Subcutaneous in vivo Sensitivity

Se

nso

r P

ho

tocu

rre

nt (

pA

)

Cy5.5 Concentration (nM)

Live Nude(Nu/Nu) Anesthetized Mouse

Dye

Control

50nM in vivo sensitivity

T. O’Sullivan et al, Opt. Express 18, (2010)


STANFORD

JSH 24

Cy5.5-RGD accumulates at tumor sites

RGDCy5.5 dye

Cancerous cell / angiogenesis

Cy5.5

Cheng et al. Bioconjugate Chem., Vol. 16 No. 6 2005

Cy5.5-RGD binds to neovasculature associated with

cancer


STANFORD

JSH 25

Application: Study binding kinetics of molecular probe in cancer tumors

• Continuously monitored the RGD-Cy5.5 and RAD-Cy5.5 probe in live anesthetized animals for 2-4 hours post-injection

• We are able to study binding kinetics with higher temporal resolution

0 1 2 3 40

10

20

30

40

50

60

AC

Ph

oto

curr

en

t (R

MS

-pA

)

Time (hr)

Tumor Control

0 1 20

10

20

30

40

50

60

AC

Ph

oto

curr

en

t (R

MS

-pA

)

Time (hr)

Tumor Control

Tumor-specific probe (RGD-Cy5.5) Not tumor-specific (RAD-Cy5.5)

T. O’Sullivan et al, in preparation (2010)


STANFORD

JSH 26

Application: Study binding kinetics of molecular probe in cancer tumors

• Continuously monitored the RGD-Cy5.5 and RAD-Cy5.5 probe in live anesthetized animals for 2-4 hours post-injection

• We are able to study binding kinetics with higher temporal resolution• Device is also sensitive to changes in anesthesia (blood

oxygenation)

0 1 2 3 40

10

20

30

40

50

60

AC

Ph

oto

curr

en

t (R

MS

-pA

)

Time (hr)

Tumor Control

Tumor-specific probe (RGD-Cy5.5)

650 700 750 800 850 900 950 10000.0

0.2

0.4

0.6

0.8

1.0

Abs

orpt

ion

(mm

-1m

M-1)

Wavelength(nm)

Tissue NIR absorbers

HHb

O2Hb

BULK LIPID

H2O

T. O’Sullivan et al, in preparation (2010)

Changes in anesthesia


STANFORD

JSH 27

Transitioning to theimplanted sensor

• The cable is a significant source of noise for analog readout

• Packaged the sensor with a low-noise readout circuit (collaboration with Roxana Heitz / Prof. Bruce Wooley Group)

• Represents first step towards realizing wireless operation

0 50 100 150 200 2501.0

2.0

3.0

4.0

5.0

Pho

tocu

rren

t (nA

)

Time (s)

Detector with analog readout Detector with digital readout


STANFORD

JSH 28

Miniaturization for Implantation

• Fabricated small (10mm x 10mm custom PCB, 0.031’’ thick) for bonding chips directly

• Ability to sample continuously at 5kHz / 5pA resolution / up to 20nA

• Device encapsulated in insulating biocompatible epoxy for direct implantation in rodents

• Weight: <1g, Size: 10x10x8mm

1cm

Suture holes


STANFORD

JSH 29

Miniaturization for implantation

Implanted sensor

Implanted sensor

Freely-moving animal subject


STANFORD

JSH 30

Outline




STANFORD

JSH 31

Compact thrombin sensors integrated ‘in-line’ could greatly improve survival:real-time hemostasis management during surgeries, hemodialysis

Beckman Coulter

Medscape.org, Rockwell Medical

Existing techniques measure clotting time or assess viscoelasticity

only provide ‘snapshots’ bulky, expensive instrumentation trained personnel

Thrombin indicates activation of the coagulation cascade, and plays an active role in hemostasis

controlledhemostasis

thrombosishemorrhage

Regulation of coagulation is crucial:

Real-time, continuous blood monitoring


STANFORD

JSH 32

Optical fiber whole blood prothrombin assay


STANFORD

JSH 33

vascular injury (blood exposed to tissue factor)

TFVIIa

Prothrombin

IXa Xa

VIIIaVa

Thrombin

Fibrinogen Fibrin Clot

XIaXIa

Coagulation cascade: thrombin molecular probes


STANFORD

JSH 34


TFVIIa

Prothrombin

IXa Xa

VIIIaVa

Cys-Gly-D-Phe-Pip-Arg - Ser-Gly-Gly-Gly-G-LysThrombin

CY5.5 quencher


XIaXIa


TFVIIa

Prothrombin

IXa Xa

VIIIaVa

Thrombin


XIaXIa

First probe modeled after C.H. Tung et al., modified with quencher dyes Probe synthesized and characterized

for IRQC-1, IRQC-2, BHQ3, QSY21, QXL680



STANFORD

JSH 35


TFVIIa

Prothrombin

IXa Xa

VIIIaVa


CY5.5 quencher


XIaXIa


TFVIIa

Prothrombin

IXa Xa

VIIIaVa

Thrombin


XIaXIa

In the absence of thrombin, Cy5.5 fluorescence is quenched



STANFORD

JSH 36

peptide cleavage


TFVIIa

Prothrombin

IXa Xa

VIIIaVa


CY5.5 quencher


XIaXIa


TFVIIa

Prothrombin

IXa Xa

VIIIaVa

Thrombin


XIaXIa

In the presence of thrombin, Cy5.5 and quencher separate fluorescence emission increases

Ser-Gly-Gly-Gly-G-Lys

quencher

CY5.5

Cys-Gly-D-Phe-Pip-Arg


quencher

CY5.5




STANFORD

JSH 37

peptide cleavage


TFVIIa

Prothrombin

IXa Xa

VIIIaVa


CY5.5 quencher


XIaXIa


TFVIIa

Prothrombin

IXa Xa

VIIIaVa

Thrombin


XIaXIa

Confirmed up to up to ~4x increases so far in 15 min incubation time, not 24 hrs

Optimizations for speed, contrast underway


quencher

CY5.5



quencher

CY5.5




STANFORD

JSH 38

Outline




STANFORD

JSH 39

Need for an Alternate TPM Source

Microscope with MML laser

Replace current LARGE and COSTLY mode locked lasers

More mobility for animals Continuous real-time monitoring Highly parallel animal studiesSchnitzer group, Stanford


STANFORD

JSH 40

Two Photon Microscopy

Non-linear deep tissue imaging Neuroscience and bio-imaging Fluorescence is excited by absorbing

two photons simultaneously (~10-16 s) 890nm < λ < 940nm

Excitation (Intensity)2

~125pJ per pulse Sub-picosecond pulse length

Advantages of TPM Localized excitation Better noise immunity Image 100s um deep 3 dimensional maps

a) One Photon b) Two-PhotonNonlinear magic: multiphoton microscopy in the biosciences,

Nature Biotechnology 2003,Warren R Zipfel, Rebecca M Williams & Watt W Webb

∆

E2

E1

E1

∆


STANFORD

JSH 41

Mark Schnitzer Lab Stanford

Two photon neural imaging


STANFORD

JSH 42

Mode-Locking Repetition Rate

• Pulse Energy = PAVG/Repetition Rate– Lower R More energy per pulse for a given power– 1GHz, 125pJ/pulse 125mW Average power

• 1GHz repetition rate requires a 42mm GaAs cavity -- impractical– Use external cavity– Increase ng to shrink cavity

f

fT 1

nL

cfR

2

Adapted from R. I. Aldaz thesis, Stanford University


STANFORD

JSH 43

Mode-Locked Repetition Rate (2)

• Longer effective cavity lower rep rate and more energy/pulse

Adapted from R. I. Aldaz thesis, Stanford University

nL

cfR

2


STANFORD

JSH 44

Monolithic Mode Locked Lasers (MMLL)

Design Challenges Passive Mode Locking for an integrated device Small and light MMLL R=2xL/c, 100Mhz requires 1.5m air or 40cm GaAs cavity 100 pJ/pulse or Average Power ~10 mW Pulse widths 1~50 ps in integrated MLL

Mode-locked quantum-dot lasers,E. U. Rafailov, M. A. Cataluna & W. Sibbett,Nature Photonics 1, 395 - 401 (2007)

QW

http://www.nature.com/nphoton/journal/v1/n7/full/nphoton.2007.120.html


STANFORD

JSH 45

Optically Pumped PCW MLL Design

• Two section photonic crystal slow light waveguide cavity– Optically pumped gain section– Electrically reverse bias saturable absorber (SA) – Sweeps out photo-generated carriers quickly– p-contact made to selectively etch p+ GaAs cap layer; n-

backside contact – Photonic crystal mirrors, one partially transmitting, form cavity.

Pump Laser

-VA

SLOW Waveguide

95% Photonic Crystal Mirror

99.99% Photonic Crystal Mirror

n-doped

p-doped

adapted from E. U. Rafailov et al, Nature Photonics, Vol. 1 (2007).


STANFORD

JSH 46

Slow Light in Photonic Crystal Waveguides

• Two Regimes:• Steep angle of incidence

– Near Γ point– E.g. DBR mirrors– Not confined in a slab

• Nearly parallel to axis– Near band edge– TIR top & bottom mirrors• Must operate below light line

for propagating modes

– Large ng (10s-100s)

– Very sensitive to fabrication– Very large dispersion

• Up to 109 ps2/kmT. Krauss, J. Phys. D: Appl. Phys. 40 (2007)


STANFORD

JSH 47

Saturable Absorber Biasing

• Lateral Biasing– Complex fabrication involving selective area ion implantation– InP regrown buried heterojunctions CW room temp lasing @

1.55um

• Vertical Biasing– Simple but inefficient, only need to sweep out carriers not inject

• Method of choice for prototype device

S. Matsuo et al., Optics Express, Jan (2011).B. Ellis et al., Nature Photonics, Apr (2011).


STANFORD

JSH 48

Light Output

QW Laser in Slow Light PC

• Combine control of electronic and photonic states– Monolithic Passively Mode Locked Edge Emitting Photonic

Crystal Waveguide Laser (MPMLEEPCWL)

AlGaAs Cladding Layer

AlGaAs Cladding Layer

GaAs Substrate

Gain RegionSaturable Absorber

GaAs SQW/MQW Layer

PC Slow Light Waveguide( in active layer)

Laser Gain Electrode

Saturable Absorber Electrode

PC Air Columns

Side View of Wafer Top View of Device


STANFORD

JSH 49

First Indication of Mode-Locking

Gain SA

p-AlGaAs Cladding

n-AlGaAs Cladding

n-GaAs Substrate

p+ GaAs Cap

1x AlInGaAs QDs

Gain Section

Saturable Absorber

Output, 920nm

22.3 GHz!!!

K. Leedle, Late News CLEO San Jose 2013


STANFORD

JSH 50

RF Spectrum and Autocorrelation

• Assume sech2 pulse shape ~4ps pulses• 18.1 GHz rep rate

Laser RF Spectrum

4.0ps

560mA, -2.5V SA

Autocorrelation

560mA, -2.5V SA

K. Leedle, Late News CLEO San Jose 2013


STANFORD

JSH 51

• Definite threshold hysteresis under most conditions• Nearly 10x jump at threshold typical, easy to tell by SA current

200 300 400 500 6000

2

4

6

8

10

12

14

16

18

Current [mA]

Pow

er [

mW

]

LI Curves

Saturable AbsorberFloating0.0V-1.0V-2.0V-2.5V-3.0V-3.5V-4.0V


STANFORD

JSH 52

Integrated sensor arrays are a foundation for bio-medical diagnostics, continuous tumor monitoring, drug development and minimally invasive in-vivo imaging

Integrated bio-sensor arrays can be easily fabricated to realize high throughput, compact, cheap bio-sensor arrays

Photonic crystal-laser integration offers unique opportunity to control both electronic and photonic states to produce new systems for high sensitivity sensing

Development of a new class of engineered, near IR fluorescent proteins is an absolute game changer for in-vivo molecular imaging

Prediction: Nanotechnology will have its greatest impact in biology and medicine

Summary


STANFORD

JSH 53

Acknowledgements

COLLABORATORS

Students Postdocs Faculty ColleaguesEvan Thrush Ofer Levi Mark SchnitzerThomas O’Sullivan Pascale El-Kallassi Sam GambhirMeredith Lee Zac Walls Jim ZehnderAltamash Janjua Ophir Vermesh Natesh ParashruamaKen Leedle Alfred ForchelElizabeth Munro

SUPPORTStanford BioX ProgramSanofiNSF

Thank You

integrated lasers for biophotonics

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