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Integrated Lasers for Biophotonics

Integrated Lasers for Biophotonics. James S. Harris Stanford University. Peking University Summer School Beijing, China July 19, 2013. Outline. Motivation and background Implantable sensor design and fabrication In vivo monitoring of a molecular probe in a tumor

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Integrated Lasers for Biophotonics

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  1. Integrated Lasers for Biophotonics James S. Harris Stanford University Peking University Summer School Beijing, China July 19, 2013

  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

  3. The NANO-BIO-TECH Revolution Integrated Circuit-1961 STM-1981 AFM-1986 Vo-Dinh, Nanobiotech1, 3 (2005)

  4. PET MRI Medical imaging From structural/anatomical… …to functional imaging PET Fluorescence MRI Bioluminescence

  5. Multimodality Imaging Strategies

  6. Light-tissue interactions Incident Light Tissue Chromophore in ground state Absorption Diffuse Reflectance Multiple elastic Scattering Chromophore in excited state Specular Reflectance Single Backscattering Photon at incident wavelength Fluorescent photon Fluorescence Fluorescence Raman-shifted photon Raman Scattering Raman Scattered Provides molecular functional information

  7. Advantages of near IR Window of operation: 650–900 nm Integrated semiconductor sensors Low intrinsic absorption & scattering mabs = 0.04 cm-1mscattering = 10 cm-1 No tissue autofluorescence Day vs night star viewing Shah. Et al, NeuroRx Review2, 215 (2005)

  8. Photonic systems integration • Integration of laser-induced fluorescence (LIF) detection LIF Detection System Example Integrated Sensor 1 mm – 100 m 1 meter • Discrete components • Integrated system • Cheap, portable and parallel • Expensive, bulky and non-portable

  9. VCSELs are key for low-cost, compact biosensors • Vertical Cavity Surface Emitting Lasers: • Miniature + can be integrated • Low-cost manufacturing/packaging • Arrayable 2 ft long, 30 lbs Bulky, expensive external light sources and delicate alignment not realistic for point-of-care/ bedside Images: Molec. Devices ,D. Armani et al, Nature 2003; B. Cunningham et al, Sens Act B 2002; Biacore Images: Logitech, M-Com

  10. AlN 6.0 ( . 2 1 m ) m 4.0 ( . 3 1 m ) m ZnS MgSe GaN VisibleSpectrum ZnSe 3.0 ( . 4 1 m ) m AlP -SiC CdS a ZnTe Bandgap energy (eV) AlAs GaP 2.0 CdSe ( . 6 2 m ) m CdTe AlSb AlSb GaAs InP Si 1.0 ( 1 . 2 4 m ) m GaSb GaSb InN BULK Ge SLE InSb InAs InAs (Ge) 3.0 3.2 3.4 5.4 5.6 5.8 6.0 6.4 Lattice Constant (Å) Materials challenges for biosensors .31 Fluorescent Proteins .41 Wavelength (µm) .62 Telecom & IT Revolution 1.2

  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

  12. Integrated sensing geometry Fluorescent Molecules Back-scattered excitation light Emission Emission Filter Excitation Absorption region (i-GaAs) Spontaneous emission p-DBR Laser Cavity n-DBR Top view GaAs Substrate 675nm VCSEL Detector Side view

  13. Implantable Fluorescence Sensor

  14. Gallium Arsenide (GaAs)-based Integrated Optical Sensor Detector Optical Filter (above 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

  15. 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* Tissue NIR absorbers H2O HHb BULK LIPID O2Hb What are we sensing? *X. Shu, et al, Science324, 804 (2009)

  16. Cyanine 5.5 (Cy5.5) dye – Many existing preclinical probes in use What are we sensing? Cy5.5 Absorption/Emission

  17. 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) Sensor components: Laser

  18. Sensor components: Detector • Detector characteristics • Area: 0.75mm2 • Internal quantum efficiency: >75% • Dark current: <5pA/mm2 (0.1V) T. O’Sullivan et al, Proc. SPIE 7173, (2009)

  19. Sensor specifications:emission filtering Emission Filter Spectral Response • Consists of thin-film interference filter and thick hybrid (absorption) filter element • 15-20% overlap with fluorescence emission

  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 Excitation blocking elements 5x10 array of sensors

  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

  22. How to monitor a cancerous tumor • 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 Cy5.5 dye ? Cancer Molecule of interest

  23. 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 Control Dye Sensor performance:In vivo sensitivity Live Nude(Nu/Nu) Anesthetized Mouse 50nM in vivo sensitivity T. O’Sullivan et al, Opt. Express 18, (2010)

  24. Cy5.5-RGD accumulates at tumor sites Cy5.5 Cy5.5 dye RGD Cancerous cell / angiogenesis Cy5.5-RGD binds to neovasculature associated with cancer • Cheng et al. Bioconjugate Chem., Vol. 16 No. 6 2005

  25. Application: Study binding kinetics of molecular probe in cancer tumors Not tumor-specific (RAD-Cy5.5) Tumor-specific probe (RGD-Cy5.5) • 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 T. O’Sullivan et al, in preparation (2010)

  26. Application: Study binding kinetics of molecular probe in cancer tumors Tissue NIR absorbers H2O HHb BULK LIPID O2Hb Tumor-specific probe (RGD-Cy5.5) Changes in anesthesia • 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) T. O’Sullivan et al, in preparation (2010)

  27. 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 Transitioning to theimplanted sensor

  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

  29. Miniaturization for implantation Implanted sensor Implanted sensor Freely-moving animal subject

  30. 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

  31. Real-time, continuous blood monitoring • Regulation of coagulation is crucial: controlled hemostasis hemorrhage thrombosis • 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 Beckman Coulter Medscape.org, Rockwell Medical Compact thrombin sensors integrated ‘in-line’ could greatly improve survival: real-time hemostasis management during surgeries, hemodialysis

  32. Optical fiber whole blood prothrombin assay

  33. Coagulation cascade: thrombin molecular probes

  34. Coagulation cascade: thrombin molecular probes • 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

  35. Coagulation cascade: thrombin molecular probes In the absence of thrombin, Cy5.5 fluorescence is quenched

  36. Coagulation cascade: thrombin molecular probes In the presence of thrombin, Cy5.5 and quencher separate  fluorescence emission increases peptide cleavage

  37. Coagulation cascade: thrombin molecular probes • Confirmed up to up to ~4x increases so far in 15 min incubation time, not 24 hrs • Optimizations for speed, contrast underway peptide cleavage

  38. 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

  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 studies Schnitzer group, Stanford

  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 E2 E1 ∆ ∆ E1 a) One Photon b) Two-Photon Nonlinear magic: multiphoton microscopy in the biosciences, Nature Biotechnology 2003, Warren R Zipfel, Rebecca M Williams & Watt W Webb

  41. Two photon neural imaging Mark Schnitzer Lab Stanford

  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 GaAscavity -- impractical • Use external cavity • Increase ngto shrink cavity Adapted from R. I. Aldaz thesis, Stanford University

  43. Mode-Locked Repetition Rate (2) • Longer effective cavity  lower rep rate and more energy/pulse Adapted from R. I. Aldaz thesis, Stanford University

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

  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. -VA p-doped SLOW Waveguide n-doped 95% Photonic Crystal Mirror 99.99% Photonic Crystal Mirror adapted from E. U. Rafailovet al, Nature Photonics, Vol. 1 (2007). Pump Laser

  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/km T. Krauss, J. Phys. D: Appl. Phys. 40 (2007)

  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 • Simplebut inefficient, only need to sweep out carriers not inject • Method of choice for prototype device B. Ellis et al., Nature Photonics, Apr (2011). S. Matsuo et al., Optics Express, Jan (2011).

  48. Saturable Absorber Gain Region AlGaAs Cladding Layer AlGaAs Cladding Layer GaAs Substrate QW Laser in Slow Light PC • Combine control of electronic and photonic states • Monolithic Passively Mode Locked Edge Emitting Photonic Crystal Waveguide Laser (MPMLEEPCWL) PC Slow Light Waveguide( in active layer) Laser Gain Electrode Light Output GaAs SQW/MQW Layer Saturable Absorber Electrode PC Air Columns Side View of Wafer Top View of Device

  49. First Indication of Mode-Locking p+ GaAs Cap p-AlGaAs Cladding Saturable Absorber n-AlGaAs Cladding n-GaAs Substrate Gain SA 22.3 GHz!!! 1x AlInGaAs QDs Gain Section Output, 920nm K. Leedle, Late News CLEO San Jose 2013

  50. RF Spectrum and Autocorrelation • Assume sech2pulse shape  ~4ps pulses • 18.1 GHz rep rate Laser RF Spectrum 560mA, -2.5V SA K. Leedle, Late News CLEO San Jose 2013 Autocorrelation 560mA, -2.5V SA 4.0ps

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