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Reconstruction in in wid ide-field in interferometric mic icroscopy for im imaging weakly scattering bio iological nanoparticles wit ith super-resolution M. Selim nl Electrical Engineering, Physics, Biomedical Engineering Graduate

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SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 1/32

Reconstruction in in wid ide-field in interferometric mic icroscopy for im imaging weakly scattering bio iological nanoparticles wit ith super-resolution

  • M. Selim Ünlü

Electrical Engineering, Physics, Biomedical Engineering Graduate Medical Sciences BUNano Photonics Center

  • O. Avci, C. Yurdakul, D. Sevenler,
  • F. Ekiz-Kanik, Lei Tian
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SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 2/32

Outline

  • Motivation – Biological Nanoparticles everywhere
  • Problem definition – contrast and size
  • Detection vs. visualization
  • Interferometric Reflectance Imaging Sensor
  • Biological Nanoparticle Detection and Sizing
  • Pupil function engineering
  • Resolution improvement by oblique illumination and reconstruction
  • Towards 100nm in label-free visible light microscopy
  • Conclusions and Future
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SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 3/32

Motivation - Nanoparticles

(Adapted from wichlab.com/research) Polymer-based Semiconductor-based Metallic-based

Artificial nanoparticles Natural nanoparticles EV and Exosomes

Artificial nanoparticles

  • Optically & physically engineered
  • Used as labels or vehicles in

diagnostics, therapeutic applications

  • Gold, polystyrene NPs, QDs

Natural nanoparticles

  • Low-index, complex-shaped
  • Hard to detect without labels
  • Virus – infectious diseases and cancer
  • Exosome – secreted from cancer cells

ADVANCED WIDE-FIELD INTERFEROMETRIC MICROSCOPY FOR NANOPARTICLE SENSING AND CHARACTERIZATION

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Extra cellular vesicles, exosomes, and viruses

Example cryo-EM images

  • f

infectious extracellular vesicle (Bullitt Lab – BU MED)

  • Viruses are the most abundant species on earth.

~1032 phages in the biosphere ~107 viruses on average in a mL of seawater SEM image of Ebola virion

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Optical microscopy can see small – but …

micro.magnet.fsu.edu/primer/

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Biological Nanoparticle Detection Challenges – size and dielectric contrast

Einc Esca

m p m p

R       2 4

3

  

Size contrast

Ziegler

Signal ~ R6

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Single nanoparticle detection / visualization

  • High-resolution imaging systems provide visualization of

nanoparticles – detailed structural information

  • Low-throughput, expensive and laborious
  • Digital detection systems provide sensing of nanoparticles without

visualization – limited or no structural information

  • High-throughput, often inexpensive and straightforward

Conventional Microscope Often undetected Digital detection Detected but not resolved Sample

Biological particle

High-resolution Detected & resolved

ADVANCED WIDE-FIELD INTERFEROMETRIC MICROSCOPY FOR NANOPARTICLE SENSING AND CHARACTERIZATION

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Current St State of f th the Art rt Technology

Zeiss Libra 200 clf.stfc.ac.uk

Electron microscopy Fluorescence microscopy (STED/PALM)

  • Laborious
  • Sample prep
  • Expensive
  • Not label-free
  • Low-throughput
  • Great resolution
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Outline – IRIS

  • Motivation – Biological Nanoparticles everywhere
  • Problem definition – contrast and size
  • Detection vs. visualization
  • Interferometric Reflectance Imaging Sensor
  • Biological Nanoparticle Detection and Sizing
  • Pupil function engineering
  • Resolution improvement by oblique illumination and reconstruction
  • Conclusions and Future
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SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 10/32

Interferometric

ic Refl flectance Imagin ing Sensor (IR (IRIS IS) a hig igh throughput bio iosensin ing pla latform

soap film Oxide coated Si Ünlü et al, ”STRUCTURED SUBSTRATES FOR OPTICAL SURFACE PROFILING,’ US Patent No: 9599611, 2017

pg/mm2 sensitivity 1,000s of spots

Protein microarray chips with 100s to 1,000s of probe spots @ $0.1/cm2

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SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 11/32

Eref Esca

 sin 2 I

2 2 det sca ref sca ref

E E E E   

m p m p

R       2 4

3

  

Size Material SiO2 Si Phase Term

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SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 12/32 Rahul Vedula(MD) and George Daaboul, PhD ‘13

Single Particle Detection Simple

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Exosome detection

Anti-CD81 capture probe image acquired before and after incubation with purified HEK293 cells derived exosomes.

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Verification by SEM and AFM – down to r=30nm dry

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Various viruses

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In-liquid detection to simplify the assay

16

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Passive Cartridge - Simple Workflow

1. Remove cartridge from package just prior to use 2. 100 uL of sample is pipetted into the bottom of the reservoir (*care needs to be taken not to introduce bubbles) 3. Luer cap (sealed with adhesive strip) is screwed down finger tight 4. When liquid reaches the pad, the luer cap is vented (adhesive strip removed) 5. Cartridge is placed in the instrument to begin acquiring data

‘15 ‘17

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Outline – Going Beyond Detection and Sizing

  • Motivation – Biological Nanoparticles everywhere
  • Problem definition – contrast and size
  • Detection vs. visualization
  • Interferometric Reflectance Imaging Sensor
  • Biological Nanoparticle Detection and Sizing
  • Pupil function engineering
  • Resolution improvement by oblique illumination and reconstruction
  • Towards 100nm in label-free visible light microscopy
  • Conclusions and Future
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SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 20/32

Reconstruction in In Interference Microscopy

?

  • bservation

imaging system in

  • ut
  • bject
  • bservation

noise

  • bject

system response convolution matrix

(J. Trueb*, O. Avci* et al., IEEE JSTQE, 2016)

ADVANCED WIDE-FIELD INTERFEROMETRIC MICROSCOPY FOR NANOPARTICLE SENSING AND CHARACTERIZATION

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Interferometric fringes – defocus profile

Changing the focus position changes the path length to the detector differently for reference reflection and scattered light

‘17 ‘17

  • D. Sevenler et al, "Quantitative interferometric reflectance imaging for the detection and

measurement of biological nanoparticles," Biomedical Optics Express, 2017

  • O. Avci, et al., "Physical Modeling of Interference Enhanced Imaging and Characterization of

Single Nanoparticles," Optics Express, 2016

  • O. Avci, et al. "Pupil function engineering for enhanced nanoparticle visibility in wide-field

interferometric microscopy," Optica2017

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‘17

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‘17

Overall of 10X enhancement

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Collection Path – Apodization and Reference Attenuation

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Registered silica particles defocus curve ~4X enhancement (2 (2.2 .2% → 8%)

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Reconstruction – fi first with defocus

  • Defocus based reconstruction

SEM

1 micron

300 nm 500 nm Top 80 nm

Si SiO2

50 nm Side (conventional) (reconstruction)

50x/0.8NA 525nm

  • Tikhonov regularization: Least-squares cost function with quadratic side-constraint
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NATURE PHOTONICS | VOL 8 | MAY 2014 |

Reconstruction – Structured Il Illumination (? (?)

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10/2/2018

  • Enhancing low-index nanoparticle resolution via reconstruction schemes

Asymmetric illumination based reconstruction for super resolution (with Lei Tian)

ADVANCED OPTICAL SCHEMES IN WIDE-FIELD INTERFEROMETRIC MICROSCOPY FOR ENHANCED NANOPARTICLE SENSING AND CHARACTERIZATION

Right Bottom Left Top

Fourier transforms of the transfer functions (H) for each asymmetric illumination configuration

Super-resolution in wide-field interferometric microscopy

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SEM raw reconstruction 50x/0.8NA 525nm

Experimental Results

300 nm Sketch 10/2/2018 SEM

100x/0.9NA 525nm 50x/0.8NA 525nm

Si

  • xide

Sample – E-beam fabricated

80 nm

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150 nm separation, 0.9 NA, 𝝻=420nm

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FWHM ~ 130nm < (𝝻 / 3)

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  • CONCLUSIONS & FUTURE
  • Optical interference is a very powerful

sensing technique.

  • Multi-disciplinary innovation
  • Single biological nanoparticle detection /

counting / size and shape discrimination / visualization

  • Goals: Down to r=20nm Biological

nanoparticle detection in liquid

  • Lateral resolution of ~100nm without

labeling