Interferometric Mic In icroscopy for Detection and Vis - - PowerPoint PPT Presentation

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Interferometric Mic In icroscopy for Detection and Vis - - PowerPoint PPT Presentation

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Interferometric Mic In icroscopy for Detection and Vis isualization of f Bio iological Nanoparticles M. Selim nl Electrical Engineering, Physics, Biomedical Engineering Graduate Medical Sciences BUNano Photonics Center CLEO 2018

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CLEO 2018 selim@bu.edu www.bu.edu/OCN 1/32

In Interferometric Mic icroscopy for Detection and Vis isualization

  • f

f Bio iological Nanoparticles

  • M. Selim Ünlü

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

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CLEO 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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CLEO 2018 selim@bu.edu www.bu.edu/OCN 3/32

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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Technologies for bio-nanoparticle characterization

Cryo-TEM

  • Fantastic resolution
  • Low throughput and difficult

Nanoparticle Tracking Analysis

  • Estimate size of particles based
  • n Brownian motion
  • Little/no molecular information

Needed: High-throughput methods to measure the size, shape and molecular profile

  • f biological nanoparticles

Fluorescence microscopy (STED/PALM)

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CLEO 2018 selim@bu.edu www.bu.edu/OCN 7/32

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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CLEO 2018 selim@bu.edu www.bu.edu/OCN 8/32

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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CLEO 2018 selim@bu.edu www.bu.edu/OCN 9/32 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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Virus characterization: size determination

SEM IRIS

Daaboul et al, ACS nano 8 (2014)

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Various viruses

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In-liquid detection to simplify the assay

13

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Virus detection for diagnostic applications

Scherr et al, ACS Nano 10 (2016) and Scherr et al, Lab on a Chip (2017) (2017)

Highly-sensitive virus detection directly from blood serum

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CLEO 2018 selim@bu.edu www.bu.edu/OCN 15/32

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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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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CLEO 2018 selim@bu.edu www.bu.edu/OCN 18/32

‘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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CLEO 2018 selim@bu.edu www.bu.edu/OCN 20/32

‘17

Overall of 10X enhancement

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Collection Path – Apodization and Reference Attenuation

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Silica particles defocus curve ~5X enhancement (3 (3% → 15%) )

a)฀full-NA฀illumination฀at฀λ=530฀nm,฀b)฀apodized฀illumination฀at฀ λ=530฀nm,฀c)฀apodized฀illumination฀at฀λ=460฀nm,฀d)฀apodized฀ illumination฀with฀amplitude฀filter฀in฀the฀collection฀path฀at฀λ=460฀nm.

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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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฀ ฀ ฀NATURE฀PHOTONICS฀|฀VOL฀8฀|฀MAY฀2014฀|฀฀

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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

FWHM ~ 130nm < (𝝻 / 3)

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Elongated polystyrene rods

Reconstructed image Full NA

Samir Mitragotri (Harvard)

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Commercialization

Nanoview

ZOIRAY

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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