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Reconstruction in wide-field interferometric microscopy for imaging weakly scattering biological nanoparticles with 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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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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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 of 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
mp
mpR
24 3
0
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 State of the Art 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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Interferometric Reflectance Imaging Sensor (IRIS) a high throughput biosensing platform
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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Eref
Esca
sin2I22
det scarefscaref EEEE
mp
mpR
24 3
0
Size Material
SiO2
Si
Phase Term
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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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Reconstruction in Interference Microscopy
?
observation
imaging system
in out
object
observation noise object 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% → 8%)
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Reconstruction – first with defocus
• Defocus based reconstruction
SEM
1 micron
300 nm
50
0 n
m
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 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
oxide
Sample – E-beam fabricated
80 nm
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150 nm separation, 0.9 NA, 𝝺=420nm
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FWHM ~ 130nm < (𝝺 / 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