for im imaging weakly scattering bio iological nanoparticles wit - PowerPoint PPT Presentation

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

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 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 2/32

A DVANCED W IDE - FIELD I NTERFEROMETRIC M ICROSCOPY F OR N ANOPARTICLE S ENSING A ND C HARACTERIZATION Motivation - Nanoparticles EV and Exosomes Natural nanoparticles Artificial nanoparticles (Adapted from wichlab.com/research) Semiconductor-based Metallic-based Polymer-based Artificial nanoparticles Natural nanoparticles • • Optically & physically engineered Low-index, complex-shaped • • Used as labels or vehicles in Hard to detect without labels diagnostics, therapeutic applications • Virus – infectious diseases and cancer • Gold, polystyrene NPs, QDs • Exosome – secreted from cancer cells SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 3/32

Extra cellular vesicles, exosomes, and viruses � SEM image of Ebola virion Example cryo-EM images of Viruses are the most abundant species on earth. infectious extracellular vesicle ~10 32 phages in the biosphere (Bullitt Lab – BU MED) ~10 7 viruses on average in a mL of seawater SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 4/32

Optical microscopy can see small – but … micro.magnet.fsu.edu/primer/ SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 5/32

Biological Nanoparticle Detection Challenges – size and dielectric contrast E inc E sca Ziegler       p m 3 4 R Signal ~ R 6    0 2 p m Size contrast SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 6/32

A DVANCED W IDE - FIELD I NTERFEROMETRIC M ICROSCOPY F OR N ANOPARTICLE S ENSING A ND C HARACTERIZATION 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 Sample High-resolution Conventional Microscope Digital detection Biological particle Often undetected Detected but not resolved Detected & resolved SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 7/32

Current St State of f th the Art rt Technology Electron microscopy Fluorescence microscopy (STED/PALM) • Great resolution • Laborious Zeiss Libra 200 • Sample prep • Expensive • Not label-free • Low-throughput clf.stfc.ac.uk SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 8/32

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 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 9/32

I nterferometric ic R efl flectance I magin ing S ensor (IR (IRIS IS) a hig igh throughput bio iosensin ing pla latform soap film Oxide coated Si Protein microarray chips with 100s to 1,000s of probe spots @ $0.1/cm 2 Ünlü et al, ”STRUCTURED SUBSTRATES FOR OPTICAL SURFACE PROFILING,’ US Patent No: 9599611, 2017 pg/mm 2 sensitivity 1,000s of spots SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 10/32

2   2   E ref I E E 2 E E sin det ref sca ref sca E sca       Phase Term p m 3 4 R    0 2 p m SiO 2 Si Size Material SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 11/32

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

Exosome detection Anti-CD81 capture probe image acquired before and after incubation with purified HEK293 cells derived exosomes. SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 13/32

Verification by SEM and AFM – down to r=30nm dry SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 14/32

Various viruses SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 15/32

In-liquid detection to simplify the assay 16 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 16/32

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 ‘17 ‘15 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 17/32

SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 18/32

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 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 19/32

A DVANCED W IDE - FIELD I NTERFEROMETRIC M ICROSCOPY F OR N ANOPARTICLE S ENSING A ND C HARACTERIZATION Reconstruction in In Interference Microscopy imaging system object observation in out (J. Trueb*, O. Avci* et al., IEEE JSTQE, 2016) ? observation noise system response object convolution matrix SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 20/32

Interferometric fringes – defocus profile Changing the focus position changes the path length to the detector differently for reference reflection and ‘17 scattered light ‘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," Optica 2017 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 21/32

‘17 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 22/32

‘17 Overall of 10X enhancement SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 23/32

Collection Path – Apodization and Reference Attenuation SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 24/32

Registered silica particles defocus curve ~4X enhancement (2 (2.2 .2% → 8%) SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 25/32

Reconstruction – fi first with defocus • Tikhonov regularization: Least-squares cost function with quadratic side-constraint • Defocus based reconstruction Top SEM (conventional) (reconstruction) 300 nm 500 nm 1 micron Side 80 nm 50 nm SiO 2 50x/0.8NA 525nm Si SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 26/32

Reconstruction – Structured Il Illumination (? (?) NATURE PHOTONICS | VOL 8 | MAY 2014 | SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 27/32

SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 28/32

A DVANCED O PTICAL S CHEMES IN W IDE - FIELD I NTERFEROMETRIC M ICROSCOPY F OR E NHANCED N ANOPARTICLE S ENSING A ND C HARACTERIZATION Super-resolution in wide-field interferometric microscopy • Enhancing low-index nanoparticle resolution via reconstruction schemes Asymmetric illumination based reconstruction for super resolution (with Lei Tian) Fourier transforms of the transfer functions (H) for each asymmetric illumination configuration Left Right Bottom Top 10/2/2018 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 29/32

Experimental Results reconstruction raw Sample – E-beam fabricated 50x/0.8NA 525nm SEM Sketch 50x/0.8NA 525nm 300 nm 100x/0.9NA 525nm SEM 80 nm oxide Si 10/2/2018 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 30/32

150 nm separation, 0.9 NA, 𝝻 = 420nm SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 31/32

FWHM ~ 130nm < ( 𝝻 / 3) SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 32/32

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 SPIE PW 2018 selim@bu.edu www.bu.edu/OCN 33/32