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Interferometric Microscopy for Detection and Visualization of Biological Nanoparticles
M. Selim Ünlü
Electrical Engineering,
Physics,
Biomedical Engineering
Graduate Medical Sciences
BUNano
Photonics Center
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OUTLINE • A bit of philosophy – some history of optics
• Optical Interference
• Interferometric Reflectance Imaging Sensor (IRIS) • Principles
• Requirements and technology
• Kinetic measurements of molecular binding
• Single bio-nanoparticle detection • Exosomes
• Viruses
• Bacteria
• Super-resolution imaging
• Single Molecule Detection
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MOTIVATION Enjoy curiosity driven research to make the world a better place for all. Training / transforming students.
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Conventional in vitro Dx (IVD) • Biomarkers indicate state of disease
• Point of care (POC) diagnostics would expedite clinical decision making
ELISA – Popular “wet lab” diagnostic tool
Pregnancy test
Glucose monitor
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Diagnostics/detection
• Uses secondary labeling for detection • Requires laboratory and skilled technicians • Time-consuming process
SPR (Surface Plasmon Resonance)
• Label-free optical detection • Large, expensive equipment • Requires laboratory environment
ELISA (Enzyme-linked immunosorbent assay)
PCR (polymerase chain reaction)
• Sample preparation, • Dark/enclosed chamber, • Known specific location of target
Single particle detection - High-Q Resonators - Digital PCR, Simoa • Small interaction volume • Fragile/complex devices, difficult light coupling
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Altruistic - MOTIVATION Global access to state-of-the-art diagnostics
March 2011, Nicaragua
Field trip with 14 BU-ENG undergraduate students.
Global health / limited resource settings
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Descartes (1596-1659)
Descartes reasoned that light must be like sound.
Is light a WAVE then ?
So he modeled light as pressure variations in a medium (aether or ether).
Optics in 17th-century Europe
Rene Descartes
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Kamāl al-Dīn al-Fārisī
كمالالدين فارسی
Farisi is known for giving the first mathematically satisfactory explanation of the rainbow, and an explication of the nature of colors that reformed the theory of Ibn al-Haytham Alhazen Farisi also "proposed a model where the ray of light from the sun was refracted twice by a water droplet, one or more reflections occurring between the two refractions." He verified this through extensive experimentation using a transparent sphere filled with water
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Willibrord Snell (1591-1626)
Willibrord Snell discovered the Law of Refraction, now named after him.
n1
n2
n
1sin(
1) n
2sin(
2)
ni is the refractive index of each medium.
Optics in 17th-century Europe
1
2
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Galileo (1564-1642) used Galilean telescope to look at our moon, Jupiter and its moons, and the sun.
Galileo’s drawings of the moon
Optics in 17th-century Europe
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"I procured me a triangular glass prism to try therewith the celebrated phenomena of colours." (Newton, 1665)
Isaac Newton (1642-1727)
Light was one of Newton’s many areas of research. After remaining ambivalent for many years, he eventually concluded that it was evidence for a particle theory of light.
Optics in 17-18th-century Europe
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Optical Interference: from basic to the ultimate
>10 orders of magnitude
0.1 atto-m displacement
~ 10 nm thickness change can be visually observed
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PhD years – semiconductor devices
15
In my comfort zone from undergrad Waves – EM PhD on Resonant Cavity Detectors and on to BU in 1992
HPT
RCE - HPT
EXP
750 800 850 900 950 10000
1
2
3
4
CO
LL
EC
TO
R C
UR
RE
NT
(arb
. uni
ts)
W A V E L E N G T H ( nm )
‘98 ‘01 ‘01 ‘03 ‘05
Prof. Kishino Sophia Univ
Let’s incorporate a
reflector at the bottom
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@ BU
EMERGING INFECTIOUS DISEASES
PHOTONICS
LIFE SCIENCE AND ENGINEERING
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OUTLINE • A bit of philosophy – some history of optics
• Optical Interference
• Interferometric Reflectance Imaging Sensor (IRIS) • Principles
• Requirements and technology
• Kinetic measurements of molecular binding
• Single bio-nanoparticle detection • Exosomes
• Viruses
• Bacteria
• Super-resolution imaging
• Single Molecule Detection
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High index absorbing substrate
Transparent Spacer
Biomass
Ref
lect
ance
, R
Wavelength, λ
Spectral shift (d) (a) (b) (c)
Interferometric Reflectance Imaging Sensor (IRIS)
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Sensor Chip Requirements
soap film Protein microarray chips with 100s to 1,000s of probe spots Oxide coated Si
Ünlü et al, ”STRUCTURED SUBSTRATES FOR OPTICAL SURFACE PROFILING,’ US Patent No: 9599611, 2017
pg/mm2 sensitivity 1,000s of spots
@ $0.1/cm2
• Multilayer reflector with no stray light • Flat and smooth surface • Chemical functionalization / glass • Manufacturable and scalable
High index absorbing substrate
Transparent Spacer
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Cartridge Requirements • Optical quality for imaging • Manufacturable and scalable • Easy assembly with chip • Cost • Multi-layer polycarbonate
laminates (7 layers) • Good prototype solution • Cost remains high for
medium volume
Scherr et al, ACS Nano 10 (2016) Scherr et al, Lab on a Chip (2017)
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Si-based Microfluidics solution • Fluidics through Si • Manufacturable / scalable – established infrastructure • Top window can be separately optimized
• AR coating • Polarization preserving
Yalcin-Ozkumur, JSTQE (2018)
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Mechanical Fixture
‘18
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Cam Operation
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Prototype Instrument
Off-the-shelf components Parts cost (BOM) under $10K
’20
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IRIS – a versatile biosensing technology platform for microarrays
Dynamic Measurements Single virus/exosome Quantitative / QC
• decade long R&D • various applications demonstrated.
• From QC to single exosome/virus detection • pg/mm2 sensitivity • Single biological nanoparticle detection and characterization • attoM sensitivity for protein and nucleic acid detection
microns
mic
rons
0 50 100 150
0
20
40
60
80
100
DN
A Im
mob
iliza
tion,
ng/
mm
2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
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OUTLINE • A bit of philosophy – some history of optics
• Optical Interference
• Interferometric Reflectance Imaging Sensor (IRIS) • Principles
• Requirements and technology
• Kinetic measurements of molecular binding
• Single bio-nanoparticle detection • Exosomes
• Viruses
• Bacteria
• Super-resolution imaging
• Single Molecule Detection
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Dynamic – Quantitative Microarrays
Ozkumur et al, “Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications,” PNAS, 2008
’09
’13
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1. Molecular binding Kinetic Measurements
• Multiplexed (100s to 1000s of probes) • Quantitative • Glass surface • Inexpensive instrumentation and
disposables • ng/ml target sensitivity • ~kDa molecular weight
’21
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OUTLINE • A bit of philosophy – some history of optics
• Optical Interference
• Interferometric Reflectance Imaging Sensor (IRIS) • Principles
• Requirements and technology
• Kinetic measurements of molecular binding
• Single bio-nanoparticle detection • Exosomes
• Viruses
• Bacteria
• Super-resolution imaging
• Single Molecule Detection
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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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Single Particle – IRIS : Digital Detection
• Label Free direct sensing of individual viruses • Digital Detection: Single molecule level detection of Nucleic Acids
and Proteins • ULTIMATE BIODETECTION PLATFORM?
Single Virus Detection (label –free)
Single Molecule Detection of Antigen proteins and DNA/RNA
SiO2
Si IRIS detection platform
nano-barcode
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Extra cellular vesicles, exosomes, and viruses
Example cryo-EM images of infectious extracellular vesicle (Bullitt Lab – BU MED)
SEM image of Ebola virion
© ALEXIS ROSENFELD/SCIENCE SOURCE
Viruses are the most abundant species on earth. >1031 phages in the biosphere ~107 viruses on average in a mL of seawater
Compare to ~1023 stars in the known universe
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Example: Engineered viruses for cancer therapy
© CREATIVE BIOLABS
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Optical microscopy can see small – but …
micro.magnet.fsu.edu/primer/
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Nanoparticle Detection and Sizing Why difficult and how we make it easy
Einc Eref
Esca
sin2I22
det scarefscaref EEEE
mp
mpR
24 3
0
Size Material
SiO2
Si
Phase Term
Ziegler
Resonators provide very high sensitivity Photonic Crystals Toroids/Disks/Spheres
High Q / Small interaction volume Evanescent optical field
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Interferometric measurements of single molecules
Pikiarik and Sandogdar, Nature Communications 5 (2014)
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Teaching Introduction to Electronics – recruiting PhDs
Rahul Vedula(MD) and George Daaboul, PhD ‘13
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Single Particle Detection Fluorescent 100nm Carboxyl modified beads immobilized on Lysine surface. Incubation time 15min
Simple
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SP - IRIS – a simple, compact Nano particle sensor
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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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Before Incubation
After Incubation
50 100 150 200 2500
20
40
60
80
Diameter(nm)F
req
ue
ncy
50 100 150 2000
20
40
60
80
Diameter(nm)
Fre
qu
en
cy
Noise particles
Virus particles
Hemorrhagic Fever Detection (VSV-pseudotypes)
Prof. John Connor
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Various viruses
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In-liquid detection to simplify the assay
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Virus detection for diagnostic applications
Scherr et al, ACS nano 10 (2016) and Scherr et al, Lab on a Chip 17 (2017)
Highly-sensitive virus detection directly from blood serum
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Real-Time in-liquid Virus Detection
‘15
‘17
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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
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E-coli Detection
’21
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5X objective imaging and processing
LOD of ~50CFU/ml. with 1000X concentration -> 1 CFU/20 ml
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Detection and Verification
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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
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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‘18
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‘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% → 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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OUTLINE • A bit of philosophy – some history of optics
• Optical Interference
• Interferometric Reflectance Imaging Sensor (IRIS) • Principles
• Requirements and technology
• Kinetic measurements of molecular binding
• Single bio-nanoparticle detection • Exosomes
• Viruses
• Bacteria
• Super-resolution imaging
• Single Molecule Detection
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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
on Brownian motion • Little/no molecular information
Needed: High-throughput methods to measure the size, shape and molecular profile of biological nanoparticles
Fluorescence microscopy (STED/PALM)
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NATURE PHOTONICS | VOL 8 | MAY 2014 |
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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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• 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
FWHM ~ 130nm < (𝝺 / 3)
’20
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Elongated polystyrene rods
Reconstructed image
Full NA
Samir Mitragotri (Harvard)
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OUTLINE • A bit of philosophy – some history of optics
• Optical Interference
• Interferometric Reflectance Imaging Sensor (IRIS) • Principles
• Requirements and technology
• Kinetic measurements of molecular binding
• Single bio-nanoparticle detection • Exosomes
• Viruses
• Bacteria
• Super-resolution imaging
• Single Molecule Detection – Digital Microarrays
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• Digital means counting
• 10,000-fold more sensitive than commercial microarrays, while maintaining all of the advantages:
1. Highly multiplexed
2. Low cost
3. Fast
• Application: molecular diagnostics
Single Virus Detection
(label –free)
Single Molecule Detection of Antigen proteins and DNA/RNA
SiO2
Si IRIS detection platform
nano-barcode
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Analog vs. Digital
From David Walt @ Tufts Quanterix
Wikipedia and twitter
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Fluorescence DNA microarrays
1. DNA chip: 100 – 40,000 of unique probes
2. Incubate with sample – RNA targets hybridize
3. Stain with a fluorescent reporter
4. Measure the fluorescence of each spot
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Performance limits of fluorescence
• Sensitivity limit: 10 fluorophores/um2
• Dynamic range: 100-1000
… but microarray spots are 10,000 um2
~100 µm diameter
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Digital microarray concept
Hesse et al, Genome Research 2006
Target Control
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Light scattering
Light scattering is advantageous:
• No saturated emission rate and no photobleaching: only limit
to speed is your input light power
• We can use a very simple instrument
• We can do dynamic measurements as well
Replace fluorescent reporters with nanoparticle conjugates
Challenge – seeing/detection small nanoparticles
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40 50 60 70 800
50
100
150
Diameter (nm)
Fre
quen
cy
40 60 800
50
100
150
Diameter (nm)
Fre
quen
cy
Protein chips by single nanoparticle counting
Size-discrimination permits sensitive, specific detection in complex biological samples
BSA Negative control
Anti- β-lactoglobulin
SiO2 Si
β-lactoglobulin
Detection complex
a)
Before detection complex
After detection complex
b) Detection of 5pM β-lactoglobulin
0 20 40 60 800
200
400
Diameter (nm)
Frequency
No filtering − Mean = 59nm, SD = 18nm, Counts = 782
0 20 40 60 800
200
400
Diameter (nm)
Frequency
Anomoly filtering − Mean = 56nm, SD = 17nm, Counts = 685
0 20 40 60 800
200
400
Diameter (nm)
Frequency
PSF filtering − Mean = 58nm, SD = 11nm, Counts = 428
0 20 40 60 800
200
400
Diameter (nm)
Frequency
Size−discrimination − Mean = 53nm, SD = 4nm, Counts = 332
i) ii)
iii) iv)
c)
60µm 60µm BSA Negative control
Anti- β-lactoglobulin
SiO2 Si
β-lactoglobulin
Detection complex
a)
Before detection complex
After detection complex
b) Detection of 5pM β-lactoglobulin
0 20 40 60 800
200
400
Diameter (nm)
Frequency
No filtering − Mean = 59nm, SD = 18nm, Counts = 782
0 20 40 60 800
200
400
Diameter (nm)
Frequency
Anomoly filtering − Mean = 56nm, SD = 17nm, Counts = 685
0 20 40 60 800
200
400
Diameter (nm)
Frequency
PSF filtering − Mean = 58nm, SD = 11nm, Counts = 428
0 20 40 60 800
200
400
Diameter (nm)
Frequency
Size−discrimination − Mean = 53nm, SD = 4nm, Counts = 332
i) ii)
iii) iv)
c)
60µm 60µm
50 70
1 hour incubation
BSA Negative control
Anti- β-lactoglobulin
SiO2 Si
β-lactoglobulin
Detection complex
a)
Before detection complex
After detection complex
b) Detection of 5pM β-lactoglobulin
0 20 40 60 800
200
400
Diameter (nm)
Frequency
No filtering − Mean = 59nm, SD = 18nm, Counts = 782
0 20 40 60 800
200
400
Diameter (nm)Frequency
Anomoly filtering − Mean = 56nm, SD = 17nm, Counts = 685
0 20 40 60 800
200
400
Diameter (nm)
Frequency
PSF filtering − Mean = 58nm, SD = 11nm, Counts = 428
0 20 40 60 800
200
400
Diameter (nm)
Frequency
Size−discrimination − Mean = 53nm, SD = 4nm, Counts = 332
i) ii)
iii) iv)
c)
60µm 60µm
’13
LODblood < 1fM
LODserum< 100aM
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Goal: Larger field of view to scan a larger area lower NA – lower contrast Answer: Nanorods as labels - polarization
a b
a b
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Circular polarization
50x, 0.8 NA
20x, 0.45 NA
10x, 0.3NA
‘17
‘19
Polarization enhancement, real-time DNA detection
miRNA detection
mRNA detection AST
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A digital microarray with IRIS 10x Objective … 50x … 100x
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A Typical Assay
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Application Areas
Detecting cancer biomarkers at ultra-low levels and rare mutations - potential to enable new options for diagnostics and treatment in cancer research. KRAS mutation detection (colorectal cancer). Work in collaboration with CNR, Milan.
Detecting minute changes in cardiac biomarkers – at-risk patients identified earlier in their disease progression to guide more personalized care. miR-451 is a cardiac biomarker. Work in collaboration with Umass Medical.
Detecting infectious disease biomarkers before the onset of an immune response. HBsAg detected at 1,000X better sensitivity than commercial assays. Funded by Aselsan.
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LEDs replace tunable laser
(2010)
36”
Bench-top IRIS (2007) 18”
Prototype system (2008)
9”
System Maturation and Prototyping
‘08 ‘14
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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: • Lateral resolution of ~100nm without
labeling
• Sub-fM multiplexed detection of RNA, DNA and proteins