Interferometric Microscopy for Detection and Visualization of ...some history of optics •Optical...

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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