QLI Lab
UIUC
Quantitative phase imagining (QPI):
metrology meets biology
Gabriel Popescu
Department of Electrical and Computer Engineering
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
Quantitative Light Imaging Laboratory
http://light.ece.uiuc.edu
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QLI Lab
UIUC
Contributors
QLI Lab
Collaborators
•Basanta Bhaduri, Postdoc
•Raj Gannavarpu, Postdoc
UIUC:
•Rashid Bashir, ECE
•Catherine Best-Popescu, BioE
•Steve Boppart, ECE
•Scott Carney, ECE
•Martha Gillette, MCB
•Xiuling Li, ECE
•Eric Pop, ECE
•Supriya Prasanth, MCB
•John Rogers, MSE
•Krishna Tangella, Christie Clinic
•Zhuo Wang,
PhD (alumnus)
•Mustafa Mir,
PhD (alumnus)
•Taewoo Kim,
PhD student
•Shamira Sridharan, PhD student
•Tan Nguyen,
PhD student
•Mikhail Kandel,
MS student
•Hassaan Majeed, PhD student
•Ruoyu Zhu,
•Ryan Tapping,
•Joonoh Lim,
•Joe Leigh,
2
undergrad
undergrad
undergrad
undergrad
•UCLA:
•UIC:
•UGA:
Alex Levine
Andre Balla
Steve Stice
QLI Lab
UIUC
Outline
1. Background and motivation
2. Quantitative phase Imaging (QPI): SLIM
a. 2D: neural network formation
b. 3D: cell tomography
3. Label-free cancer diagnosis
4. Summary
QLI Lab
UIUC
Outline
1. Background and motivation
2. Quantitative phase Imaging (QPI): SLIM
a. 2D: neural network formation
b. 3D: cell tomography
3. Label-free cancer diagnosis
4. Summary
QLI Lab
Antonie van Leeuwenhoek
“Father of cell biology”
Motility of bacteria- 1683
Red blood cells- 1682
UIUC
QLI Lab
Two challenges
2. Contrast
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•17th century
UIUC
QLI Lab
Cells are transparent
UIUC
•Phase objects
Phase contrast
•20th century
F. Zernike (1935)
-1953 Nobel Prize
QLI Lab
Phase changes
•PC and DIC use phase to create intensity contrast
PC and DIC qualitative phase methods
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UIUC
QLI Lab
GOALS
•Develop a new type of microscopy
for quantitative biological studies
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Quantitative phase imaging (QPI)
Bright field Image
Phase contrast image
QPI image
200
0
Phase shift [nm]
-Quantify phase shift
-Nanometer sensitivity
-Stable
-Noninvasive
-No sample preparation
UIUC
QLI Lab
From phase to biology

n
n0
  2  n  n0  h / 
 1 mrad  h  1 nm!
 1 mrad  n  n0  0.00001
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UIUC
QLI Lab
Fluctuations
  n h  h n
UIUC
QLI Lab
UIUC
McGraw Hill (2011)
QLI Lab
UIUC
Outline
1. Background and motivation
2. Quantitative phase Imaging (QPI): SLIM
a. 2D: neural network formation
b. 3D: cell tomography
3. Label-free cancer diagnosis
4. Summary
QLI Lab
SLIM: back to white light
Spatial Light Interference Microscopy
UIUC
(Boulder
Nonlinear)
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Z. Wang et al., OpEx (2011).
QLI Lab
SLIM and AFM
UIUC
(with Prof. J. Rogers)
 ( x, y) 
2

 n( x, y, z)dz
Δn  z
•Sub‐nanometer spatial sensitivity
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LASER
512x512 pixels
‐8
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Z. Wang et al., Opt. Exp. 2011
QLI Lab
Spatiotemporal sensitivity
UIUC
•No speckles
0.3 nm spatially •Common path 0.03 nm temporally 16
Z. Wang et al., Opt. Exp. 2011
QLI Lab
Environmental Control
•
•
•
•
http://www.zeiss.de
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UIUC
Perfusion Chamber
Temperature Control
Humidity
CO2
QLI Lab
UIUC
QLI Lab
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SLIM and fluorescence
‐mitochondria in colon cancer cells (w/ Prof. Ruskin)
UIUC
‐in prep.
QLI Lab
UIUC
Outline
1. Background and motivation
2. Quantitative phase Imaging (QPI): SLIM
a. 2D: neural network formation
b. 3D: cell tomography
3. Toward label-free single molecule imaging
4. Summary
QLI Lab
Phase shift during osmolarity
UIUC

n
n0
•E.g. hypertonicity
Thickness
Refractive index
M '    '( x, y )dxdy M '  M (1  f 2 )
Dry mass ~ constant
G. Popescu et al. Am J Physiol Cell Physiol, (2008).
QLI Lab
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Weighing E. coli with SLIM
With Ido Golding, Baylor
UIUC
QLI Lab
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Weighing E. coli with SLIM
With Ido Golding, Baylor
M. Mir*, Z. Wang*, et al, PNAS (2011)
UIUC
QLI Lab
Neuron Differentiation
with Steve Stice, University of Georgia
•
SLIM: differentiation of neural progenitor cells (hNp1 from Aruna
Biomedical) to mature neural network.
• GOALS:
• Measure changes in dry mass during differentiation
• Intracellular transport as they differentiate
• Motility of cell bodies
• Quantify communication in the network.
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UIUC
Differentiation: Day 0
QLI Lab
UIUC
0.5 Hz
40x
5 min
Mir, Kim, et al., Sci. Reports (2014)
Differentiation: Day 3
QLI Lab
UIUC
0.5 Hz
40x
5 min
Mir, Kim, et al., Sci. Reports (2014)
Differentiation: Day 5
QLI Lab
UIUC
0.5 Hz
40x
5 min
Mir, Kim, et al., Sci. Reports (2014)
Differentiation: Day 11
QLI Lab
UIUC
0.5 Hz
40x
5 min
Mir, Kim, et al., Sci. Reports (2014)
Differentiation: Day 13
QLI Lab
UIUC
0.5 Hz
40x
5 min
Mir, Kim, et al., Sci. Reports (2014)
Neural Network Formation 24 hrs
QLI Lab
UIUC
3x3
10X
0.75
x1
mm2
1/15
min
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Mir, Kim, et al., Sci. Reports (2014)
QLI Lab
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UIUC
Mir et al., Sci. Reports (2014)
QLI Lab
Spatial organization
300 μm
Untreated
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UIUC
15 mM LiCl
M. Mir*, T. Kim*, et al., Scientific Reports, 4, 4434 (2014)
QLI Lab
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UIUC
Mir et al., Sci. Reports (2014)
QLI Lab
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UIUC
Mir, Kim, et al., Sci. Reports (2014)
QLI Lab
UIUC
Outline
1. Background and motivation
2. Quantitative phase Imaging (QPI): SLIM
a. 2D: neural network formation
b. 3D: cell tomography
3. Label-free cancer diagnosis
4. Summary
Coherence gating
1.0
1.0
0.8
0.5
0.6
[c]
Spectrum
QLI Lab
0.4
0
0.2
-0.5
0
-1.0
400
500
600
700
UIUC
-2
-1
0
1
2
c [m]
 [nm]
( x, y; )  ( ) ei0
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•Z. Wang et al., Opt. Exp. (2011)
•Z. Wang et al. APL (2010).
QLI Lab
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Depth sectioning with SLIM
UIUC
QLI Lab
SLIM  3D
UIUC
z
1.2 µm
x
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Z. Wang et al., Op. Ex. 2011
Scattering problem
QLI Lab
Scattering
potential
Plane wave
ki
UIUC
Scattered
wave
Figures
x
y
z
,
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,
,
,
,
,
,
→
,
→
,
,
,
; Direct problem, easy
; Inverse problem, hard
QLI Lab
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X-ray diffraction
UIUC
QLI Lab
X-rays: “phase” problem
UIUC
Emil Wolf, on his 90th Birthday
“Not all the guesses have been successful. This is clear, for example, from
the following: Two different structures were predicted for the mineral bixbyite,
one by L. Pauling, the other by W. H. Zachariasen. It is not known which, if
either, is correct. “
•
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E. Wolf, in Advances in Imaging and electron physics, ed. Hawkes, P. W. E.), Academic Press,
San Diego, 2011.
QLI Lab
Light phase: no problem!
1971 Nobel Prize
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D. Gabor, A new microscopic principle, Nature, 161, 777 (1948).
UIUC
QLI Lab
UIUC
Diffraction tomography
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QLI Lab
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UIUC
Kim et al., Nature Photonics (2014)
WDT Theory
QLI Lab
UIUC
• Helmholtz Equation
,
,
,
0
• Diffraction tomography
eiqz
  k  , q     
U s  k  , z;      A  
2q
2
o
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WDT Theory
QLI Lab
UIUC
• Generalize to white light and imaging
Measurement
, ;
Coherent transfer function
∼
⊗
Numerical Aperture
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Reconstruction
,
Power Spectrum
Kim et al., Nature Photonics (2014)
QLI Lab
E. Coli in 3D
UIUC
• Subcellular helical structure in E. Coli
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Kim et al., Nature Photonics (2014)
QLI Lab
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HT 29 cell in 3D
UIUC
Kim et al., Nature Photonics (2014)
QLI Lab
UIUC
Outline
1. Background and motivation
2. Quantitative phase Imaging (QPI): SLIM
a. 2D: neural network formation
b. 3D: cell tomography
3. Label-free cancer diagnosis
4. Summary
QLI Lab
Prostate Cancer: Diagnosis
UIUC
CK5/6
H&E
Biopsy
slide
Stained slide
Microscopy image
Z. Wang, K. Tangella, A. Balla and G. Popescu, Tissue refractive index as marker of disease, J. Biomed. Opt. 16 (11), 116017 (2011).
V. Molinie, G. Fromont, M. Sibony, A. Vieillefond, et. al. Diagnostic utility of a P63/α-methyl-CoA-racemase (p504s) cocktail in atypical foci in prostate, Modern Pathology, 17,
1180 1190 (2004)
SLIM Images of Prostatectomy Samples
UIUC
QLI Lab
Stained
SLIM
Grade 3
Grade 4
Grade 5
Sridharan, S., Macias, V., Tangella, K., Kajdacsy-Balla, A., Popescu, G. Nature Scientific Reports (in press).
QLI Lab
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Apps
UIUC
QLI Lab
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Real time SLIM
UIUC
QLI Lab
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Fully loaded
UIUC
QLI Lab
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Breast Cancer Diagnosis
UIUC
Majeed et al.
In preparation
QLI Lab
Colon Cancer Progression
Normal
Hyperplasia
200μm
200μm
200μm
200μm
Dysplasia
200μm
200μm
UIUC
Cancer
200μm
200μm
Sridharan et al. In preparation
QLI Lab
Machine learning for automatic
diagnosis/prognosis
UIUC
 800 cores previously imaged are now manually segmented for stromal and epithelial
components
Capture QPI
image
Image segmentation
Calculate features of cores from glands
Diagnosis results
Cancer/Benign,
Cancer grade etc
Ground truth: human labeling
Nguyen et al. In preparation
Three classes:
• Gland
• Stroma
• Lumen
QLI Lab
Phi Optics, Inc.
Launched at Photonics West
Feb 1st, 2014
CellVista Q1000, Q100
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Disclosure: G. Popescu has financial interest in Phi Optics.
UIUC
QLI Lab
Summary
•QPI enables nanoscale dynamics studies
-out-of-plane membrane fluctuations (h)
-in-plane mass transport (n)
•Cell growth
•Intracellular transport
•Cell tomography
•Diagnosis
•…
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UIUC
QLI Lab
UIUC
Thank y u