ECSE-6963 Introduction to Subsurface Sensing and Imaging Systems

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ECSE-6963Introduction to Subsurface Sensing and
Imaging Systems

• Lecture 16 Phase Imaging, Spectral Imaging
• Kai Thomenius1 Badri Roysam2
• 1Chief Technologist, Imaging technologies,
• General Electric Global Technology Center
• 2Professor, Rensselaer Polytechnic Institute

Center for Sub-Surface Imaging Sensing
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Recap

• Use of Phase in Imaging
• Doppler ultrasound
• Phase-contrast optical imaging
• Today
• Differential interference contrast
• Spectral imaging

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Recap Interferometric Architectures
Sensing wave
object
Coherent Source
interference
Reference wave
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Polarization
Polarization can be achieved with crystalline
materials which have a different index of
refraction in different crystal planes. Such
materials are said to be bi-refringent or doubly
refracting.
http//micro.magnet.fsu.edu
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Anisotropic Media
Birefringent media split the incoming light into
two rays One of the light waves, termed the
ordinary ray, travels straight through the
crystal The other extraordinary ray is
refracted to a significant degree. The ordinary
and extraordinary light rays are polarized
in mutually perpendicular directions. have
different speeds of propagation.
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Forms of Birefringence
Birefringence is an asymmetry of properties that
may be optical, electrical, mechanical,
acoustical, or magnetic in nature. 1. Intrinsic
birefringence such as in calcite. 2. Structural
birefringence sensitive to refractive index
fluctuations or gradients in the surrounding
medium e.g., biological macromolecular
assemblies such as chromosomes, muscle fibers,
microtubules, liquid crystalline DNA, and fibrous
protein structures such as hair, many synthetic
materials such as fibers, long-chain polymers,
resins, and composites. 3. Stress and strain
birefringence occur due to external forces and/or
deformation acting on materials that are not
naturally birefringent. e.g., stretched films
and fibers, deformed glass and plastic lenses,
and stressed polymer castings. 4. Flow
birefringence can occur due to induced alignment
of materials such as asymmetric polymers that
become ordered in the presence of fluid flow.
e.g., Rod-shaped and plate-like molecules and
macromolecular assemblies, such as high molecular
weight DNA and detergents.
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Polarized Light Microscopy
Basic Idea Some transparent materials are
anisotropic The specimen will appear different
as it is rotated
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Differential Interference Contrast (DIC)

• A single ray of light is split into two rays
  that are close to each other (??x )
• one traversing the specimen (specimen ray)
• the other missing the specimen, but interacting
  with the background (reference ray)
• The rays are then recombined at the image plane,
  where wave interference may occur.
• Detects differences in phase between nearby
  points

interference
Reference ray
Specimen
Specimen ray
Splitter
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The Wollaston Splitter
Two polarized rays at known angle
The Wollaston prism is a polarizing beam
splitter/combiner that is used in DIC
microscopes. It is made of two prisms with
optical axes that are perpendicular to each
other. This is a key component of CD players!
Wollaston
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Differential Interference Contrast (DIC)
1
2
2
Wollaston prism 1 separates the incoming light
into two beams that are shifted spatially by a
few tenths of a micrometer (?x), and
perpendicularly polarized (shown in red and
blue). Wollaston prism 2 is complementary to the
first prism that recombines the two
waves. Interference occurs at the analyzer, and
we can see the image as an intensity variation.
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The DIC Math
We have two beams that are separated spatially by
a tiny distance dx. If the transmission function
of the object is f(x,y), the interference image
intensity is where p(x,y) is the Airy point
spread function (PSF) of the microscope
objective, with incoherent illumination being
assumed. For a phase object neglect the second
term and get The image highlights the phase
gradients in the direction of the displacement
vector dx !!
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DIC Images
There is no halo as in phase contrast
microscopy. The DIC images have a false
three-dimensional relief appearance due to the
directional differentiation. The
three-dimensional appearance does not represent
reality. In other words the 3-D relief of DIC
imaged specimens is an optical rather than a
geometric relief.
Direction of ?x
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Phase Contrast vs. DIC
http//micro.magnet.fsu.edu
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Limitations of DIC
Absolute Measurement
DIC
Assumed Index Profile Before Compaction
M10026.m
M10026.m
Assumed Index Profile During Compaction
M10026.m
M10026.m
Thanks to Prof. Charles DiMarzio, NU
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Quadrature Interferometric Microscopy
Thanks to Prof. Charles DiMarzio, NU
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Unwrapped Phase Image of a Mouse Oocyte
Unwrapped Phase
Amplitude
Phase
10027.jpg
3993.jpg
10028.jpg
Thanks to Prof. Charles DiMarzio, NU
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QTM Overcomes Limitations of DIC
QTM Measurement
DIC
Assumed Index Profile Before Compaction
M10026.m
M10026.m
Assumed Index Profile During Compaction
M10026.m
M10026.m
Thanks to Prof. Charles DiMarzio, NU
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Mouse Embryo Growth Stages
oocyte
zygote
2-cell
8-cell
morula
blastocyst
Embryonic Stem cells
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Recap EM Interaction with Matter
Wavelength Range Type of interaction Comment
10m 1meter (Radio Frequency). Change of nuclear spin (magnetic component of EM wave more important) Nuclear Magnetic Resonance Nucleons absorb/emit based on their spin property
1m 1 cm (Radio Frequency) Change of electron spin (magnetic component of EM wave more important) Electron Spin Resonance Electrons absorb/emit based in their spin property
1cm 100?m (Microwaves) Change of orientation (rotation) (electric component of EM wave more important) Mostly rotational effects
100?m 1?m (Infrared) Change of configuration (electric component of EM wave more important) Mostly vibrations, rotations, and bending of molecules while it still remains in its electronic ground state. The molecule must be asymmetric. Vibrations need more energy than rotations (20 ?m or shorter).
1?m 10nm (visible - ultraviolet) Change of electron distribution (electric component of EM wave more important) Changes in electronic states of atoms in the molecule produce changes in electric dipoles of the atoms, that interact with the applied wave
10nm 100pm (X-ray) Change of electron distribution At these shorter wavelengths, photons can actually disrupt the absorbing molecule by photodissociation or even produce photoionization of individual atoms. (1000-angstrom photons will photoionize electrons in the outer shells, whereas 100-angstrom or shorter photons will photoionize electrons in the inner shells.)
100pm and smaller (gamma rays) Change of nuclear configuration Mostly passes through
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Classification of SSI Systems Based on probe and
Detector Spectra
Detectors
Most specific
Narrow-band
Wide-band


Narrow-band (e.g. laser)
Probes
Wide-band (e.g white light)
Least specific
Spectral channels

• Interesting case
• Multi-spectral (2-50 channels) and
• hyper-spectral (50 or more channels) imaging
  systems

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Wideband Probe, Narrow Detector Example

• Estimation of composition of substances with
  known spectral signatures at each position
• Feature detection at each position
• Wavelength ratiometric imaging

Courtesy Luis Jiminez at UPRM
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Narrow Probe, Narrow Detector Example
Blue blobs DAPI (DNA Stain) Red clouds Lewis X
(LeX) Green tubes GFAP Tourquoise (tubes and
blobs) Laminin (stains vessels and bulbs)
Data Sally Temple, AMC
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Value of Molecule-Specific Imaging The adult
neural stem cell niche
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Value of Molecule-Specific Imaging The adult
neural stem cell niche
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Summary

• We introduced some interference-based imaging
  technologies
• Optical Coherence Tomography (more later)
• Phase contrast (not limited to optics!)
• Differential Interference Contrast
• Spectral response provides substance-specificity
  to imaging
• Fluorescence provides molecular specificity

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Homework Assignment

• Visit the following website describing Green
  Fluorescent Proteins (GFPs)
• http//www.microscopyu.com/articles/livecellimagin
  g/fpintro.html
• In a couple of sentences, describe why the
  discovery of GFPs has revolutionized biological
  imaging
• Molecular Probes (www.probes.com) is a maker of
  fluorescent markers, with an informative web
  handbook
• Search their website for a fluorescent stain DAPI
  that highlights the nuclei of cells (i.e., a
  nuclear stain). Figure out the typical excitation
  and emission wavelengths for this stain,
  specifications for the barrier filter, and
  identify the type of imaging (single or
  multi-photon) that it is suited for.
• Find a fluorescent marker for mitochondria
  these are parts of cells that are involved in
  energy production. Figure out the typical
  excitation and emission wavelengths for this
  stain, specifications for the barrier filter, and
  identify the type of imaging (single or
  multi-photon) that it is suited for.

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Instructor Contact Information

• Badri Roysam
• Professor of Electrical, Computer, Systems
  Engineering
• Office JEC 7010
• Rensselaer Polytechnic Institute
• 110, 8th Street, Troy, New York 12180
• Phone [phone]
• Fax [phone]
• Email roysam_at_ecse.rpi.edu
• Website http//www.ecse.rpi.edu/roysabm
• NetMeeting ID (for off-campus students)
  128.113.61.80
• Secretary Laraine, JEC 7012, [phone],
  michal_at_.rpi.edu

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Instructor Contact Information

• Kai E Thomenius
• Chief Technologist, Ultrasound Biomedical
• Office KW-C300A
• GE Global Research
• Imaging Technologies
• Niskayuna, New York 12309
• Phone [phone]
• Fax [phone]
• Email thomeniu_at_crd.ge.com, thomenius_at_ecse.rpi.edu
  
• Secretary TBD