ECSE4962 Introduction to Subsurface Sensing and Imaging Systems

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ECSE-4962Introduction to Subsurface Sensing and
Imaging Systems

• Lecture 7 Basics of Biological Microscopy
• Kai Thomenius1 Badri Roysam2
• 1Chief Technologist, Imaging Technologies,
• General Electric Global Research Center
• 2Professor, Rensselaer Polytechnic Institute

Center for Sub-Surface Imaging Sensing
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Outline of Course Topics

• THE BIG PICTURE
• What is subsurface sensing imaging?
• Why a course on this topic?
• EXAMPLES THROUGH TRANSMISSION SENSING
• X-Ray Imaging
• Computer Tomography
• Intro into Optical Imaging
• COMMON FUNDAMENTALS
• propagation of waves
• interaction of waves with targets of interest 

• PULSE ECHO METHODS
• Examples
• MRI
• A different sensing modality from the others
• Basics of MRI
• MOLECULAR IMAGING
• What is it?
• PET Radionuclide Imaging
• IMAGE PROCESSING CAD

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Recap

• Several details involving CT scanner operation
  were reviewed
• Distinctions among the major methods for image
  reconstruction.
• In particular, the role of the convolution filter
  in FBP was considered.
• CT related resources on the web were identified.
• The various generations of CT scanners were
  defined.

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Basics of Biological Microscopy
Specimen Preparation
Microscopy
Image Capture
Image Analysis

• Why bother?
• Biomedical sciences overall morphological
  features of specimens quantitative tool
• Explosive growth in physical and materials
  sciences semiconductor industry
• Forensic scientists hairs, fibers, clothing,
  blood stains, bullets, and other items associated
  with crimes

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What we are after

• Structure of specimens
• The micro-anatomy of biological objects, as
  revealed by a variety of physical properties
  (transmissivity, reflectivity, phase,)
• Function of parts of specimens
• The location activity of specific substances
  within cells and tissue
• Combined structural and functional imaging tells
  us where the function is happening relative to
  the specimen anatomy
• Most powerful and useful

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Basic Components of a Microscope

• Light source generates light
• Condenser forms a light cone concentrated on
  specimen.
• Light passes thru the specimen and into the
  objective.
• Objective gathers light from the various parts of
  the specimen.
• Ocular further magnifies the real image projected
  by the objective.
• Total magnification is the product of the
  magnifications of the objective ocular.

eye
eyepiece ocular
objective
specimen condenserlight source
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Imaging Methods

• Light Microscopy
• Transmitted light (brightfield) microscopy
• Dark objects visible against a bright background
• Transmitted light (darkfield) microscopy
• Light reflected off specimen enters the objective
• Confocal microscopy
• Phase contrast microscopy
• Emphasizes diffraction for photons which have
  passed thru specimen.
• DIC microscopy
• Fluorescence Microscopy
• Fluorescent dyes absorb UV light and emit other
  wavelengths

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• Standard Light Microscope (Olympus BH2)

Objective lens The most important part
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Excellent Microscopy Tutorial

• http//microscopy.fsu.edu/primer/index.html
• Numerous interactive Java simulations
• Quite comprehensive
• Excellent 50-page intro to optical microscopy
• And entertaining
• For example, for ray tracing the lens makers
  equation
• Simple Bi-Convex Thin Lenses.mht

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Numerical Aperture (N.A.)
n refractive index Ref scienceworld.wolfram.c
om
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Airy Pattern Resolution
Resolution The ability of a microscope to allow
one to distinguish small, closely situated objects
To reveal finer details, we must use smaller
wavelengths of light, and/or a higher numerical
aperture (N.A.)
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More on Resolution

• Ref FSU Rayleigh disks
• Ref scienceworld.wolfram.com on Fraunhofer
  diffraction

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Resolution Wavelength
mid-spectrum wavelength 550nm
To reveal finer details, we must use smaller
wavelengths of light, and a higher numerical
aperture (N.A.)
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Still More on Resolution

• Airy Disk
• The intensity distribution produced by Fraunhofer
  diffraction due to a circular aperture.
• Intensity distribution is proportional to

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Diffraction-limited spot width

• While the PSF extends to infinity, the "width" of
  the PSF can be described as the radial location
  of the first minimum.
• J1 is zero at approximately 3.8. Thus, the r
  where PSF first equals zero is given by ra
  3.8.
• With a little rearrangement, we can estimate the
  diameter D of the PSF as given on the right.
• or around 1 ? for high NA objectives. This is a
  good estimate the spot formed by
  diffraction-limited focus.

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Fluorescence Microscopy
Excitation Light
Emitted Light
Fluorescence ? Intensity2

• Main Advantages
• Specificity Fluorescent substances are usually
  very specific about excitation emission
  wavelengths, and only fluoresce when the
  excitation is ON.
• We attach fluorescent molecules (fluorochromes)
  to the molecules we want to study.
• Sensitivity Using highly sensitive detectors and
  carefully chosen filters, one can image as few as
  50 molecules per square micrometer.

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Fluorescence Microscopy
Generally, preferable to illuminate from above
(epi-illumination) rather than from below
(transmitted fluorescence illumination).
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Practical Issues

• Need to choose fluorophore molecule carefully
• Smaller molecules penetrate specimen better
• Need to tradeoff image brightness with specimen
  damage
• Brightness of image
• E.g., other things being equal, a 40X objective
  with an N.A. of 1.0 will yield images more than
  five times brighter than a 40X objective with a
  numerical aperture of 0.65.
• Electronic sensors give much higher sensitivity
  compared to film
• Photon noise usually a problem
• Fading - These are conditions that may affect the
  re-radiation of light and thus reduce the
  intensity of fluorescence.
• Known as photobleaching, and quenching
• The fluorophore gets tired and damaged under
  intense excitation light

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Electronic Detectors

• PMT photo multiplier tubes
• CCD charge coupled device

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The Airy Pattern in 3-D
Also known as the point-spread function (PSF)
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Achieving Fine Axial Resolution

• Finer Physical Sectioning
• Limited in scope
• Distorts specimen
• Destructive 3-D imaging possible
• Optical Sectioning
• Method 1 (hardware method) Build a microscope
  that only extracts the light from the best-focus
  plane, and rejects light from above and from
  below
• Method 2 (software method) Develop algorithms
  that attempt to eliminate the light from above
  and below based on a mathematical model of the
  point spread function
• Method 3 combine 1 and 2.

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Confocal Microscopy

• Key distinctions
• Single point illumination
• Scanned illumination
• Pinhole
• Photomultiplier tube

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Effect of Pinhole
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Confocal Microscopes

• The diagram on the previous page shows how to
  image one point in the specimen
• The specimen is stepped along x, y and z
  directions to capture a full 3-D digital image
• Elaborate, precise, expensive (about 100K)
  computer-controlled instruments, usually shared
  by many users

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Fluorescence Confocal Imaging A Great
Combination!
XY
YZ
Alexa Dye Injected Neuron Image Dimensions
512x480x301
XZ
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Deconvolution The Software Method to Optical
Slicing
Point-Spread Function h(x, y, z)
True Image i(x,y,z)
Observed Image y(x,y,z)
In practice, its complicated by the fact that H()
has zeroes (more later!).
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Deconvolution Example (software method)
Before
Rat Pyramidal Neuron Stained with HRP
After
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Confocal Deconvolution Example
Before
After
Top View
Top View
Side View
Side View
Rat CA3 Hippocampal neuron image (Data Courtesy
Dr. James Turner, Wadsworth Center, Albany, New
York)
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Which Method to Use?

• Widefield Valuable whenever 3-D measurements are
  needed
• Confocal We see from the confocal neuron example
  that the software deconvolution method has the
  greatest impact on axial resolution the lateral
  resolution is not improved much.
• The software method is computationally intensive
  (lots of 3-D Fourier Transforms)
• So, when axial resolution is critical, the
  software method is valuable worth the
  computation.

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Summary

• Basics of biological microscopy
• Transmission light microscopy
• Fluorescence light microscopy
• Confocal microscopy
• Combined methods
• Multiple fluorophores, in combination with other
  modalities to provide structural and functional
  imaging
• References
• http//micro.magnet.fsu.edu/primer/index.html
• www.bli.uci.edu/lammp/powerpoints/
  ECE_176/Microscopy_lecture.ppt
• www.cyto.purdue.edu/flowcyt/ educate/confocal/524L
  ec1/524Lec1.ppt

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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.rpi.edu/roysab
• NetMeeting ID (for off-campus students)
  128.113.61.80
• Secretary Laraine Michaelides, JEC 7012, (518)
  276 8525, 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 Laraine Michaelides, JEC 7012, (518)
  276 8525, michal_at_rpi.edu