ECSE6963 Introduction to Subsurface Sensing and Imaging Systems

1
ECSE-6963Introduction to Subsurface Sensing and
Imaging Systems

• Lecture 17 Spectral Imaging Fluorescence
• Kai Thomenius1 Badri Roysam2
• 1Chief Technologist, Imaging technologies,
• General Electric Global Technology Center
• 2Professor, Rensselaer Polytechnic Institute

Center for Sub-Surface Imaging Sensing
2
Recap

• Use of Phase in Imaging
• Doppler ultrasound
• Phase-contrast optical imaging
• Differential interference contrast
• Spectral imaging
• Fluorescence and contrast agents

3
Recap EM Interaction with Matter
4
Recap Fluorescence Imaging
Courtesy William Shain Lab
5
Light Absorption
Absorption can only occur when
If the energy of the probing photon is mismatched
to the energy difference between the quantum
states, the material is transparent to the probe.
6
Emission Fluorescence

• Opposite of absorption
• Can be coherent (Raman)/incoherent (Fluorescence)
• Typically, there is some loss in the molecule, so
  the emitted energy is lower than the absorbed
  energy
• The difference is the Stokes Shift

Molar Concentration of fluorophore
Excitation intensity
Path length
Fluorescence intensity
Quantum Efficiency ( molecules that emit)
Molar absorptivity
7
Multi-photon Excitation

• Use 2 or more infrared photons to excite
  fluorophores ordinarily excitable with higher
  frequencies
• Much more detail in images collected deeper in
  the sample.
• No sample photobleaching outside focal plane.
• Dramatic improvement in longevity of living
  cells, tissues and organisms.
• Ready determination of co-localising fluorescent
  probes.
• No need for confocal apertures.
• Ability to image autofluorescence
• UV flourophores may be excited using a lens that
  is not corrected for UV as these wavelengths
  never have to pass through the lens.
• MPE also offers enhanced photoselection in
  spectroscopy.

3-photon works the same way
8
Multi-photon Microscopy

• Practical issues
• The two photons need to arrive simultaneously a
  low probability event
• Use ultrafast, mode-locked near-infrared lasers
  (i.e. Tisapphire 100-200 femtosecond pulse
  duration, 76MHz repetition rate).
• Under the appropriate conditions, these lasers
  produce short duration pulses with the high peak
  power required for a multi-photon effect and an
  average power low enough to make specimen damage
  negligible.
• Such lasers are tunable over a range of
  700nm-1000nm which permits optimal wavelength
  selection to elicit an efficient multi-photon
  effect.
• Probability of simultaneous absorption falls off
  steeply away from the focal volume

9
Skin Tumor Example
10
Observing Changes Over Time
Successive images are 24 hours apart
11
Limitations of Multi-photon

• Slightly lower resolution with a given
  fluorophore when compared to confocal imaging.
• This loss in resolution can be eliminated by the
  use of a confocal aperture at the expense of a
  loss in signal.
• Thermal damage can occur in a specimen if it
  contains chromophores that absorb the excitation
  wavelengths, such as the pigment melanin.
• Only works with fluorescence imaging.
• Currently rather expensive.

12
Second-harmonic Generation

• Similar to 2-photon, but essentially lossless
• Great for live imaging
• E.g., collagen
• Great for
• Deep-tissue imaging
• quantification along the frequency axis!

Virtual state
13
SHG Example
Extra-cellular matrix
Muscle filament lattice structure
Extra-cellular and intracellular structures
within native muscle tissue
An optical section at a depth of 250 µm into the
sample
http//www.biophysj.org/cgi/content/full/82/1/493
14
Improving Light Microscopy

• Its Not Just About Resolution
• Resolution Limited by Diffraction
• Its About What Is Measured
• Transmission, Reflection, Phase, Fluorescence,
  Polarization, Non-Linear Properties
• Minimizing specimen damage
• And About How Data Are Processed
• Registration, Deconvolution, Tomography,
  Parameter Estimation
• And About Measuring Everything at Once

15
What is Sensed by the Different Modes?
LSCM
SHG
Epi F
DIC
TPLSM
QTM
Staring
Scanning
Phase (optical path length)
16
What can be measured
Morphological Dynamics

• Sensor Fusion
• Different sensors have different strengths and
  weaknesses
• Combine them to take advantage of each

Space (x, y, z)
Time (t)
Structure
Dynamics
Function
Signaling Paths, Molecular Transport
Chemical Dynamics
Fluorescent labels
Other Physical Properties
e.g., Refractive index, strain
17
(Phase Fluorescence) Example
Data courtesy Gary Banker (U. Oregon)
18
Staring Modes Similar Embryos, Diverse Images
Thanks to Bill Warger
19
Multi-Modal Imaging of Mouse Embryos
2-photon
Confocal Fluorescence
Differential Interference Contrast
Quadrature Microscopy
20
Multi-Modal Imaging of Mouse Kidney Cells Using
a 3D Fusion Microscope
2-photon DAPI
2-photon Alexa Fluor 488
QTM
DIC
Thanks to Dan Townsend
21
Accessing Wider Regions
22
Current State of the Art

• Fusion Microscopy Put Multiple modalities on
  same platform

23
Current Trends

• Ever increasing numbers of structural and
  functional endpoints can be observed
  simultaneously in 3-D
• Growing libraries of organic fluorophores
  quantum dots
• Multi- and hyper-spectral microscopes
• Spectral unmixing tools
• Support for complex fluorescence phenomena
• Easier to work with live cells
• Sensitive, high-resolution, 3-D imaging
• Minimally-damaging (MP, SHG), time-resolved
  imaging
• Better instrumentation better understanding of
  biology
• Fusion of multiple microscopy modalities
• High-extent high-resolution high-throughput
  imaging
• High-throughput tissue prep imaging hardware

24
Summary

• Spectral response provides substance-specificity
  to imaging
• Fluorescence and Multi-photon imaging are
  powerful tools
• Thanks to Prof. Charles DiMarzio for slides
  regarding QTM and Fusion Microscopy
• Lets talk about Projects!!

25
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

26
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