Optical Microscopy

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Optical Microscopy

• Widefield Microscopy - Brightfield, Darkfield,
  DIC, Phase Contrast, Fluorescence
• Total Internal Reflection (TIR and TIRF)
  Microscopy
• Confocal Microscopy - fluorescence and
  reflection.
• Multiphoton or Nonlinear Microscopy
• Nearfield Microscopy (NSOM)
• 4-Pi Microscopy
• STED Microscopy (STimulated Emission Depletion)
• Structured illumination microscopy (SIM) and
  saturated structured illumination microscopy
  (SSIM)
• Selective plane illumination microscopy

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Optical Sectioning in Biological Microscopy
Fluorescently labeled sea urchin eggs
Conventional light microscopy doesnt work well
on thick (gt few microns) specimens
Fixation and Physical Sectioning
Widefield Fluorescence
Deconvolution Methods (Computational)
Widefield Fluorescence
Live specimens
Confocal Microscopy
Confocal Aperture
Multiphoton Microscopy
nonlinear processes
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Laser scanning microscopy
The focused laser is raster scanned across the
sample and the fluorescence is detected,
amplified and digitized.
Objective lens
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Confocal Microscopy produces optical sections by
excluding light from outside of the focal plane.
Fluorescence
emission
excitation
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Two-Photon, Multiphoton or Nonlinear Microscopy
uses nonlinear optical processes to create
contrast and obtain optical sectioning.
The two most common nonlinear processes are Two
photon fluorescence and second harmonic
generation (SHG)
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In vivo imaging - example transgenic mouse
models of Alzheimer's disease.
3D projection of b amyloid plaque stained with
Thio-S, excitation at 760 nm.
Christie, R. H., Bacskai, B. J., Zipfel, W. R.,
Williams, R. M., Kajdasz, S. T., Webb, W. W.
Hyman, B. T. (2001) J Neurosci 21,
858-64. Bacskai, B. J., Kajdasz, S. T., Christie,
R. H., Carter, C., Games, D., Seubert, P.,
Schenk, D. Hyman, B. T. (2001) Nat Med 7,
369-72.
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Transgenic mouse models of ovarian cancer based
on p53 and Rb inactivation
Histology
MPM
Intrabursal injections of AdCre into mice
carrying conditional p53, Rb1 or both alleles
results in 100 epithelial neoplasms
With Alexander Niktitins laboratory, Biomedical
Sciences
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Is it possible to use nonlinear laser scanning
microscopy to image a cm field of view as an
aid, for example, to better define tumor borders?
Advantages may be 1. Better 3D view. 2.
Maximum optical resolution could still be on the
order of 4 microns and the system would be able
to zoom to the cellular level. 3. Ability to
excite both targeted contrast agents (example
5-ALA -gt protoporphyrin IX) and use intrinsic
signals for an overall tissue view.
Disadvantages 1. A more complex instrument. 2.
Since it would be used with a conventional
surgical microscope, image registration may be
difficult to achieve.
Typical field of view in a laser scanning
(confocal or multiphoton) microscope is 0.5 x
0.5 mm
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Intraoperative Fluorescence microscope from Zeiss
OMPI Pentero
Glioblastoma IV under white light and under BLUE
400 illuminationWalter Stummer, M.D., University
of Düsseldorf, Düsseldorf, Germany
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Multiphoton imaging with a 2x lens (0.14 NA) -
field of view is 7 mm (movie is of the word
Cornell in 12 pt font from my business card)
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Ascites tumor model (transformed p53/Rb ovarian
epithelial cells injected IP)
Widefield fluorescence image (cells also express
GFP)
tumor
White light image of small (3 mm diameter)
metastasis on small instestine
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Two color multiphoton imaging of tumor on the
small intestine in an ascites tumor model
7 mm