Optical Microscopy
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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
Optical Sectioning in Biological Microscopy
Conventional light microscopy doesn’t work
well on thick (> few microns) specimens
Live specimens
Widefield
Fluorescence
Widefield
Fluorescence
Fixation and
Physical
Sectioning
Deconvolution
Methods
(Computational)
Confocal
Microscopy
Confocal
Aperture
Multiphoton
Microscopy
nonlinear processes
Fluorescently
labeled sea
urchin eggs
Laser scanning microscopy
The focused laser is raster scanned across the sample and the fluorescence is
detected, amplified and digitized.
Objective lens
Confocal Microscopy produces optical sections by excluding light from
outside of the focal plane.
Fluorescence
excitation
emission
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):
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.
Transgenic mouse models of ovarian cancer based on p53 and Rb
inactivation
Intrabursal injections of AdCre into
mice carrying conditional p53, Rb1
or both alleles results in ~100%
epithelial neoplasms
MPM
With Alexander Niktitin’s laboratory, Biomedical Sciences
Histology
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 ->
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
Intraoperative Fluorescence microscope from Zeiss – OMPI Pentero
Glioblastoma IV under white light and under BLUE 400 illumination
Walter Stummer, M.D., University of Düsseldorf, Düsseldorf, Germany
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)
Ascites tumor model (transformed p53/Rb ovarian epithelial cells injected IP)
tumor
White light
image of
small (~3 mm
diameter)
metastasis on
small
instestine
Widefield fluorescence
image (cells also express
GFP)
Two color multiphoton imaging of tumor on the small intestine in an ascites
tumor model
7 mm