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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

Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

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Page 1: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

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

Page 2: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

Optical Sectioning in Biological Microscopy

Widefield Fluorescence

Multiphoton Microscopy

ConfocalMicroscopy

Deconvolution Methods

(Computational)

Confocal Aperture

nonlinear processes

Widefield Fluorescence

Fixation and Physical

Sectioning

Liv

e s

pec

ime

ns

Conventional light microscopy doesn’t work well on thick (> few microns) specimens

Fluorescently labeled sea urchin eggs

Page 3: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

Laser scanning microscopy

Objective lens

The focused laser is raster scanned across the sample and the fluorescence is detected, amplified and digitized.

Page 4: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

Fluorescence

excitation emission

Confocal Microscopy produces optical sections by excluding light from outside of the focal plane.

Page 5: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

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):

Page 6: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

In vivo imaging - example: transgenic mouse models of Alzheimer's disease.

3D projection of 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.

Page 7: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

MPM Histology

With Alexander Niktitin’s laboratory, Biomedical Sciences

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

Page 8: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

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

Page 9: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

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

Page 10: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

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)

Page 11: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

White light image of

small (~3 mm diameter)

metastasis on small

instestine

Ascites tumor model (transformed p53/Rb ovarian epithelial cells injected IP)

tumorWidefield fluorescence image (cells also express GFP)

Page 12: Optical Microscopy Widefield Microscopy - Brightfield, Darkfield, DIC, Phase Contrast, Fluorescence … Total Internal Reflection (TIR and TIRF) Microscopy

Two color multiphoton imaging of tumor on the small intestine in an ascites tumor model

7 mm