BME6260: Biomedical Imaging Optics and Spectroscopy

Matthew Mancuso BEE 7600, Professor Dan LuoDepartment of Biomedical Engineering, Cornell University Presented Thursday February 3rd, 2011

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/fluoromicroanatomy.html

“Measure what is measurable, and make measurable what is not so.” --Galileo Galilei

in 15 Minutes or Less

What is and is not imaging?

Imaging Techniques Other Characterization Techniques

An image (from Latin imago) is an artifact, for example a two-dimensional picture, that has a similar appearance to some subject—usually a physical object or a person.

--Wikipedia, http://en.wikipedia.org/wiki/Image

•Optical Microscopy• Widefield Microscopy

• Bright Field/Dark Field• DIC/Phase Contrast• Fluorescent• photo-activated localization

microscopy (PALM), STORM• Laser Scanning Microscopy

• Confocal • Multiphoton

•Electron Microscopy• Scanning (SEM)• Transmission (TEM)

•Atomic Force Microscopy

•Optical and E&M Techniques• Spectroscopy• X-ray Scattering• Ellipsometry

•Elementary Particle Techniques• Neutron Scattering

•Force Techniques• Profilometry

We’ll cover some of these (and more!) on Tuesday!

Optical Techniques:Widefield

Basic Microscopy Imaging an entire Field of View at a time

(“When I was a kid, we had to….”)

http://www.tutornext.com/help/optical-microscope

Bright Field Microscopy

Condenser

Objective

Tube Lens

Eye Piece

Specimen

“Infinity Space”

Usefulness / Purpose

Shortcomings / Limits

“Kohler Illumination”

•Only useful on dark and strongly refracting materials•Diffraction Limited (approximately half wavelength)•Convoluted from other planes ( thick samples hard)

•Simple(Quick to do, Easy, Cheap, Reliable)• FAST (High Frame Rate, Great for videos)

Dark field Microscopy

Condenser

Objective

Tube Lens

Eye Piece

Specimen

Direct IlluminationBlock

Usefulness / Purpose

Shortcomings / Limits

Darkfield Filter

• Easy set-up• Impressive Contrast and Quality• Relatively good for live samples• Small things not light sensitive (nanotechnology)

• Low light Intensities•Diffraction Limited(approximately half wavelength)• Strong illumination can damage samples

Differential Interference Contrast and Phase Contrast

Microscopy

Objective

Tube Lens

Eye Piece

Wollaston Prism

Polarizer

Wollaston Prism

Usefulness / Purpose

Shortcomings / Limits

• Great for biological samples• High contrast with less light than darkfield• Also good for thin films in nanotechnology

• Thin samples• Similar refractive indices•Diffraction Limited(approximately half wavelength)

http://www.olympusmicro.com/primer/techniques/dic/dicphasecomparison.html

Polarizer

EmissionFilter

Fluorescent Microscopy

Objective

Tube Lens

Eye Piece

Specimen

Usefulness / Purpose

Shortcomings / Limits

• Excellent in biological samples• Can label certain sub-cellular features• Coupled with GFP provides a powerful tool

• Requires Fluorescently labeled specimen• Diffraction limited (mostly)• Weak signals often an issue

ExcitationFilter

Dichroic Mirror

http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html

Epi-illumination

PALM / STORM

http://www.microscopyu.com/tutorials/flash/superresolution/storm/index.html

Usefulness / Purpose Shortcomings / Limits

•Ultra high Resolution• Optical Technique which overcomes Diffraction limit

•Very slow (hours)• Requires fluorescent sample• Fitting PSFs requires computational ability

Optical Techniques:Laser Scanning

“Scanning Microscopy” Raster scans through one small pixel(point) at a time

(“Here at Cornell, Watt Webb invented…”)

hvE

http://web.cecs.pdx.edu/~jeske/litho/scanning.html

Laser Scanning Confocal Microscopy

Usefulness / Purpose

Shortcomings / Limits

•Resolution increase over widefield• Excellent in biological samples• Allows optical sectioning

• slower (hard for dynamic systems)• Diffraction limited • complicated compared to previous techniques

http://www.vcbio.science.ru.nl/en/image-gallery/laser/

Fluorescent Confocal

http://www.frontiersin.org/human_neuroscience/10.3389/fnhum.2010.00044/full

Two Photon Microscopy

http://belfield.cos.ucf.edu/one%20vs%20two-photon%20excitation.html

Usefulness / Purpose Shortcomings / Limits

•Long wavelength enables deep imaging• Resolution increase over confocal• Simpler, faster than STORM/PALM

•Developed at Cornell!

• Overall still complex, slow• Requires expensive femto-second laser

Electron Techniques

“Replacing Light with Electrons” Bigger Particles Diffract less

(“To see smaller, throw something bigger at the problem”)

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Transmission Electron Microscopy

Usefulness / Purpose

Shortcomings / Limits

•Ultra high resolution (single to tens of nm)• Electron Diffraction << Photon Diffraction• Developed in 1930s, huge resolution for the time

• Extensive Preparation, Electron Transparent (Thin)• Small field of view• Can damage samples, hard for biology

Scanning Electron Microscopy

Usefulness / Purpose

Shortcomings / Limits

http://www.purdue.edu/rem/rs/sem.htm

•Ultra high resolution (single to tens of nm)• Electron Diffraction << Photon Diffraction•Can scan over 5 to 6 orders of magnitude

• Often requires covering sample in metal• Most SEMs operate in vacuum• Hard to use on live/sensitive samples

Mechanical Techniques

“Seeing by Feeling” Touches one small point at a time

(“Nanoscale Braille”)

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21

Atomic Force Microscopy

http://www.imagemet.com/index.php?id=12&main=products&sub=applications http://www.phy.duke.edu/research/bio_physics/

Usefulness / Purpose

Shortcomings / Limits

•Can provide three dimensional images• Doesn’t require vacuum; can work in water!•Can reach true atomic resolution

• Limited Scan size and Field of View• Slow compared to Optical/ Electron Techniques

Thanks!References

1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).

2. W. Denk, J. Strickler, and W. Webb, "Two-photon laser scanning fluorescence microscopy," Science 248, 73-76 (1990).

3. F. J. Giessibl, "Advances in atomic force microscopy," Reviews of Modern Physics 75, 949 (2003).

4. B. Huang, W. Wang, M. Bates, and X. Zhuang, "Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy," Science 319, 810-813 (2008).

5. C. W. Oatley, W. C. Nixon, and R. F. W. Pease, "Scanning Electron Microscopy," in Advances in Electronics and Electron Physics, L. Marton, ed. (Academic Press, 1966), pp. 181-247.

6. D. B. Williams and C. B. Carter, "The Transmission Electron Microscope," in Transmission Electron Microscopy (Springer US, 2009), pp. 3-22.