1. BME6260: Biomedical Imaging Optics and Spectroscopy Matthew Mancuso BEE 7600, Professor Dan Luo Department of Biomedical Engineering, Cornell University Presented Thursday February 3 rd, 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

2. 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.Latinsubjectperson --Wikipedia, http://en.wikipedia.org/wiki/Imagehttp://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!

3. 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

4. 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)

5. Dark field Microscopy Condenser Objective Tube Lens Eye Piece Specimen Direct Illumination Block 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

6. Differential Interference Contrast and Phase Contrast Microscopy Objective Tube Lens Eye Piece Wollaston Prism Polarizer 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.htmlPolarizer

7. 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 DichroicMirror http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.htmlEpi-illumination

8. 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

9. Optical Techniques: Laser Scanning “Scanning Microscopy” Raster scans through one small pixel(point) at a time (“Here at Cornell, Watt Webb invented…”) http://web.cecs.pdx.edu/~jeske/litho/scanning.html

10. 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

11. 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

12. Electron Techniques “Replacing Light with Electrons” Bigger Particles Diffract less (“To see smaller, throw something bigger at the problem”)

13. 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

14. 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

15. Mechanical Techniques “Seeing by Feeling” Touches one small point at a time (“Nanoscale Braille”)

16. 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

17. 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.