2009 Lab 2

From CellBiology
Inverted Biological Microscope

Microscopy Methods

This Lab is an introduction to cell biology methods using microscopy. It includes a brief historic background and relevant modern technological advances. The focus is more on the application of these techniques in cell biology, rather than a comprehensive understanding of the physics and technology underlying the techniques.


"Diffraction inevitably limits the resolution of microscopy to around half the wavelength of light" Ernst Abbe (1873, German physicist)

This rule has recently been bent, not broken.....


Also take the time to look at the Textbook References and some of the Cell Biology Images on this Wiki. Later in the course we will be visiting the Confocal Microscope Facility, so spend some time reading about this technique.


MBoC Figure 9-8. Four types of light microscopy

Figure 9-2. Resolving power

Microscopy Timeline

  • 1665 - Robert Hooke publishes Micrographia, a collection of biological micrographs.
  • 1674 - Anton van Leeuwenhoek improved simple microscope for biological specimens.
  • 1833 - Brown published a microscopic observation of orchids, describing the cell nucleus.
  • 1898 - Golgi first saw and described the Golgi apparatus by staining cells with silver nitrate.
  • 1931 - Ernst Ruska first transmission electron microscope, TEM).
  • 1934 - Frits Zernike describes phase contrast microscopy.
  • 1957 - Marvin Minsky patents principle of confocal imaging.
  • 1953 - Frits Zernike receives the Nobel Prize in Physics for invention of the phase contrast microscope.
  • 1955 - George Nomarski theoretical basis of Differential interference contrast microscopy.
  • 1981 - Gerd Binnig and Heinrich Rohrer develop the Scanning Tunneling Microscope (STM).
  • 1981 - Daniel Axelrod develop Total Internal Reflection Fluorescence Microscope (TIRFM).
  • 1981 - Allen and Inoué perfected video-enhanced-contrast light microscopy.
  • 1986 - Ernst Ruska, Gerd Binnig and Heinrich Rohrer receive the Nobel Prize in Physics for invention of the electron microscope (ER) and scanning tunneling microscope (GB and HR).
  • 2000 - Hell and collaborators develop Stimulated Emission Depletion Microscopy (STED)
  • 2008 - Freudiger and Wei Min develop Stimulated Raman Scattering Microscopy (SRS)

Microscopy Techniques

Inverted Biological Microscope
  • Light Microscopy
    • normal - transmitted brightfield illumination of fixed and stained specimens
    • inverted - overcome focal length problems, combine with special optical techniques
    • optics - Phase contrast, Nomarski Differential Interference Contrast (DIC)
  • Electron Microscopy
    • transmission
    • scanning
    • tunneling
  • Fluorescent Microscopy
  • Confocal Laser Scanning Microscopy (CLSM)
  • Total Internal Reflection Fluorescence Microscopy (TIRFM)
  • Live Cell Imaging Timelapse

Light Microscopy

Transmission Microscopy

Useful for fixed and histologically stained cells or tissue sections. Histology Stains

Phase Contrast Microscopy

  • refractive index differences within cellular components and between cells and their surrounding aqueous medium
  • enhances contrast in transparent specimens


Links: MBoC Figure 9-8. Four types of light microscopy

Differential Interference Contrast (DIC) Microscopy

Used to observe structure and motion in unstained, transparent living cells and isolated organelles. This method produces a monochromatic shadow-cast image of optical phase gradient.

Polarized Light Microscopy

This generates structural anisotropy due to form birefringence, intrinsic birefringence, stress birefringence. For example, birefringent microtubules in the mitotic spindle.

Fluorescence Microscopy

  • Fluorescence The process where an atom or molecule is transiently excited by absorption of external radiation at the proper energy level (usually ultraviolet or visible light) to then release the absorbed energy as a photon having a wavelength longer than the absorbed energy.
  • Autofluorescence The generation of background fluorescence by endogenous metabolites and organic or inorganic fluorescent compounds present in cells (catecholamines, cytochromes, fatty acids, flavins, flavin proteins and nucleotides (FAD and FMN), lipofuchsin pigments, porphyrins, reduced pyridine nucleotides (NADH and NADPH), serotonin, vitamin B)

Confocal Laser Scanning Microscopy (CLSM)

  • optical microscopy technique based on wide-field fluorescence microscopy
  • a laser beam is focussed into the sample and using electronic lenses and apertures (pinhole) only the fluorescence light that comes directly from the confocal plane is detected by a photomultiplier
  • fluorescence from outside this plane is cancelled out by a pinhole

See also Laboratory 7 - Confocal Microscopy

Links: Introduction to Confocal Microscopy | Figure 9-19. Conventional and confocal fluorescence microscopy compared

Total Internal Reflection Fluorescence Microscope (TIRFM)

TIRFM Optics.gif

  • A microscopy method which views a very thin region (200 nm) of cell.
  • This can be used for example to examine structures and features associated with the membrane contact region(s) of a cell and a substrate.
  • occurs when light passes from a high-refractive medium (glass) into a low-refractive medium (cell, water)
  • evanescent field produced by total internally reflected light excites fluorescent molecules at the cell-substrate interface
    • evanescent means "tends to vanish"- evanescent waves are formed when waves traveling in a medium suffer total internal reflection at its boundary because they hit it at an angle greater than the so-called critical angle.


Links: Nature Methods Primer: fluorescence imaging under the diffraction limit

Live Cell Imaging Timelapse

This technique views cells growing in culture by a video camera linked to an inverted phase microscope. The cells also need to be maintained at physiological temperature, usually by a heated stage or container and carbon dioxide level, either by a sealed tissue culture flask or gassed container. Note that light levels must be very low, or shuttered, and a still camera can also be set to take an image at regular intervals, these images can then be put together as a movie.


Laser Capture Microscopy

Laser-capture microdissection cartoon.gif

Laser Capture Microscope is also called Laser Capture Microdissection (LCM). A technique which uses light microscopy in combination with a laser dissection (cutting) of subpopulations of tissue cells. The cells of interest can then be harvested (collected) or unwanted cells removed to give histologically pure enriched cell populations.



Links: Laser capture microdissection of mammalian tissue. Edwards RA. J Vis Exp. 2007;(8):309. Epub 2007 Oct 1. PMID: 18989416 | Combining laser capture microdissection and proteomics techniques. Mustafa D, Kros JM, Luider T. Methods Mol Biol. 2008;428:159-78. Review. PMID: 18287773


http://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.85.html

Electron Microscopy

Transmission Electron Microscopy

Links: UNSW Electron Microscope Unit - TEM

Scanning Electron Microscopy

Links: UNSW Electron Microscope Unit - SEM | Wiki - SEM | Iowa State University - Material Science

Correlative Light/Electron Microscopy (CLEM)

Combines light microscope technique with later electron microscope analysis of the same visualised structure.

Correlative light-electron microscopy

Note there is different form of microscopy that uses the same acronym Controlled Light Exposure Microscopy (CLEM) for prolonged live-cell imaging and strongly reduced photobleaching.

Scanning Tunneling Microscope (STM)

Scanning tunneling microscope Scanning tunneling microscope (STM) is a type of electron microscope that shows three-dimensional images of a sample. In the STM, the structure of a surface is studied using a stylus that scans the surface at a fixed distance from it.

The Nobel Prize in Physics 1986

  • "for his fundamental work in electron optics, and for the design of the first electron microscope"
  • "for their design of the scanning tunneling microscope"

A related technique Atomic Force Microscopy (ATF) works best with hard materials and is used in mainly non-biological components.

Links: Online Tutorial | Wiki - STM

New Laser Microscopy

There are several techniques which will allow the identification of specific molecules in space and their state. These methodologies will not be part of this current laboratory, but are included for information purposes and to show the current directions of research in this area. Many of these techniques use the unique properties of laser light.


Stimulated Emission Depletion Microscopy (STED)

  • fluorescence microscopy technique
  • uses two lasers in a confocal scanning microscope
  • the second laser generates a bleached "donut" around the object of interest in the centre illuminated by the first laser.

Photoactivation Localization Microscopy (PALM)

Nuclear envelope imaging
  • fluorescence microscopy technique
  • activated fluorophores are illuminated during image acquisition
  • all of them are bleached and then a new subpopulation is photoactivated to begin the next cycle

Stochastic Optical Reconstruction Microscopy (STORM)

  • fluorescence microscopy technique
  • related to PALM
  • a second photoactivation laser is used
  • switches the photoactivated molecules back to their starting state after the desired number of photons has been collected

Raman Microscopy

  • laser beams illuminate a sample resulting in a characteristic shift in wavelength caused by chemical bonds
  • used to identify and locate molecules (used for lipid research)

Coherent Anti-Stokes Raman scattering (CARS) Microscopy

  • uses two laser beams to excite molecular vibrations and generates a stronger signal

Stimulated Raman Scattering Microscopy (SRS)

  • excites molecules with two laser beams
  • calibrated so that the difference between the laser frequencies of the beams matches the vibrational frequency of the molecule to be imaged

References



Textbook References

Molecular Biology of the Cell 4th ed. Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co. ; c1999

The Cell - A Molecular Approach Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc. ; c2000

PubMed

  • PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed
  • PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC
  • Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM
  • Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

Reviews

  • Structure and function of mammalian cilia. Satir P, Christensen ST. Histochem Cell Biol. 2008 Jun;129(6):687-93. Epub 2008 Mar 26. Review. PMID: 18365235


Articles

  • Klar TA, Jakobs S, Dyba M, Egner A, Hell SW. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8206-10. PMID: 10899992
  • D. Axelrod, Cell surface contacts illuminated by total internal reflection fluorescence, J. Cell Biol. 89 (1981), pp. 141–145 PMID: 7014571

External Links

2009 Course Content

Lectures

Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells | Stem Cells | Development | Revision

Laboratories

Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture 1 | Tissue Culture 2 | Microarray Lab visit

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G