2014 Lab 2

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

ASCB iBioSeminars

These are a series of online presentations describing a range of microscopy techniques.

• Fluorescence Microscopy
• Confocal Microscopy
• Introduction to Electron Microscopy
• TIRF microscopy

Light Microscopy

Transmission Microscopy

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

Phase Contrast Microscopy

Phase contrast optical pathway

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

• “phase halo” - can be either bright around dark objects or dark surrounding bright objects
• diffracted light passes through the phase ring as well as the nonphase areas and interacting at the image plane
• light diffraction and interference and not of the optical path of the sample

Now complete the exercise phase contrast microscope alignment

Links: MBoC Figure 9-8. Four types of light microscopy | Principles of Phase Contrast Microscopy | [Phase Contrast 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.

Links: DIC Microscopy

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)

Links: Fluorescence Microscope

Confocal Laser Scanning Microscopy (CLSM)

Confocal Microscopy 2 Methods

See also Laboratory 7 - Confocal Microscopy

• 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

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

Total Internal Reflection Fluorescence Microscope (TIRFM)

• 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
  • carbon dioxide level, either by a sealed tissue culture flask or gassed container.
  • light levels must be very low, or shuttered,
• 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 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

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"

New 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.

Superresolution Microscopy

Conventional light microscopy resolution is limited by the diffraction of light (Ernst Abbe, 1873).

Links: diffraction barrier

three-dimensional structured illumination microscopy (3D-SIM)

Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy

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)

• 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

Imaging Software

Originally images from microscopy were captured with the eye and interpreted by drawings based on the observed images. This imaging was then replaced by photographic techniques using cameras and film collection of images. Film cameras were gradually replaced by digital cameras as their resolution and sensitivity increased and these could also be programmed to collect image sequences or video to capture timelapse information.

These collected images can then be analysed by a range of different software packages. Microscope manufacturers and independent software developers have now created a range of image analysis software packages. Note that some microscope and camera manufacturers have developed modified image formats that have been tailored to their own software that allow saving of microscope and camera settings at image collection.

Many high resolution charge-coupled device (CCD) scientific cameras collect greyscale images and use filters or look up tables (LUT) to convert to colour images. Some CCD cameras are colour, generally using a 3 colour CCD detector.

• Fluorescent images are best viewed and compared using greyscale, as this removes the eye's varied sensitivity to different colours.
• For scientific imaging it is important that the original image and any modifications carried out on that image are carefully identified.
• "Photoshopping" of scientific imaging, other than for layout and labeling, can be an unethical modification of data.

Links: JCB - What's in a picture? The temptation of image manipulation | NYT - Clone Scientist Relied on Peers and Korean Pride | NIH - Image J

Image Formats

The resolution and sensitivity of cameras has significantly improved in the last 10 years, with this has come a huge increase in image file sizes. The bit depth and format of how an image is collected and saved is important to how the image will be later be analysed. Many image formats compress files to reduce their size, and some compression algorithms are "lossless" while others are "lossy".

• Lossless formats will allow measurement of specific pixel "values" and comparison with other similarly collected images.
• Lossy formats may modify specific pixel values or pool image regions.

Tagged Image File Format (TIFF, TIF)

• can saves 8 bits or 16 bits per color (red, green, blue) for 24-bit and 48-bit totals
• can be either lossy and lossless
  • LZW compression algorithm is used for lossless storage
• image format is not supported by all cameras and web browsers

Joint Photographic Experts Group (JPEG, JPG, JFIF)

• is a lossey image compression method
• supports 8 bits per color (red, green, blue) for a 24-bit total, producing relatively small files
• files suffer generational degradation when repeatedly edited and saved

Portable Network Graphics (PNG)

• free open-source successor to the GIF
• format supports truecolor (16 million colors)
• can be either lossy and lossless
• useful for images has large uniformly colored areas

Graphics Interchange Format (GIF)

• limited to an 8-bit palette, or 256 colors
• can be either lossy and lossless
• not suitable for scientific images but for graphics or simple diagrams
• format supports animation

Windows bitmap (BMP)

• handles graphics files within the Microsoft Windows OS
• files are uncompressed and can be large

Flexible Image Transport System (FITS)

• digital file format used to store, transmit, and manipulate scientific and other images
• most commonly used digital file format in astronomy Wiki - FITS

Digital Imaging and Communications in Medicine (DICOM)

• standard for handling, storing, printing, and transmitting information in medical imaging
  • developed by American College of Radiology (ACR) and National Electrical Manufacturers Association (NEMA)
• enables integration of scanners, servers, workstations, printers, and network hardware from multiple manufacturers into a picture archiving and communication system (PACS)

Lab 2 Individual Assessment

1. On your own student page and the Lab 1 uploaded image update the reference using the Pubmed formatting shown in the practical class tutorial (see also 2013 Group Test Project).
2. Identify a recent research article (not review) that uses either confocal microscopy or super-resolution microscopy as one of the study's techniques. Explain briefly (1 paragraph) how the microscopy technique specifically contributed to the article's findings.

ASCB iBioSeminars

These are a series of online presentations describing a range of microscopy techniques.

• Fluorescence Microscopy
• Confocal Microscopy
• Introduction to Electron Microscopy
• TIRF microscopy

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

• MBoC 9. Visualizing Cells
• MBoC Figure 9-8. Four types of light microscopy
• Search MBoC for microscopy

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

• 5.1. Microscopy and Cell Architecture
• Search MCB for microscopy

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

• Tools of Cell Biology
• Search The Cell for microscopy

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

• "microscopy" Entrez all databases
• "microscopy method" Entrez all databases

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

External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.

• Cell Image Library
• History History of Microscopy
• Scanning Electron Microscopy The Early History and Development of The Scanning Electron Microscope
• Journal Microscopy Today
• Light Microscope Images UWA Blue Histology
• Electron Microscope Images Dennis Kunkel's Microscopy
• Cellular Imaging Microscopy & Imaging Resources on the WWW K-12 Educational Resources
• WWW Virtual Library Microscopy
• Nature Specials Microscopy
• Journal Cell Biology Education
• Protocols Online Cell Biology
• Molecular Expressions Optical Microscopy Resources on the Web | Laser Scanning Confocal Microscopy
• Introduction to Confocal Microscopy Nikon's MicroscopyU
• Wiki TIRF
• Introduction to confocal and fluorescence microscopy Emory’s Physics Department
• Confocal high res images The Science Creative Quarterly's overview of confocal microscopy
• Confocal Microscope Capabilities Programmable Array Microscope
• Physics Department, University of Michigan Daniel Axelrod
• University of Arizona Fluorescent Spectra

2014 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 - Intermediate Filaments | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | 2013 Revision | Development | 2014 Revision

Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Tissue Culture | Microarray Visit | Stem Cells Lab

2014 Projects: Group 1 | Group 2 | Group 3 | Group 4

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