Microscopes & Microscopy

Microscopes & Microscopy by C. Grier Sellers, M.S. Doctoral Candidate in Biology NSF GK12 Fellow Temple University

Basic Principles: What do Microscopes do? • Magnification: objects are made to appear larger than they are • Resolution: the ability to see close together objects as distinct • Microscopes are an important tool of biologists, and are used to study cells, tissues, and microorganisms

Different Kinds of Microscopes: • Compound Light Microscope (transmitted light) • Dissecting Light Microscope (reflected light) • Transmission Electron Microscope (transmitted electron beam) • Scanning Electron Microscope (reflected electron beam)

Measurement Units in Microscopy: • In light microscopy, objects are measured in micrometers (μm) • 1 μm = 1/1000th of 1 mm, or 1 x 10-6 meters • In electron microscopy, objects are measured in nanometers (nm) • 1 nm = 1/1000th of 1 μm, or 1 x 10-9 meters

Early Light Microscopes: http://micro.magnet.fsu.edu

A Compound Light Microscope: (with parts labeled)http://commons.wikimedia.org/wiki/File:Labelledmicroscope.gif

Lenses of the Compound Light Microscope & their Functions: - Oculars (eyepieces): provide some magnification, focus image at eye (can be 1 or 2 of them) • Objectives: located on rotating nosepiece, provide magnification and resolving power (may be 3 to 6 of them) - Condenser: located under stage, focuses light on the specimen. It may also contain an iris diaphragm that controls the size of the cone of light entering the condenser. (our ‘scopes don’t have a condenser)

Close-up of an Objective Lens: http://www.bhphotovideo.com/ • Example: 10x / 0.25 • 10x: means that it magnifies the object 10 times. • 0.25 is the Numerical Aperture (N.A.) of this objective. The higher the N.A., the greater the resolving power of an objective.

Total Magnification: • Easy to calculate ! • Simply multiply the ocular magnification by the magnification of the objective you are using • Example: Ocular magnification (15x) X Objective magnification (45x) = 675x • Hint: the magnification of each ocular and objective lens is usually written on it

Resolving Power: dmin = 0.61  / N.A. • dmin is the minimum distance between objects that can be seen as distinct (in μm) •  is the wavelength (for light, 380 -760 nm = 0.38 - 0.76 μm) • N.A. is the Numerical Aperture of the objective lens • Example: green light ( = 0.52 μm): dmin = (0.61)(0.52)/1.4 = 0.23 μm (Hence, the wavelength of light limits the resolving power of light microscopes !)

Resolving Power: • Human eye: about 0.2 mm • Compound Light Microscope: about 0.2 μm • Transmission Electron Microscope: about 0.2 nm

Greatest Useful Magnification: Compound Light Microscope: around 1500x Transmission Electron Microscope: around 250,000x (biologists usually use less) Although you could easily make lenses for light microscopes to magnify more, the images would be blurry due to the lower resolution

Contrast Enhancing Methods: • What is Contrast?: The apparent difference in brightness between objects • These methods are important for studying cells that lack much contrast, such as animal cells • Chemical Methods: • Stains (dyes) that color parts of cells (though many stains kill cells) • Optical Methods (can be used with living cells): − Closing down the iris diaphragm (but you lose some resolution) • Dark Field Illumination • Phase Contrast Microscopy • Differential Interference Contrast (DIC)

Cheek Cells: Bright Field Image (unstained): http://www.angelfire.com/de/nestsite/modbiolab02.html

Cheek Cells: Bright Field Image (stained): http://www.scuddlebutt3.co.uk

Cheek Cells: a) Bright Field & b) Phase Contrast & c) DIC Compared:

Phase Contrast Image: (Flagellate Protozoa from Termite Intestines)http://visualsunlimited.photoshelter.com/

Fluorescence & Microscopy: • Fluorescence: when a molecule is excited by light at a shorter wavelength, and emits light at a longer wavelength • Autofluorescence: some substances, such as chlorophyll, are naturally fluorescent • Fluorescence Microscope: a microscope that excites and detects fluorescent materials • Fluorescent Dyes: are widely used in biology to label cell organelles, molecules, and genes in cells

Chloroplast Autofluorescence: (Paramecium bursaria) http://starcentral.mbl.edu/

Confocal Laser Scanning Microscopy (CLSM): • The higher the magnification the less “depth of field” - only a narrow slice is in focus • The CSLM gets around this problem • Lasers scan the specimen at different depths • A computer reconstructs a clear 3D image • Usually used with fluorescent specimens

Confocal Scanning Laser Microscope (CLSM):http://pict-ibisa.curie.fr

Myrionecta rubra & Geminigera cryophila: a & b): Fluorescence images, c) TEM image, d) Confocal FISH image(Johnson et al., Nature, 445:426-428, 2007)

Transmission Electron Microscope (TEM): • Is capable of much higher resolution than the light microscope • An electron beam is transmitted through a very thin section • Instead of glass lenses, magnetic lenses are used, which bend and focus the electron beam much like a glass lens bends and focuses light • Specimen preparation is time-consuming • Only fixed and stained (dead) specimens can be examined

Transmission Electron Microscope (TEM): http://www.botany.unimelb.edu.au/botany/em/tem.html

Comparison of Light Microscope & TEM:http://www.lab.anhb.uwa.edu.au

TEM Image: (thin section of Myrionecta rubra) (Hansen & Fenchel, Mar. Biol. Res., 2: 169-177, 2006)

Scanning Electron Microscope (SEM): • Is capable of higher resolution than the light microscope • An electron beam is “bounced off” the specimen to a detector, instead of being passed through it • It produces a detailed image of the surface of the specimen, but not its internal structure

Scanning Electron Microscope (SEM): http://www.engr.uky.edu/emc/facilities/sem.html

SEM Image: (Emiliania huxleyi, a haptophyte alga)http://starcentral.mbl.edu/

Acknowledgements: Temple University “Scientists as Teachers / Teachers as Scientists Program” National Science Foundation GK12 Grant

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Microscopes & Microscopy