Measurement & Microscopes

Sizes of Things Size relative Prefix Meaning to a meter Length Kilo- thousand 103 kilometer (km) Centi- hundredth 10-2 centimeter (cm) Milli- thousandth 10-3 millimeter (mm) Micro- millionth 10-6 micrometer (µm) Nano- billionth 10-9 nanometer (nm) 4 mm 2 mm 200 µm With the unaided eye, you can see down to about 200 µm (that’s 0.2 mm). With a quality light microscope, you can see something as small as 200 nm clearly (that’s 0.0002 mm). With an electron microscope, you can visualize something as small as 0.2 nm clearly (that’s 0.0000002 mm or otherwise stated, 2x10-7 mm). 200 nm Check out: http://www.cellsalive.com/howbig.htm

Sizes of Things & Photomicrography Check out: How Big? http://www.cellsalive.com/howbig.htm Scale of the Universe http://htwins.net/scale2/ Olympus Bioscapes Competition winners http://www.olympusbioscapes.com/gallery/2012/ Nikon Small World Competition (photo & video) http://www.nikonsmallworld.com/

Microscopes: Tools of Biologists Different microscopes have different advantages and disadvantages. Biologists use different microscopes depending upon what they want to look at / observe. ________________________: also called a stereo microscope 2 eyepieces 2 lenses, __________ and ___________ (in the eyepiece) 2 lamps, one above and one below the specimen Advantages: Specimen can be alive No special preparation of specimen needed Easy to use! Relatively inexpensive Disadvantages: Low magnifying power (up to 60x usually) Dissecting Microscope objective ocular lens

Compound Light Microscope ______________________________ 2 lenses, __________ and ___________ (aka eyepiece) On student ‘scope, usually have 10x ocular lens & several objective lenses: 2x (scanning), 10x (low power), & 40x (high power) Sample often placed in _______ on a slide Some specimens are stained or “fixed” with dyes -- this enhances contrast & helps preserve specimen (but kills it too) Light passes _________ sample and is “bent” by lenses to produce a magnified image Advantages: Specimen can be alive Easy specimen preparation Relatively easy to use Fairly inexpensive for a basic ‘scope Can magnify up to _______ clearly Disadvantages: Cannot see anything smaller than ______________ (note: a small cell is about 10 µm across) objective ocular lens water through 1000x 0.2 µm (200 nm)

Modern Optical Microscopes with Digital Camera attachments http://www.microscopyu.com/museum/e600pol.html http://www.microscopyu.com/museum/smz1500.html

Transmission Electron Microscopy http://en.wikipedia.org/wiki/Transmission_electron_microscopy http://www.fei.com/products/tem/tecnai/

TEM Specimen Preparation In TEM, the specimen must be thin enough to allow electrons to travel through the tissue. You can cut very thin slices of your specimen either by fixing it in plastic or freezing it. The specimen is cut into thin sections by a diamond knife in an instrument called ultramicrotome. Each section is only 50-100 nm thick. A thin section of your sample is placed on a copper grid and stained with a heavy metal, like uranium & lead, which scatters electrons well and improves the contrast in the microscope. The slice of tissue can now be studied under the electron beam. Alternately, a solution of isolated material (can be a solution with bacteria or a solution with isolated molecules) is spread on a support grid coated with plastic. A solution of heavy metal salt is added. The metal salt solution does not bind to the material but forms a "shadow" around it on the grid. The specimen will appear as a negative picture when viewing it in the TEM. An ultra-microtome slices samples as fine as 70 nm thin so they can be viewed in a transmission EM. http://www.eicn.ucla.edu/UTCultramicrotome http://www.nobelprize.org/educational/physics/microscopes/tem/preparation.html http://en.wikipedia.org/wiki/Transmission_electron_microscopy

Scanning Electron Microscopy http://www.nist.gov/cnst/nanofab/toolset-metrology.cfm http://en.wikipedia.org/wiki/Scanning_electron_microscope

• Electron Microscopes Sends beam of _________ over or through specimen This beam is bent by large _______________ to produce a magnified image There are two main types of electron microscopes: _____________ Electron Microscope (TEM) Beam of electrons passes ________ an ultra-thin section of a specimen and onto a fluorescent screen or photographic film Images produced are ________________ of specimens __________ Electron Microscope (SEM) Beam of electrons passes over the _______of a specimen, electrons bounce off at different angles & are “read” to produce image Images are ____ pictures of the ________ of samples (no inside views) electrons electromagnets Transmission through cross-sections Scanning surface 3D surface

TEM ____ Micrographs Pancreatic cell Golgi body Herpes virus http://en.wikipedia.org/wiki/Transmission_electron_microscopy Bacillus subtilis bacterium http://www.histology-world.com/photoalbum/displayimage.php?album=55&pid=509#top_display_media Herpes virus Nucleus surrounded by rER Mitochondria (in lung cells) http://www.histology-world.com/photoalbum/thumbnails.php?album=55&page=2

____ Micrographs SEM Hair shaft Can you guess what this is? http://micro.magnet.fsu.edu/primer/java/electronmicroscopy/magnify1/index.html ____ Micrographs SEM Hair shaft Can you guess what this is? Ebola Viruses (one w/ false color) The fossilized shell of a microscopic ocean animal, magnified 392 times its actual size. The ancient creature, called Radiolarian, lived in the waters off Antarctica and is now used to study such things as climate and ocean circulation. http://bepast.org/dataman.pl?c=lib&dir=docs/photos/ebola/

What is this? http://www.aecom.yu.edu/aif/gallery/sem/sem.htm

White blood cell (B-cell) with viruses budding out of it. http://www.aecom.yu.edu/aif/gallery/sem/sem.htm

TEM (above) and SEM (below) of viruses budding from a mammalian cell http://www.aecom.yu.edu/aif/gallery/sem/sem.htm

• Electron Microscopes Advantages: Can be used to view smaller things like ________ and _____________________ that cannot be seen with light ‘scopes Can magnify over 100,000x and allows you to see objects as small as _______ Disadvantages: Preparation of specimen can be _________ Ex) Cannot view living organisms Much more complex & difficult to use/operate than light ‘scope Extremely expensive to buy and maintain Only produces ______________ images (any color electron micrographs have false color added afterwards by computer) viruses sub-cellular organelles 0.2 nm difficult Freezing or embedding in resin, slicing ultra-thin sections black & white

Virtual Electron Microscopy http://micro.magnet.fsu.edu/primer/java/electronmicroscopy/magnify1/index.html