Microscopy

UNITS OF MEASUREMENT 1000 mm (millimeters) in 1 meter (m) 1000 µm (microns) in 1 mm Bacteria are about 1µm or smaller 1000 nm (nanometers) in 1 µm Viruses are about 1nm 1000 viruses can fit into one bacterium Protozoa are fairly large single-celled animals. You can see them with the naked eye. That is called being “macroscopic” Bacteria are so small, they are measured in µm. They are the smallest things you can see under a microscope with the oil immersion lens. Viruses are even smaller, so they are measured in nm.

Limits of Resolution 3 Figure 4.3

VOCABULARY Immersion oil: keeps light from bending and allows lens to be refracted. Parfocal: focused in all lenses. Depth of field: how much of the background is in focus at the same time that the foreground is in focus.  Resolution: ability of two lenses to distinguish two points. RESOLUTION: the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.4 nm can distinguish between two points greater than or equal to 0.4 nm. When you go to the eye doctor, you look at the chart (Snellen chart) and read it from 20 feet away. If you can read what a normal sighted person can read from 20 feet away, it is called 20/20 vision. If you can’t read it well, your eyesight has less resolution than normal.

TYPES OF MICROSCOPES SIMPLE MICROSCOPE: Has only one lens, like an ocular (eyepiece) COMPOUND MICROSCOPE: More than one lens, like an ocular and an objective. An example is the Brightfield microscope. There are two main types of compound microscopes: Light Microscopes and Electron Microscopes.

SIMPLE MICROSCOPE

COMPOUND MICROSCOPE: One Eyepiece

COMPOUND MICROSCOPE: Two Eyepieces

Types of Compound Microscopes Dissecting Brightfield Darkfield Phase-contrast Differential Interference contrast Fluorescence

Dissecting Microscope: For looking at large objects

BRIGHTFIELD ILLUMINATION: For live organisms with no stain

BRIGHTFIELD ILLUMINATION: Can also use for stained tissues

DARKFIELD ILLUMINATION For live organisms with no stain

DARKFIELD ILLUMINATION For fluorescent organisms

Brightfield vs Darkfield

PHASE CONTRAST MICROSCOPY For seeing organelles in live organisms

DIFFERENTIAL INTERFERENCE CONTRAST For seeing organelles in live organisms in three dimensions

DIFFERENTIAL INTERFERENCE CONTRAST

DIFFERENTIAL INTERFERENCE CONTRAST

Fluorescence Microscopy Cells are stained with fluorescent dyes called fluorochromes. UV Light is shined on the specimen. Fluorescent substances absorb UV light and emit visible light. Figure 3.6b

FLUORESCENCE MICROSCOPY

FLUORESCENCE MICROSCOPY

Transmission Electron Microscope Much higher resolution than microscopes. Can only be used on dead cells

Transmission Electron Microscope

Transmission Electron Microscope: Inside of a Plant Cell

Scanning Electron Microscope Very high resolution like a Transmission Electron Microscope except makes images in three dimensions.

Scanning Electron Microscope

Scanning Electron Microscope: Flea

Scanning Electron Microscope: Pollen

Scanning Probe Microscope A scan is passed over the specimen, line by line. The surface dimensions are recorded and sent to a computer, which shows the image in false color.

Scanning Probe Microscope: Red Blood Cells

Scanning Probe Microscope: Chromosomes

COMPARISON OF MICROSCOPES BRIGHTFIELD Dark objects are visible against a bright background. Light reflected off the specimen does not enter the objective lens Not for looking at live cells Maximum resolution is 0.2µm and maximum magnification is 2000x Stains are used on specimens DARKFIELD Light objects are visible against dark background Used for live cells, cilia, flagella Especially good for spirochetes Uses special condenser with an opaque disc that eliminates all light in the center PHASE-CONTRAST No staying required Accentuates diffraction of the light that passes through a specimen Good for live cells; good contrast Most sensitive; cilia shows up Not three-dimensional DIFFERENTIAL INTERFERENCE CONTRAST Uses two beams of light Shows three dimensions Has a prism to get different colors Good for live cells (unstained) Best resolution

COMPARISON OF MICROSCOPES FLUORESCENCE Uses ultraviolet light Stained cells with fluorescent dye; energizes electrons and creates visible light No live cells Quick diagnosis of TB and syphilis TRANSMISSION ELECTRON Get flat images Have vacuum pumps to allow electrons to float better Stain with heavy metal salts Shows sections of cell, revealing organelles Requires an ultramicrotome Best resolution of all microscopes SCANNING Surface view only Needs a vacuum Three-dimensional view SCANNING PROBE Physical probe scans the specimen Raster scan: image is cut up into pixels and transmitted to computer Not limited by diffraction Slower in acquiring images Maximum image size is smaller

For the next 20 minutes…. Everyone who does not have a lab group, stand to the side of the room. Decide what people to join with to form a lab group. Discuss ideas for your group project. How will you conduct it? What supplies will you need, and how will you measure the results? Will you count how many of each different colony you get?