Lecture 4: Microscopy Flashcards
Understand the difference between resolution and contrast.
Resolution is the ability to distinguish 2 objects based on distance. Very specific to distance, not if you can distinguish the objects better.
Contrast ability to distinguish 2 objects based on intensity (shading/coloring).
Explain how resolution and contrast can be altered to improve the quality of an image.
Contrast can be altered to improve the quality of an image by staining the specimen or background with color, using different angle of illuminating light (dark field microscope), change light phase (phase contrast microscope and fluorescence).
Resolution can be altered to improve the quality of an image by using a lower wavelength of light (blue). Increase the numerical aperture (adding immersion oil causes less detracted light).
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Light Microscopes- Bright Field
Light passes evenly through the specimen. It is the most commonly used type of microscope.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Light Microscopes- Dark Field
It has a special condenser that blocks direct light. Specimen is illuminated at an angle, and viewed by reflected light. Used to illuminate very thin bacteria, or other unstained specimens that stand out better against a dark background.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Light Microscopes- Phase Contrast
The phase plate retards some light and advances other light so that refractive index differences are amplifies. It is especially useful for viewing the very slight contrast differences between internal structures.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Light Microscopes- Differential Interference Contrast (DIC, Normarski)
Light is separated by a Wollaston prism and polarized. To 90 degree polarized light beams pass simultaneously through the specimen and recombine. It is useful for viewing protozoans so that they appear to be 3D.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Light Microscopes- Flurescence
UV light strikes a fluorescently labeled specimen, which emits light of a visible wavelength. It is useful when specimens are stained with fluorescent dyes (ex. species-specific dyes linked to antibodies).
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Light Microscopes- Confocal Scanning Laser
A laser is scanned across a specimen. Light from all but a thin plane is blocked by an adjustable pinhole. Images from many planes are combined by computer. It is used to view thick specimens and still keep ll parts of the specimen in focus.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Electron Microscope- Transmission Electron Microscope
Like the bright field light microscope, except illumination used electrons (shorter wavelength) rather than light. It takes high resolution pictures of very thin specimens at high magnification.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Electron Microscope- Scanning Electron Micrscope
Electron beam scans across a specimen. Reflected electrons and X-rays are detected to produce a 3D image. High resolution pictures of prepared, fixed surfaces at variable magnification.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Electron Microscope- Electron Cryotomography
Multiple TEM images are taken from different angles and combined to produce a 3D internal image. Extremely high resolution of internal cell structures in 3D.
Be able to describe the basic function (how they work) and microbiological use (what you would use them to see) of the following types of microscopes: Atomic Force Microscope
Electromechanical probe scans across a surface, deflecting as it encounters electron clouds around atoms and molecules. It is used to visualize surfaces at atomic resolution. Also used to push atoms and molecules around on a surface.
Understand the difference between a simple stain, a differential stain, and a negative stain.
Simple Stain: Individual cell; dye (charged molecule with color), oppositely charged from the structure.
Differential Stain: Using multiple simple stains in a particular order (peptidoglycan), acid-fast: tells if their is mycelia acid in the outer membrane.
Negative Stain: Stains background, ink stain (particles that are not charged).
