Lecture 3 Flashcards
Why is staining important
improves CONTRAST between different cells/features
resolution
The ability to distinguish between two adjacent objects as distinct and separate
resolution is a function of..
the physical properties of light
resolution is determined by..
the wavelength of light used and numerical aperture of lens
resolution is _________________ to wavelength
inversely proportional
shorter wave length =
better resolution
Limit of resolution for light microscope is about…
o.2 um
Compound light microscopes use….. to illuminate cells
visible light
4 types of light microscopy
- bright-field
- phase contrast
- dark-field
- fluorescence
bright field microscopy
specimens are visualized because of differences in contrast (density) between specimen and surroundings
total magnification =
objective magnification x ocular magnification
Maximum useful magnification is…
~2000x
Phase-Contrast Microscroscopy was invented in 1936 by…
Frits Zernike
Phase Contrast Microscopy
- amplifies differences in the refractive index of cell and surroundings;
- improves contrast without use of stain
- allows for visualization of live samples
- dark cells on a light background
dark field microscopy
light reaches the specimen from the sides
- good for observing motility
- appears light on a dark background
fluorescence microscopy
used to visualize specimens that fluoresce
- may fluoresce naturally of after staining
- widely used for enumerating bacteria
differential interference contrast microscopy
uses a polarizer to create two distinct beams of polarized light
-gives structures such as endospores, vacuoles, and granules a 3D appearance
atomic force microscopy
-a tiny styles is placed close to a specimen and it measures weak repulsive forces between it and the specimen
confocal scanning laser microscopy
-computerized fluorescence microscope coupled with a laser source to generate a 3D image
electron microscopy
electron microscopes use electrons instead of photons to image cells and structures
transmission electron microscopy
operates in a vacuum and is used to examine cells and cell structure at very high magnification and resolution
- visualization at a molecular level
- specimen must be very thin and stained
scanning electron microscopy
for observing external features
-specimen is coated with a thin film of heavy metal and an electron beam scans object to form image
morphology
cell shape
coccus
spherical
