ch 2 microscopy Flashcards
light microscopes
first microscopes invented and still widely used today
resolution limit is 0.2um
refractive index
a measure of how much a substance slows the velocity of light
lenses
focus light rays at a specific place called the focal point
- distance between center of lens and focal point is the focal length
- short focal length = more magnification
objective lens
the lens closest to the specimen
forms a magnified image that is further enlarged by one or more additional lenses
bright-field microscope
produces a dark image against a brighter background
resolution
ability of a lens to distinguish two objects from each other, rather than a single larger object
shorter wavelength = greater resolution
numerical aperture
n sin theta
n = refractive index of the medium in which the lens works
theta = 1/2 the angle of the cone of light entering an objective lens
working distance
distance btw the front surface of lens and surface of cover glass or specimen when it is in sharp focus
dark-field microscope
produces detailed images of living, unstained cells and organisms by producing a bright image against a dark background
- used to observe living, unstained preparations
- used to observe internal structures in eukaryotic microorganisms
phase-contrast microscope
produces an image of a darker microbe against a lighter background
- observe microbial movement and detecting bacterial structures
- usually have a heated stage so the specimen doesn’t die
differential interference contrast microscope
creates image by detecting differences in refractive indices and thickness of different parts of the specimen
- used to observe cell walls, endospores, granules, vacuoles, nuclei
- produces a more 3D image
fluorescence microscope
produces image by exciting a specimen with a wavelength of light that triggers the object to emit fluorescence light
- stained with fluorochromes
- used for localization of specific proteins in cells
fluorochromes
absorbs light energy from the excitation light and emits visible fluorescent light that travels up through the objective lens into the microscope
confocal microscopy
uses a laser beam to illuminate fluorescently labeled specimen
- creates sharp, composite 3D image of specimens by using an aperture that eliminates stray light outside of the plane of focus
- used to study biofilms
fixation
the process by which the internal and external structures of specimens are preserved and fixed in position
heat fixation
preserves overall morphology but destroys subcellular structures
chemical fixation
protects fine cellular substructures and morphology
chromophore groups
chemical moieties with conjugated double bonds that give the dye its color
simple staining
a single stain is used
- used to determine size, shape, and arrangement of bacteria
differential staining
distinguishes organisms based on their staining properties
- used to detect presence or absence of structures
gram stain
most widely used differential staining procedure
positive - stay purple
negative - pink
acid-fast staining
used for staining members of the genus mycobacterium
- have cell walls with lipids that prevent dyes from binding to cells
loses stain = not acid fast
retains stain = acid fast
capsule staining
reveals the presence of capsules
- negative staining - capsule itself doesn’t get stained
flagella staining
revels presence and distribution pattern of flagella
electron microscope
electrons replace light as the illuminating beam that is focused
resolution is 0.2nm
transmission electron microscope (TEM)
uses a heated tungsten filament in the electron gun to generate a focused beam of electrons on the specimen
electrons that pass through the specimen form the image
- can only see very thin slices
shadowing
preparation method
specimen is coated with a heavy metal on one side
freeze-etching
rapidly freeze cells in liquid nitrogen
- allows for observations of shapes of intracellular structures
very brittle
scanning electron microscope (SEM)
produces a realistic 3D image of specimens surface features
uses electrons excited from the surface of a specimen to create detailed image
cryo-electron microscopy
used to visualize biomolecules and generate high resolution structures
samples are rapidly frozen, then multiple TEM images are captured at different angles
each protein gets a specific color
scanning probe microscopy
measures surface features of an object by moving a sharp probe over an objects surface
scanning tunneling microscope
view atoms on the surface of a solid
magnification of 100 million times
- type of SPM
atomic force microscope
up and down movement of sharp probe as it maintains constant distance from specimen
deflection of a laser beam that strikes the level holding the probe is measured
- used to image protein interactions
- used on surfaces that don’t conduct electricity well
- type of SPM
