microscopy Flashcards
chapter 2
what does visible light consist of
electromagnetic waves that behave like other waves
what is a wavelength
the distance between one peak of a wave and the next peak
what is amplitude
the height of each peak or depth of each trough
what is frequency
the rate of vibration of the wave or the number of wavelengths within a specified time period
when does reflection occur
when a wave bounces off of a material.
what is absorbance
when material captures the energy of a light wave
what is transmission
when a wave travels through a material, like light through glass
what is interference
creating complex patterns of motion
what is diffraction
when light waves interact with small objects or openings by bending or scattering
when is diffraction larger
when the object is smaller relative to the wavelength of the light
when does refraction occur
when light waves change direction as they enter a new medium
what is refractive index
the extent to which a material slows transmission speed relative to empty space
what does large differences between refractive indices of two material result in
a large amount of refraction when light passes from one material to the other
what happens when light crosses a boundar
into a material with a higher refractive index
its direction turns to be closer to perpendicular to the boundary
what is the principle behind lenses
refractive index
how can you think of a lense
an object with a curved boundary that collects the light that strikes it and refracts it so it all meets at the image point
why can a convex lens be used to magnify
it can focus at a closer range than the human eye producing a larger image
how are concave lenses and mirrors used in microscopes
to redirect light the light path to the focal point
what is the focal length
distance to the focal point
what is the relationship between higher wavelengths and shorter frequency
more oscillations per unit time than lower-freqeuncey waves
how can white light be separated into its component colors
refraction
what is dispersion
separation of colors in the rainbow like spectrum
What is phosphorescence
when photons are emitted following a delay after absorption
what is magnification
the ability of a lens to enlarge the image of an object when compared to the real object
what is resolution
the ability to tell that two separate points or objects are spearate
what affects resolution
wavelength
numerical aperture
how does wavelength affect resolution
shorter wave lengths resolve smaller objects
what is numerical aperture
the measure of a lens ability to gather light the higher the better the resolution
what is leeuwenhoek referred to as
the father of microbiology
what type of microscope did Galileo use
compound to inspect insect parts
what kind of microscope did Leeuwenhoek use
simple microscope
how many lenses in a compound microscope
2
how many lenses in a simple microscope
1
what type of microscope did Hooke use
compound
what did Hooke look at through his microscope
cork cells
what did lister create
modern light microscope
what are examples of light microscopes
brightfieqld microscope
dark field microscope
phase contrast microscope
differential interference contrast microscopes
fluorescence microscope
confocal scanning laser microscopes
two photon microscopes
what is a brightfield microscope
a compound microscope with 2 or more lenses that produce a dark image on a bright background
where is the ocular lens
in each eyepiece
what is the magnification of the ocular lens
10
how do you calculate total magnification
the product of the ocular magnification times the objective magnification
what is the stage
where the specimen is placed
how is the specimen positioned over the light
x-y mechanical stage knobs
what is the coarse focusing knob used for
large scale movements
what is the fine focusing knob used for
small-scale movements
why do images become dimmer when magnified
less light per unit of area of image
what lights up a microscope
illuminator
what does light from the illuminator pass through
condenser lens
what does a condenser lens do
focuses all of the light onto the specimen to maximize illumination
how can the amount of light hitting specimen be adjusted
opening or closing a diaphragm
what are chromophores
pigments that absorb and reflect particular wavelengths of light
why is resolution compromised at higher magnification
the large difference between the refractive indices of air and glass, air scatter the light before they can be focused by the lens
how is refractive index fixed at large magnifications
using an oil immersion lens
how does an oil immersion lens help
oil has a refractive index similar to glass increasing maximum angle at which light can strike the lens
what is a dark field microscope
a brightfield microscope with an opaque disk between the illuminator and the condenser lens
what does the opaque light stop do
blocks most light from the illuminator producing a hollow cone of light that is focused on the specimen showing bright objects on a dark background
what are the benefits of darkfield microscopy
it can create high-contrast, high resolution images of specimens without the use of stains
What do phase-contrast microscopes do
use refraction and interference caused by structures in a specimen to create high contrast, high resolution images without staining.
how do phase-contrast microscopes work
by altering wavelengths of light rays passing through the specimen to create altered wavelength paths.
what is in the condenser of a phase contrast microscope
an annular stop
what does an annular stop do
produces a hollow cone of light that is focused on the specimen before reaching the objective lens
what does the objective lens contain in a phase contrast microscope
a phase plate with a phase ring
what does the phase rang cause
light traveling directly from the illuminator passes through the the place causing waves traveling through to be one-half of a wavelength out of phase with those passing through the plate.
what is a differential interference contrast microscope
similar to phase-contrast in that they use interference patterns to enhance contrast
what happens in a DIC microscope
two beams of light are created in which the direction of wave movement differs,
what are the benefits of a DIC microscope
it gives high contrast images of living organisms in a three-dimensional appearance, they allow you to structure in live unstained specimens
how does a fluorescence microscopework
it uses fluorochromes that are capable of absorbing energy from a light source and then emitting this energy as a visible light.
what does a fluorescence microscope transmit
an excitation light generally a form of EMR with a shot wavelength toward the specimen, chromophores absorb the excitation light and emit visible light with longer wavelengths
what are fluorescence microscopes useful for
identifying pathogens or the locations of particular molecules and structures within a cell and to determine living from dead cells
what is immunofluorescence used for
identifying certain disease causing microbes by observing if abx bind to them
what does a confocal microscope do
uses a laser to scan multiple z-plances successively
what does a confocal microscope produce
numerous two-dimensional images that can be constructed into a three dimensional image
what are confocal microscopes are useful for
examining thick specimens such as biofilms, which can be examined alive and unfixed.
what does a two-photon microscope do
uses a scanning technique, fluorochromes, and long-wavelength light to visualize specimens
what is a two-photon microscope useful for
examining living cells within intact tissues
what does an electron microscope do
uses short-wavelength electron beams rather than light to increase magnification and resolution
how much can an electron microscope magnify
up to 100,000x
what can an electron microscope be used for
sub cellular structures such as single strands of DNA
what are the two types of electron microscopes
transmission electron microscope
scanning electron microscope
can an electron microscope be used on living cells
no
what are the basic types of preparation for a light microscope
wet mount and fixed mount
what is a wet mount
when the specimen is placed on a slide in a single drop of liquid, includes urine
what is fixation
attaching cells to slide through heat or chemical treating.
what happens to a fixed specimen
the microorganism is killed while preserving the integrity of cellular components
how is a sample heat fixed
it is spread on the slide and briefly heated over a heat source
when are chemical fixatives preferred
for tissue specimens
what chemicals are used to chemically fix
a chemical agent such as acetic acid, ethanol, methanol, or formaldehyde
what happens to a chemically fixed specimen
it kills microorganisms in the specimen stopping degradation of tissues and preserving their structure
what do stains and dyes contain
salts with a positive ion or the negative ion
what is a positive ion classified as
basic dye
what is a negative ion classified as
acidic dye
what is a positive stain
a dye that will be absorbed by the cells or organisms being observed adding color to objects of interest to make them stand out against the background
what is a negative stain
absorbed by the background but not the cells producing an outline or silhouette of the organisms against a colorful backgroiund
what is the typical charge of a cell wall
negative
what normally sticks to cell walls
basic dyes making them positive stains
what are the commonly used basic dyes
basic fuchsin
crystal violet,
malachite green
methylene blue
safranin
what are common acidic dyes
acid fuchsin
eosin
rose bengal
what happens in simple staining
a single dye is used to emphasize a particular structure, all of the organism appear to be the same color even if more than one type of organism
what does differential staining do
distinguishes organisms based on interactions with multiple stains
what are types of differential stains
gram staining
acid-fast staining
endospore staining
flagella staining
capsule staining
what does gram staining do
differentiates between bacteria with different types of cell walls
what is the first step in a gram stain
crystal violet a primary stain is applied to a heat fixed smear giving it a purple color
what is the second step in a gram stain
grams iodine a mordant is added
what is a mordant
a substance used to set or stabilize stains or dyes
what does grams iodine do in gram staining
acts like a trapping agent that complexes with crystal violet making a complex clump and stay contained in thick layers of peptidoglycan in the cell walls
what is the 3rd step in gram staining
a decolorizing agent is added
what is the decolorizing agent
ethanol or acetone
what happens when decolorizing agent is applied
in cells with a thick peptidoglycan layers it stays purple
in thick walls the purple is washed away making them colorless
what is the last step in gram staining
secondary counterstain is aded staining decolorized cells pink
what is the counter stain
safranin
what are the purple, stains cells referred to as
gram positive
what are the red safranin dyed cells called
gram negative
what can affect gram stain results
older bacterial cells may have weak cell walls showing false gram negative
errors such as leaving decolorized on too long
what is the clinical significant of gram staining
helps classify bacterial pathogens to treat
what is acid fast staining able to do
differentiate between two types of gram positive cells, those with a waxy mycolci acid and those that do not
what are the methods for acid fast staining
ziehl-neelsen technique and the kinyoun technique
what is the primary stain in acid fast staining
carbolfuchsin
how do cells react to carbolfuchsin
waxy cells retain even after decolorizing
what is applied to non acid fast stains
a secondary counterstain methylene blue
what is the difference between acid fast staining techniques
whether heat is used during the primary staining process (ziehl-neelsen method)
what makes acid fast important
acid fast bacterial ill show red or pink on a blue background of surrounding tissue cells
what happens in capsule staining
a negative stain is used
does capsule staining need to be heat fixed
no
what are endospores
structures produced by bacterial cells that allow them to survive harsh conditions
how do endospores appear during gram staining
clear
what does endospore staining do
uses two stains to differentiate endospores from the rest of the cell.
what happens in endospore staining
heat is used to push the primary stain into the endospore, endospore retains the green stain after decolorization. it is then counterstained with safranin
what does flagella staining do
thickens flagella by applying mordant which Coates the flagella then is stained with pararosaniline
how are cells cut without damage
by being embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions
