Module 02: Microscopy Flashcards
What are the parts of a light microscope?
(1) Bright field microscopes
(2) Darkfield microscopes
(3) Phase-contrast microscopes
(4) Differential interference contrast (DIC) microscopes (also known as Nomarski optics)
(5) Fluorescence microscopes
(6) Confocal scanning laser microscopes
(7) Two-photon microscopes
This type of microscope uses electron beams that pass through a specimen to visualize small images; useful to observe small, thin specimens such as tissue sections and subcellular structures.
Transmission Electron Microscope
This type of microscope uses electron beams to visualize surfaces; useful to observe three-dimensional surface details of specimens.
Scanning Electron Microscope
This type of microscope uses electron beams focused with magnets to produce an image.
Electron Microscope
What is the magnification of an electron microscope?
20-100,000x or more
This type of light microscope is commonly used in wide variety of laboratory applications as the standard microscope; it produces an image on a bright background.
Brightfiled Microscope
This type of light microscope increases contrast without staining by producing a bright image on a darker background, especially useful for viewing live specimens.
Darkfield Microscope
This type of light microscope uses refraction and interference caused by structures in the specimen to create high-contrast, high-resolution images without staining, making it useful for viewing live specimens and structures such as endospores.
Phase Contrast Microscope
This type of light microscope uses fluorescent stains to produce an image; can be used to identify pathogens, to find particular species, to distinguish living form dead cells, or to find locations of particular molecules within a cell.
Fluorescence Microscope (or Immunofluorescence)
This type of light microscope uses a laser to scan multiple z-planes successively, producing numerous 2-dimensional, high-resolution images at various depths that can be constructed into a three-dimensional image by a computer, making this useful for examining thick specimens.
Confocal Microscope
This type of light microscope uses a scanning technique, fluorochromes and long-wavelength light (such as infrared) to penetrate deep into thick specimens such as biofilms
Two-photon microscope
This uses visible or ultraviolet light to produce an image.
Light Microscope.
This microscope uses very short probes that are passed over the surface of a specimen and interact with it directly
Scanning Probe Microscope
What is the calculated magnification of scanning probe microscopes?
100-100,000,000x or more
This scanning probe microscope uses a probe passed horizontally at a constant distance just above the specimen while the intensity of the current is measured, can map the structure of surfaces at the atomic level; works best on conducting materials but can also be used to examine organic materials such as DNA, if fixed on a surface.
Scanning Tunneling Microscope (STM)
This scanning probe microscope is used in several ways including a laser focused on a cantilever to measure the bending of the tip or a probe passed above the specimen while the height needed to maintain a constant current is measured; useful to observe specimens at the atomic level and can be more easily used with nonconducting samples.
Atomic Force Microscope (AFM)
What are the components of a Brightfiled Microscope?
(1) Oculars (10X)
(2) Body Tube
(3) Arm
(4) Coarse Adjustment Knob
(5) Fine Adjustment Knob
(6) Power Cord
(7) Base
(8) Power Switch/Light Adjustment Knob
(9) Mechanical Stage Knobs
(10) Light Source or illuminator
(11) Condenser with Iris Diaphragm
(12) Mechanical Stage
(13) Objectives Lenses:
(a) Scanning Power (red 4X)
(b) Low Power (yellow 10X)
(c) High Power (blue 40X)
(d) Oil Immersion (white 100x; not always present)
(14) Nosepiece
(15) rheostat
This part of a Brightfiled Microscope is used to hold multiple objective lenses.
Revolving Nosepiece
This part of a Brightfiled Microscope is used to move the slide.
X-Y mechanical stage knobs
This part of a Brightfiled Microscope is used to adjust the light.
Rheostat
This part of a Brightfiled Microscope is used to hold the specimen.
Stage
This microscope renders a darker image on a lighter background, producing a clear image of these Bacillus anthracis cells in cerebrospinal fluid (the rod-shaped bacterial cells are surrounded by larger white blood cells)
Brightfield Microscope
This microscope increases contrast, rendering a brighter image on a darker background, as demonstrated by this image of the bacterium Borrelia burgdorferi, which causes Lyme disease.
Darkfield Microscope
This is the product of the ocular magnification times the objective magnification:
Total Magnification
How do you solve for the total magnification?
Total Magnification = ocular magnification × objective magnification
For example, if a 40× objective lens is selected and the ocular lens is 10×, the total magnification would be (40×)(10×)=400×
These objective lenses are used to improve resolution.
Oil Immersion objective lenses
Why are Oil Immersion objective lenses used to improve the resolution of the specimen?
Because immersion oil and glass have very similar refractive indices, there is a minimal amount of refraction before the light reaches the lens. Without immersion oil, light scatters as it passes through the air above the slide, degrading the resolution of the image.
This microscope allows us to view living, unstained samples of the spirochete Treponema pallidum.
Darkfield Microscope
This microscope is comparable to the darkfield microscope because spirochetes appear bright against a dark
background.
Photographic negative Microscope
This is inserted into a brightfield microscope is used to produce a darkfield image.
Opaque Light stop
Why is the Opaque Light stop inserted in a light microscope?
The light stop blocks light traveling directly from the illuminator to the objective lens, allowing only light reflected or refracted off the specimen to reach the eye.
What do unstained cells look like under a brightfield microscope?
The unstained cells in the brightfield image are almost invisible against the background,
What do unstained cells look like under a phase-contrast microscope?
They appear to glow against the background, revealing far more detail.
The the irregular item above the cells is ___________.
acellular debris
What are the phase differences of a phase-contrast microscope?
(1) Annular stop in the condenser produces a cone of light focused on the specimen.
(2) Object or specimen refracts or reflects light
(3) Light traveling directly from the condenser lens and light traveling from the specimen are out of phase when they pass through the objective and phase plates
(4) Wavelengths in phase or out of phase either add together or cancel out each other.
The differences of a phase-contrast microscope is produced by what?
These differences are produced by passing the rays through different parts of a phase plate. The light rays are superimposed in the image plane, producing contrast due to their interference.
This fungus
causes chromoblastomycosis.
Fonsecaea pedrosoi grown
on modified Leonian’s agar
This is a chronic skin infection common in tropical and subtropical climates.
chromoblastomycosis
These microscopes are similar to phase-contrast microscopes in that they use interference patterns to enhance contrast between different features of a specimen.
Differential interference contrast (DIC)
How does a Differential interference contrast (DIC) work?
In a DIC microscope, two beams of light are created in which the direction of wave movement (polarization) differs. Once the beams pass through either the specimen or specimen-free space, they are recombined and effects of the specimens cause differences in the interference patterns generated by the combining of the beams.
What is the result when two beams in a DIC microscope are created in which the direction of wave movement differs?
This results in high-contrast images of living organisms with a three-dimensional appearance. These microscopes are especially useful in distinguishing structures within live, unstained specimens.
This light microscope uses fluorescent chromophores called fluorochromes, which are capable of absorbing energy from a light source and then emitting this energy as visible light. This produces an image of the specimen in bright colors against a dark background.
Fluorescence microscope
These are used to absorb energy from a light source and then emit this energy as visible light.
fluorochromes
What is the importance of a fluorescence microscope in clinical microbiology?
Used in clinical microbiology to identify pathogens, to find particular species within an environment, or to find the locations of particular molecules and structures within a cell.
This is used to identify certain disease causing microbes by observing whether antibodies bind to them.
Immunofluorescence
These are protein molecules produced by the immune system that attach to specific pathogens to kill or inhibit them
Antibodies
what are the two (2) approaches of immunofluorescence?
(1) Direct immunofluorescence assay (DFA)
(2) Indirect immunofluorescence assay (IFA)
What are the parts of a Fluorescent Microscope?
(1) Detector
(2) Ocular
(3) Emission filter
(4) Dichroic mirror
(5) Excitation filter
(6) Light Source
(7) Objective
This is a parasitic worm that
causes schistosomiasis, an intestinal disease common in the tropics
larvae of Schistosoma mansoni,
This is the bacterium that causes
gonorrhea.
Neisseria gonorrhoeae
In this approach of immunofluorescence, the stain is absorbed by a primary antibody, which binds to the antigen.
Direct immunofluorescence,
In this approach of immunofluorescence, the stain is absorbed by a secondary antibody, which binds to a primary antibody, which, in turn, binds to the antigen
indirect immunofluorescence,
Explain the fluorescence of bovine pulmonary endothelial cells.
The fluorescent dyes absorbed by these bovine pulmonary artery endothelial cells emit brilliant colors when excited by ultraviolet light under a fluorescence microscope. Various cell structures absorb different dyes. The nuclei are stained blue with 4’,6-diamidino-2-phenylindole (DAPI); microtubules are marked green by an antibody bound to FITC; and actin filaments are labeled red with phalloidin bound to tetramethylrhodamine)
This can be used to visualize structures such as this roof-dwelling cyanobacterium biofilm.
Confocal Microscopy
This is a complex community of one or more microorganism species, typically forming as a slimy coating attached to a surface.
Biofilm
What is the downside of biofilms?
Because biofilms are thick, they cannot be observed very well using light microscopy; slicing a biofilm to create a thinner specimen might kill or disturb the microbial community.
Why is confocal microscopy used for biofilms?
Confocal microscopy provides clearer images of biofilms because it can focus on one z plane at a time and produce a three-dimensional image of a thick specimen.
What are the stages of biofilms?
(1) Initial Attachment
(2) Irreversible Attachment
(3) Maturation I
(4) Maturation II
(5) Dispersion
What are the two (2) types of electron microscopy?
(1) Transmission electron microscope (TEM)
(2) Scanning electron microscope (SEM)
Most light microscopes used in a college biology lab can magnify cells up to approximately _________________________.
400 times and have a resolution of about 200 nanometers.
What makes the electron microscope different from the light microscope?
Electron microscopes provide a much higher magnification, 10,0000x, and a have a resolution of 50 picometers.
Electron microscopes use ____________to focus electron beams similarly to the way that light microscopes use lenses to focus light.
magnets
Difference of TEM and SEM
(a) This TEM image of cells in a biofilm shows well-defined internal structures of the cells because of varying levels of opacity in the specimen.
(b) This color-enhanced SEM image of the bacterium Staphylococcus aureus illustrates the ability of scanning electron microscopy to render three-dimensional images of the surface structure of cells
What are the two (2) types of scanning probe microscopes)?
(1) Scanning tunneling microscope (STM)
(2) Atomic force microscope (AFM) - IBM
These are used to view images at an atomic level,
Scanning Probe Microscopy
What is the difference between STM and AFM?
(a) This STM image of a pure gold surface shows individual atoms of gold arranged in columns.
(b) This AFM image shows long, strand-like molecules of nanocellulose, a laboratory-created substance derived from plant fibers.
This property of light is the height of a wave, whereas the wavelength is the distance between one peak and the next.
Amplitude
Light waves have different frequencies, or rates of vibration, which wave has the lowest frequency and why>
The wave at the top has the lowest frequency, since it
has the fewest peaks per unit time
What are the different phenomena in terms of transparency of light?
(1) Reflection
(2) Absorbance
(3) Transmittance
(4) Interference
(5) Refraction
Explain the property of transparency.
(a) A Petri dish is made of transparent plastic or glass, which allows transmission of a high proportion of light. This transparency allows us to see through the sides of the dish to view the contents.
(b) This slice of an iron meteorite is opaque (i.e., it has opacity). Light is not transmitted through the material, making it impossible to see the part of the hand covered by the object.
This occurs when light passes from one medium, such as air, to another, such as glass, changing the direction of the light rays
Refraction
What illusion does a straight pole appearing to bend at an angle as it enters water? Describe the phenomena
This straight pole appears to bend at an angle as it enters the water. This optical illusion is due to the large difference between the refractive indices of air and water.
(1) Light passes slower through water
(2) Light passes faster through air
This is like a collection of prisms, such as the one shown here.
Lens
What happens when light passes through convex lens?
When light passes through a convex lens, it is refracted toward a focal point on the other side of the lens. The focal length is the distance to the focal point
What happens when light passes through concave lens?
Light passing through a concave lens is refracted away from a focal point in front of the lens.
This ranges from high-frequency gamma rays to low-frequency radio waves. Visible light is the relatively small range of electromagnetic frequencies that can be sensed by the human eye.
electromagnetic spectrum
On the electromagnetic spectrum, visible light falls between
ultraviolet and infrared light.
In this, photons may be emitted following a delay after absorption
Phosphorescence or Bioluminescence
