Ch 3: Observic Microorganisms Through a Microscope Flashcards
One Angstrom (Å) is equivalent to ____ or ____.
10^-10 m
0.1 nm
What are the typical magnification ranges for objective and ocular lenses in a compound light microscope?
Objectives: 5 - 100x
Oculars: 10 - 20x
What does it mean when a microscope has a resolving power of 0.2 nm?
It can distinguish two points ≥0.2 nm
Total magnification = _____?
Objective lens magnification x ocular lens magnification
The ultimate limit to the resolution of a light microscope is set by __________?
The type of radiation it utilizes
Do shorter or longer wavelengths of light provide greater resolution?
Shorter
Resolution = ______
Resolution = (0.61*λ) / (n*sinθ)
λ = wavelength of light used
n = refractive index of medium
θ = half the angular width of the cone of rays by the objective lens from a typical point in the specimen
In an electron microscope, with an accelerating voltage of 100,000 V, the wavelength of an electron is ______?
0.004 nm
What is the refractive index of air?
1
For white light, the wavelength that is commonly assumed is _____.
0.53 μm
Define “refractive index”
A measure of the light-bending ability of a medium; dependent on how much the speed of light is reduced in the medium
Why does using immersion oil produce a better resolution in a microscope?
Immersion oil has the same refractive index as glass, thus does not bend the light
What are the six types of light microscopy?
- Brightfield
- Darkfield
- Phase-contrast
- Differential interference contrast (DIC/Nomarski)
- Fluorescence
- Confocal
In _____ microscopy, dark objects are visible against a bright background.
Brightfield
True or false. Brightfield microscopy is only useful for stained biological specimens.
True. Unstained cells are virtually invisible
What are some benefits of darkfield microscopy?
Useful for live organisms not visible with light, can’t be stained, or distorted by staining
Can be used to visualize very thin microorganisms (ex: Treponema pallidum)
Phase-contrast microscopy is useful because ___________
internal structures of a cell become more sharply defined, permitting detailed examination of living tissue
What advantage does DIC microscopy have over phase-contrast?
Allows for visualization of depth
Both phase-contrast and DIC microscopy _____. (3)
- Can be used with living cells
- Don’t require cell fixation/attachment slides
- Don’t require staining
True or false. Fluorescence microscopy takes advantage of fluorochromes which absorb long wavelengths of light (UV or near UV) and emit another, shorter wavelength (visible).
False. They absorb short and emit long
Describe the path of light in fluorescence microscopy
- Barrier filter lets only one wavelength of light through
- Beam-splitting mirror reflects light of below one wavelength but transmits light above another
- Second barrier filter cuts out unwanted noise before reaching eyepiece
Describe how immunofluorescence works
Antibodies conjugated to fluorochromes bind to specific antigens in the specimen of interest
What advantage does confocal microscopy have over standard fluorescence?
Illuminate individual planes of light in order to generate a 3D image and visualize depth
What two conditions can cause photobleaching of a specimen stained with a fluorochrome?
Exposure to high wavelengths of light
Long exposure times
