Imaging And Microscopes Flashcards
What is the estimated number of “healthy” bacteria in the human body
1 trillion
What are the metric prefixes associated with microbiology
- Milli (m)
- Micro (u)
- Nano (n)
- Pico (p)
- Fernto (f)
What is the range of a light microscope and what is its dividing line around
0.25 micrometers (um)
Dividing line is between 7-8 um
What is the range of an electron microscope
100um to 1nm
What is the range of the scanning tunneling microscope
0.1nm to 1nm
Explain what the lens of the human eye does
Bends and focuses light onto the Retina (sensory structure) at the back of the eye.
What does the Retina do with the light?
Converts light into an Electra signal that carries to the brain.
Explain what happens to the ciliary muscle of the eye for distance and near vision.
For distance vision, the ciliary muscle is relaxed.
For near vision, the ciliary muscle is contracted.
Define refraction
The bending of light as it passes from one substance to another.
All imaging works because light, when it passes through objects, undergoes refraction.
Allows eyes and microscopes to focus light into a visual image.
Why does refraction happen?
Light interacts with the materials that it passes through, causing it to slow down slightly.
Reduce speed leads to bending.
The amount of light that slows down in a material is proportional to the materials density.
What happens when a beam of light moves between 2 densities?
The beam will bend.
What happens to beam of light when the densities move from low to high?
The beams will bend toward the normal line
What happens to the beam of light when the densities move from high to low?
Beams bend away from the normal line.
What is the Refractive index (n)?
The measure of how strongly a material can bend light.
How is the refractive index calculated?
As a ratio, speed of light in the material divided by the speed of light in a vacuum.
What is the lowest possible refractive index?
1 in a pure vacuum
What is the refractive index of the eye?
1.35 to 1.4 (similar to water)
What does refraction tell us?
How the eye or the microscopes focus light to create an image.
What is the quality of an image determined by?
Its resolution.
Define Resolution (d)
Smallest distance at which 2 different objects can be seen as separate from each other.
The higher the resolution = _________________________
Smaller number
What the 3 factors that affect resolution?
- Wavelength
- Refractive index
*the higher the index, the better the resolution - Lens half angle (theta)
*measure half of the size of a lens and how much light it can gather to form an image
What is the formula for resolution?
d= 0.5 wavelength / n sin (theta)
What is the average size of the lens of the eye’s resolution?
0.1mm OR 100 micrometers
What is the limit of the human vision?
100 um
Define magnification
Ability of an imaging system to make an object appear larger than it actually is in an image
Define resolution of microscopes
Ability of an imaging system to distinguish very small, fine details
What is the normal line?
Imaginary line that is drawn perpendicular to the surface where the 2 mediums meet (point where light is entering)
What are the size parameters of microbial cells?
1-100 micrometers
What are the parameters in size of viruses?
10-100 nanometers
What are the parameters in size of atoms/molecules?
0.1-10 nanometers; often measured in a unit called an Angstrom (0.1nm or 1 x 10^-10 m)
Standard light microscopes use _______________________ to focus visible light and magnify images.
A series of glass lenses
What kinds of lenses focus and aligns light into a single beam in light microscopes?
Condenser lenses
After the condensor lenses the light passes through a microbial sample, which is usually referred to as __________________.
A specimen.
What happens to light as it passes through a specimen?
Refracts
What happens to the light after it refracts through a specimen in a light microscope?
A pair of lenses used refracted light to magnify and focus light onto the retina of the eye
What are the pair of lenses that magnify refracted light in a light microscope?
- Objective lens focuses refracted light to create a magnified image.
- Ocular lens further magnifies image made by the objective lens.
What is the numerical aperture (NA)?
Combined n sin (theta) part of the resolution into a single number.
The higher the NA= the better the resolution
Remember the lower the # of resolution=the better
What is contrast?
The darkens of the image background relative to the specimen
What are the types of ways to add contrast?
Manipulation of light and chemical treatment
What is staining and what are the 2 types of staining?
Staining uses chemical dyes to give an image both color and contrast.
Simple: single dye that chemically binds to cellular structures (e.g crystal violet, most common)
Differential: uses ultimate dyes that bind differently to cellular structures, allows us to classify microbes on their staining properties. (E.g gram stain, stains bacteria based on their cell wall structure)
What is the major drawback of staining?
The process kills microbes and cannot visualize live cells.
List 4 types of staining
- Gram stain
- Acid fast stain
- Negative stain
- Flagellar stain
What can we do to provide contrast to living cells?
We can change the optical system of the microscope to allow us to manipulate the light that passes through the specimen.
List the 3 ways that we can manipulate light that passes through a specimen in a light microscope to provide contrast to living cells.
- Dark-field microscopy
- Interference microscopy
- Fluorescence microscopy
Explain dark field microscopy
*Differs from regular light microscopy
*Dark-field stop: creates a hollow cone of light
*Abbe condenser: Focuses that cone of light at an angle rather than directly onto the specimen
*Result: the only light that enters the objective is the light that passes through the specimen itself.
Explain interference microscopy.
Uses a modified optical system to create contrast using constructive and destructive interference.
*Optical system uses 2 beams of light
1. That passes through the specimen
2. One that does not pass through the specimen and is not slowed down.
Exactly aligned = brighter
Not aligned = darker
***No staining just light manipulation
What are the 2 major types of interference microscopy?
- Phase contrast: produces strong 2D images with very bright edges (shows phase halo)
- Differential interference contrast: produces a slightly weaker contrast that appears 3D (does not show phase halo)
What is phase halo?
Bright, fuzzy edge to an image
What is fluorescence microscopy?
Uses dyes that give off (emit) light when struck by visible light of a specific color.
The filters in the microscope allow only the emitted light from the specimen to reach the eye.
What are the advantages of fluorescence microscopy?
Typically doesn’t kill living cells (living cells=green, dead=red)
Dyes can be attached to antibodies to identify and stain specific proteins.
What are the limits of light microscopes?
Maximum limit:
*resolution: 0.2 micrometers
*magnification: 1000x-1500x
Why are there limits to light microscopes?
Any optical system in the universe has a physical limit of resolution
Limit = 1/2 the wavelength of the light being used (avg wavelength of visible light ~ 0.4 micrometers)
Any light microscope has a maximum resolution of 0.2 micrometers
Explain Electron microscopy
Replaces visible light with electrons and is remarkably similar to light microscopes.
What is the approximate wavelength of an electron?
1 angstrom or 0.0001 micrometers
In electron microscopy, rather than using glass lenses to focus, they use what?
Electromagnets: focus electrons to provide both magnification and resolution.
Transmission electron microscopes (TEM)
Produce 2D images that are a slice through a microbe and allows us to visualize the internal structures of microbial cells and viruses.
Magnifies structures 100,000x to 1,000,000x+
Scanning electron microscopes (SEM)
Uses an electron beam to scan the surface of a microbe.
Produces 3D images of the outer surface of a microbe.
What are the pros and cons of TEM
*Can produce images with higher magnification and better resolution.
*Limited in that it can only visualize small sections of a microbe.
What are the pros and cons of the SEM
*SEM can visualize entire microbes or microbial communities
*Limited in that it has lower magnification and worse overall resolution.
Which term best describes “the smallest distance at which 2 objects can be seen as separate from each other in an image”?
Resolution
Which term best describes “the difference between the darkness of the image background and the brightness of the specimen”?
Contrast
Which type of imaging technology produces light microscope images with a dark background that only show colored light where a light-emitting dye can be found?
Fluorescence microscopy
Which type of imaging technology produces light microscope images with a dark background that typically shows only the white light that passes through the specimen?
Dark-field microscopy
The resolution of the human eye is approximately:
100 micrometers
Which of the following would improve the resolution of an imaging system? (Hint resolution equation)
Using a source of light with a shorter wavelength
Why does electron microscopy have much better resolution than light microscopy?
The wavelength of an electron is much shorter than visible light
The theoretical maximum possible limit of resolution of any imaging system that uses some form of light or electron energy is _____________
1/2 the wavelength of the light being used.
Rank the following from largest to smallest
A. Average size of a eukaryotic cell
B. Average size of a virus
C. Average size of a bacterial or archaeal cell
D. Average size of a molecular structure like a protein or ribosome
A, C, B, D
When a beam of light asses from an extremely low density medium to a high density medium, how will the path of that beam of light change?
The path will bend toward the “normal” line.
the stronger a material will bend a beam of light, the __________________________
Greater the material’s refractive index is
How are electrons focused and magnified in an electro microscope?
Electromagnet rings focus/magnify electrons as they pass through
What is the purpose of simple and differential stains in light microscopy?
they provide greater contrast and color to microbes
How do dark-field microscopes alter light beams in order to create contrast?
It uses hollow cone of light and a condenser that focuses the cone on the specimen at an angle
How do technologies like phase contrast and DIC microscopes alter light beams in order to create contrast?
It uses 2 beams of light to create constructive and destructive interference.
Which type of microscopy would be the most effective choice for viewing the internal structures within living eukaryotic cells?
Phase contrast microscopy
What are 2 types of imaging technologies that would be able to capture a high resolution image of most types of viruses?
Cryo-electron microscopy (Cryo-EM) and Scanning electron microscopy (SEM)
