Lecture 2: Microscopy Flashcards
What is Magnification, Resolution and Contrast mean?
Magnification
- Enlarge the image Resolution
- Ability to distinguish 2 adjacent items Contrast
- Ability to distinguish items from background
What does resolution determine?
Useful magnification level
What is Numerical Aperature (NA)?
A measure of light capturing ability
-Increased NA = Increased Resolution
How does wavelength affect resolution?
Shorter wavelength = Increased resolution
What are the specs of a light microscope?
Magnification = 1-1500X Resoultion = 0.2 um (200 nm)
What are the types of light microscopes?
Simple and Compound
-Examples are Bright-field, Phase Contrast, Differential Interference Contrast (DIC), Dark-Field, Flourescence
How do you calculate the total magnification of a Bright-Field Microscope?
TM = Ocular Lens x Objective Lens
What is the Oil-Immersion technique?
Place drop of oil on slide and examine with 100X objective Lens to increase resolution by increasing the numerical aperature. The oil focuses more light into the objective
How do we increase the contrast when using improved light microscopy?
- Staining
- Phase-Contrast Microscopy
- Dark-Field Microscopy
- Fluorescence Microscopy
When staining what kinds of Dyes do we use?
Basic Dyes
-Crystal Violet binds to DNA
Differential Dyes
-Gram Stain
How does Gram staining differentiate?
Gram (+) = Purple (Thick Peptidoglycan Cell Wall)
Gram (-) = Pink (Thin Peptidoglycan Cell Wall)
What are the Advantages/Disadvantages of Staining?
Advantages
- Increase contrast
- Differentiate Cell types Disadvantages
- Kills Cells
- Can distort features
What is Phase-Contrast Microscopy?
Uses phase ring (refractive index) to improve contrast
-Used to view live cells
What is Dark-Field Microscopy?
Lights specimen from side to create dark background
-Used to view live cells especially when looking at motility and flagella
What is Fluorescence Microscopy?
Uses light emitted from cells (either autofluorescence or Fluorescent stains)
-Used to view live and fixed cells (especially useful when counting cell numbers)
What are the types of 3-D Microscopy?
Differential Interference Contrast (DIC), Atomic Force, and Confocal Laser Scanning
What is Differential Interference Contrast (DIC) Microscopy?
Uses two beams of polarized light to enhance cell structure
-Used to view cell structure (nucleus, vacuoles etc)
What is Atomic Force Microscopy?
Uses a cantilever and laser detector to make 3D image
-Used to view live specimens with no fixative or coating
What is Confocal Scanning Laser Microscopy?
Uses Laser+Fluorescence to focus on different layers
-Used with thick specimens for multi-layer image
What are the key characteristics of Electron Microscopy?
Uses Electron Beam and Electromagnetic Lens
-Operates in Vacuum
Why use electron Microscopy?
Lower wavelength = higher resolution Light Microscope can view whole microbial cells Electron Microscope can view inside microbial cells (Higher magnification and Greater Resolution)
What are the types of Electron Microscopy?
Transmission Electron Microscope
- Higher Resolution
- 2D Images
- Internal Features
Scanning Electron Microscope (uses gold covering)
- Lower Resolution
- 3D Images
- External features
Name the parts of the Bright Field Light Microscope
