Lecture 2: Microscopy

Question 1

What is Magnification, Resolution and Contrast mean?

Answer 1

Magnification

• Enlarge the image Resolution
• Ability to distinguish 2 adjacent items Contrast
• Ability to distinguish items from background

Question 2

What does resolution determine?

Answer 2

Useful magnification level

Question 3

What is Numerical Aperature (NA)?

Answer 3

A measure of light capturing ability

-Increased NA = Increased Resolution

Question 4

How does wavelength affect resolution?

Answer 4

Shorter wavelength = Increased resolution

Question 5

What are the specs of a light microscope?

Answer 5

Magnification = 1-1500X Resoultion = 0.2 um (200 nm)

Question 6

What are the types of light microscopes?

Answer 6

Simple and Compound

-Examples are Bright-field, Phase Contrast, Differential Interference Contrast (DIC), Dark-Field, Flourescence

Question 7

How do you calculate the total magnification of a Bright-Field Microscope?

Answer 7

TM = Ocular Lens x Objective Lens

Question 8

What is the Oil-Immersion technique?

Answer 8

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

Question 9

How do we increase the contrast when using improved light microscopy?

Answer 9

• Staining
• Phase-Contrast Microscopy
• Dark-Field Microscopy
• Fluorescence Microscopy

Question 10

When staining what kinds of Dyes do we use?

Answer 10

Basic Dyes

-Crystal Violet binds to DNA

Differential Dyes

-Gram Stain

Question 11

How does Gram staining differentiate?

Answer 11

Gram (+) = Purple (Thick Peptidoglycan Cell Wall)

Gram (-) = Pink (Thin Peptidoglycan Cell Wall)

Question 12

What are the Advantages/Disadvantages of Staining?

Answer 12

Advantages

• Increase contrast
• Differentiate Cell types Disadvantages
• Kills Cells
• Can distort features

Question 13

What is Phase-Contrast Microscopy?

Answer 13

Uses phase ring (refractive index) to improve contrast

-Used to view live cells

Question 14

What is Dark-Field Microscopy?

Answer 14

Lights specimen from side to create dark background

-Used to view live cells especially when looking at motility and flagella

Question 15

What is Fluorescence Microscopy?

Answer 15

Uses light emitted from cells (either autofluorescence or Fluorescent stains)

-Used to view live and fixed cells (especially useful when counting cell numbers)

Question 16

What are the types of 3-D Microscopy?

Answer 16

Differential Interference Contrast (DIC), Atomic Force, and Confocal Laser Scanning

Question 17

What is Differential Interference Contrast (DIC) Microscopy?

Answer 17

Uses two beams of polarized light to enhance cell structure

-Used to view cell structure (nucleus, vacuoles etc)

Question 18

What is Atomic Force Microscopy?

Answer 18

Uses a cantilever and laser detector to make 3D image

-Used to view live specimens with no fixative or coating

Question 19

What is Confocal Scanning Laser Microscopy?

Answer 19

Uses Laser+Fluorescence to focus on different layers

-Used with thick specimens for multi-layer image

Question 20

What are the key characteristics of Electron Microscopy?

Answer 20

Uses Electron Beam and Electromagnetic Lens

-Operates in Vacuum

Question 21

Why use electron Microscopy?

Answer 21

Lower wavelength = higher resolution Light Microscope can view whole microbial cells Electron Microscope can view inside microbial cells (Higher magnification and Greater Resolution)

Question 22

What are the types of Electron Microscopy?

Answer 22

Transmission Electron Microscope

• Higher Resolution
• 2D Images
• Internal Features

Scanning Electron Microscope (uses gold covering)

• Lower Resolution
• 3D Images
• External features

Question 23

Name the parts of the Bright Field Light Microscope

Answer 23