Lecture 2: Microscopy

Question 1

General size the human eye can detect

Answer 1

0.1mm/100 micrometers

Question 2

Range of resolution for the human eye

Answer 2

100-200 micrometers

Question 3

Range of resolution for light microscopy (Conventional Light Microscopy)

Answer 3

200 nm

Question 4

Range of resolution for light microscopy (Super-Resolution)

Answer 4

50 nm

Question 5

Atomic Force Microscopy

Answer 5

Enables visualization of untreated cells by detecting Van der Waals forces. SCANS THE SURFACE

Question 5

Fluorescence microscopy overlap zone

Answer 5

Region where absorption and fluorescence overlap preventing a clear image

Question 6

Ways to improve contrast in light microscopy

Answer 6

Staining, fluorescence, dark-field, and phase-contrast

Question 7

Sample Requirement for Scanning Electron Microscopy

Answer 7

Sample must be dried and cells need to be fixed

Question 8

Resolution

Answer 8

The smallest distance between 2 objects that allows separation (How clearly we can see an image)

Question 8

Magnification

Answer 8

The increasing of an objects apparent dimensions

Question 9

Atomic Force Microscopy Range of Resolution

Answer 9

1-2 Angstroms

Question 9

Scanning Electron Microscopy Range of Resolution

Answer 9

2 nm

Question 10

X-Ray Crystallography Range of Resolution

Answer 10

1-2 Angstroms

Question 10

Transmission Electron Microscopy Range of Resolution

Answer 10

2 nm

Question 11

Cons of light microscopy

Answer 11

Poor contrast due to transparency of bacterial cells

Question 12

Cryo-electron microscopy resolution

Answer 12

1-2 Angstroms

Question 13

Consequence of higher frequency light in microscopy

Answer 13

Higher energy causing higher resolution

Question 14

What bright field microscopy image quality depends on

Answer 14

Wavelength, magnification power, and focus

Question 14

Numerical Aperture

Answer 14

Value directly linked to better resolution (High NA = Low R= High Resolution) (High NA = Low R)

Question 15

Numerical Aperture Formula

Answer 15

NA = n*sinθ
n=refractive index (medium/solution holding the sample)

Question 16

Consequence of a large n value on resolution

Answer 16

Large n values increase NA values causing a smaller R value. Causes an overall better resolution.

Question 17

Gram positive bacteria

Answer 17

Single membrane & thick cell wall containing peptidoglycan

Question 18

Gram negative bacteria

Answer 18

Inner and outer membranes with thin cell wall

Question 19

Gram staining result in gram-negative cells

Answer 19

Safranin counterstains and binds to nucleic acid causing pink appearance in cells.

Question 20

Gram staining result in gram-positive cells

Answer 20

Absorbs safranin but remains dark purple

Question 21

Acid fast stain

Answer 21

Carbolfuchsin used to stain mycobacterium

Question 22

Endospore staining

Answer 22

Malachite green specifically binds to an endospores coat

Question 23

Process of fluorescence microscopy

Answer 23

-Light excites electrons -Electrons move to more stable high energy state -Electron begins to drop to a lower orbital to release energy -As electron drops fluorescence is released

Question 24

Absorption and fluorescence terms

Answer 24

-Absorption/Excitation -Fluorescence/Emission

Question 25

Benefits of fluorescence microscopy

Answer 25

-Can study bacterial cells inside a complex such as biofilms -Can be used to study bacterial motion

Question 26

Considerations for choosing fluorescence microscopy

Answer 26

-Excitation and emission -Orthogonality between fluorescent proteins (ability for proteins to be used simultaneously in the same system without interference) -Monomeric structure (Easier to experiment with, higher resolution, monomeric proteins are less likely to disrupt the cellular function of what is being observed) -Maturation time (Events or functions that occur fast enough may not be observable since fluorescence takes 10-15 mins) -Brightness (Visibility) -Autofluorescence (Some cells have weak fluorescent ranges)

Question 27

Single-Molecule localization in light microscopy

Answer 27

Approximation of location in order to calculate the best probability of fluoroform

Question 28

Single-molecule tracking in light microscopy

Answer 28

Tracks consecutive images of the same molecules in order to gain unique frames to reconstruct a path

Question 29

Dark field microscopy

Answer 29

Central aperture above the light source allowing visibility of cellular structures

Question 30

Cryo-electron microscopy

Answer 30

Rapid freezing of samples to preserve native structure

Question 31

Sample requirement for transmission electron microscopy

Answer 31

An electron dense negative stain from salt or heavy metal

Question 32

Benefit of scanning electron microscopy

Answer 32

Effective for visualizing cells in complex communities such as biofilms

Question 33

Benefit of cryo-electron microscopy

Answer 33

Maintains native structure of protein