Lab 4: Microscopy

Question 1

condenser lens

Answer 1

gathers the diffuse rays from the light source and illuminates the specimen with a small cone of bright light.

Question 2

Objective lens

Answer 2

collects light rays focused on specimen by condenser lens

Question 3

Magnification

Answer 3

ocular x objective magnification

Question 4

Empty magnification

Answer 4

no further detail produced by an increase in magnification because there is no further detail to enhance

Question 5

Resolving power

Answer 5

defined in terms of the ability to see two neighbouring points in the visual field as two distinct entities—limited by the wavelength of the illumination.

Question 6

Limit of resolution for light microscope

Answer 6

0.2 um (200 nm) —sufficient to resolve larger cellular organelles (nuclei or mitochondria)

Question 7

Best ways to make thin, translucent specimen visible? Downsides?

Answer 7

• staining with dye (specific dyes bind with certain biological molecules)
• usually kills cell

Question 8

Bright-Field Microscopy

Answer 8

cone of light that illuminates the specimen is seen as bright background and specimen is dark (good for specimens of high contrast)

Question 9

Whole mount

Answer 9

intact object (living or dead)

Question 10

section

Answer 10

very thin slice of specimen (cells must be killed by immersing tissue in fixative which essentially immobilizes cell in as close to its live state as possible. Then embedded in paraffin wax so it can be sliced, then slide with wax is immersed in toluene to dissolve wax).

Question 11

Common fixatives for light microscopy

Answer 11

formaldehyde, alcohol, acetic acid

Question 12

Phase-Contrast Microscopy

Answer 12

Makes highly transparent objects more visible. Extremely useful in teaching situation (observing live cells). Haloes around objects.

Question 13

Differential Interference Contrast Microscopy

Answer 13

DIC or Nomarski

- very similar to phase contrast microscopy; delivers image with apparent 3D quality

Question 14

Fluorescence Microscopy

Answer 14

Allows viewers to observe location of certain molecules. Can be used to study dynamic processes in cell like tagging DNA or RNA with specific sequences.

Question 15

Fluorophores

Answer 15

absorb photons of a specific wavelength and re emit portion of energy in longer wavelength (fluorescence).

Question 16

GFP

Answer 16

Green fluorescent protein

• doesn’t require an additional cofactor to absorb and emit light; instead the light absorbing/emitting chromophore is formed by self modification of three of the amino acids that make up primary structure of the GFP polypeptide.
• coding region of GFP is joined to coding region of gene for protein under study

Question 17

FRET

Answer 17

Fluorescence Resonance Energy Transfer

• can measure distances between fluorophores in nanoscale range. Usually used to measure changes in distance between two parts of a protein.
• uses idea that excitation energy can be transferred from one fluorescent group to another as long as bodies are close enough (1 to 10 nm)—efficiency of the transfer depends on distance

Question 18

WHat can FRET be used for?

Answer 18

can follow protein folding or association and dissociation of components within a membrane

Question 19

FRAP

Answer 19

Fluorescence recovery after photobleaching

- used to study rate of protein turnover in living cells

Question 20

Confocal Microscopy

Answer 20

Produces image of thin plan situated within much thicker specimen. Laser beam rapidly scans specimen at single depth thus illuminating only a thin plane (optical section)

Question 21

Light Sheet Fluorescence Microscopy

Answer 21

Noninvasive imaging of large, three-dimensional samples (whole embryos or organisms)

Question 22

TEM

Answer 22

Transmission Electron Microscopy
- forms images using electrons transmitted through specimen
- much higher res images that any light microscope because wavelengths are that much smaller
-

Question 23

SEM

Answer 23

Scanning EM

- uses electrons that have bounced off surface of specimen

Question 24

Limit of resolution for TEM

Answer 24

practical limit is 3 to 5 angstroms (tenth of nm) but actual limit when observing cellular structures is more typically 10 to 15 angstroms

Question 25

The scattering of electrons by a part of the specimen is proportional to

Answer 25

the size of the nuclei of the atoms that make up the specimen.

Question 26

Specimen preparation for TEM

Answer 26

Heavy metals used to increase the electron scattering capabilities of biological tissue. Fixatives must stop life of cell without altering the structure of the cell.

Question 27

Artifact

Answer 27

Structure that isn’t present in living cell, was a result of fixative chemicals changing cell. Must prove structure isn’t an artifact by other imaging methods (live cell imaging ideally)

Question 28

Common fixatives for EM

Answer 28

glutaraldehyde
- has aldehydes which react with amino groups in proteins and cross link proteins into insoluble networks
osmium textroxide
- heavy metal that reacts with fatty acids to preserve cellular membranes

Question 29

Thickness of wax sections for EM

Answer 29

0.1 um (equiv to thickness of four ribosomes)

Question 30

Stains for EM samples

Answer 30

heavy metal solutions

- uranyl acetate and lead citrate

Question 31

Cyrofixation

Answer 31

rapid freezing of a sample

• less likely to lead to formation of artifacts because it doesn’t alter macromolecules but formation of ice crystals is an issue
• much faster prep

Question 32

After fixative, samples must be…

Answer 32

dehydrated

Question 33

Electron microscopy is well suited to study

Answer 33

very small particulate materials like viruses, ribosomes, multi subunit enzymes, cytoskeletal elements, and protein complexes.

Question 34

Goal of specimen prep. for SEM

Answer 34

To produce object that has the same shape and surface properties as living state, but is devoid of fluid (so it can be observed in vacuum). Air drying samples results in destruction largely from surface tension at air-water interfaces.

Question 35

Critical point drying

Answer 35

Used to prepare samples for SEM

• takes advantage of the fact that every solvent has a critical point where gas and liquid density are equal thus no surface tension exists between the two states which can alter 3D structure
• solvent of specimen replace with liquid CO2 which is vaporized under pressure

Question 36

Difference between electron beams in SEM and TEM

Answer 36

TEM - electron beam is focused by condenser lenses to simultaneously illuminate entire viewing field (image = electrons that pass through specimen)

SEM - electrons are accelerated as fine beam that scans the specimen (image = electrons that are scattered back aka indirect image formation)

Question 37

Resolving power of SEM

Answer 37

related to diameter of electron beam - resolutions less than 5 nm

Question 38

What can you observe with SEM?

Answer 38

Structure of outer cell surface and all various processes, extensions, and extracellular materials.