Unit 2: How Cells are Studied

Question 1

Optical pathway of compound optical microscope:
1. Iris diaphragm

Answer 1

Restricts amount of light entering lens

Question 2

Optical pathway of compound optical microscope:
2. Condenser lens

Answer 2

Focuses the light onto the specimen – no magnification

Question 3

Optical pathway of compound optical microscope:
3. Objective lens

Answer 3

Picks up the light transmitted by the specimen and focus it on the focal plane of the objective lens, creating a magnified image

Question 4

Optical pathway of compound optical microscope:
4.

Answer 4

Reflecting prism

Question 5

Optical pathway of compound optical microscope:
5. Ocular lens

Answer 5

(eyepiece)
The image on the objective focal plane is further magnified by the ocular lens, or eyepiece, and projects it onto the human eye

Question 6

Total magnification = ____*_____

Answer 6

Objective lens * Ocular lens

Question 7

Most important property of microscope is NOT it’s ______, but it is it’s _____. Why?

Answer 7

Magnification,
Resolving power

Because resolution can differentiate between structures at a certain magnification. The clearer the image (more resolution).

Question 8

Resolution (D) is the

Answer 8

ability to see two nearby points as distinct images. The smaller the value of D, the better the resolution.

Question 9

How is resolution determined?

Answer 9

Resolution is determined by the objective lens and its ability to gather the “cone of light” coming from the specimen. The light comes into the objective lens as a cone due to diffraction by the specimen:

D = (0.61* lambda)/(n*sin(alpha))
* lambda is the wavelength of incident light in nm
* n is the refractive index of the medium between the specimen and the objective
* alpha represents half the angle of the cone of light
objective lens specimen

Question 10

Why does electron microscope have more resolution than a light microscope?

Answer 10

Because of electrons wavelength being super tiny, much smaller than light wavelengths.

Question 11

The lower the wavelength, the better the

Answer 11

Resolution

Question 12

The resolution of such an electron microscope is times greater than that of the light microscope.

Answer 12

~100,000

Question 13

fundamental limitation on all microscopes:

Answer 13

A given type of radiation cannot be used to probe details smaller than its own wavelength (l).

Question 14

The best alpha is ___degrees, why?

Answer 14

70 degrees, because it can take 140 degrees of scattered light

Question 15

1 of 2 ways to partially circumvent the fundamental limitation on all microscopes:

Answer 15

By increasing alpha, which will decrease D. Best alpha is thus 70deg

Question 16

2 of 2 ways to partially circumvent the fundamental limitation on all microscopes:

Answer 16

increase the refractive index of the medium between the specimen and the objective lens (n).
(e.g. n = 1.0 for air, n = 1.5 for oil). Thus, using oil increases resolution by 50%.

Question 17

No matter how many times the image is magnified, the light microscope can never resolve objects that are less than

Answer 17

~ 0.2 μm in size (not good enough to see proteins interacting in a cell)

Question 18

3 types of light microscopy:

Answer 18

1. Brightfield: no contrast. can put a stain but are usually toxic and can only see dead cells. Good for counting cells and see overall observations
2. Phase-contrast: requires phase plate (light and dark) can play with emergent light
3. Differential interference contrast (DIC) or Nomarski interference: polarized light used, gives it more dimensions (one side more illuminated than the other). Good for thicker samples.

Question 19

TEM vs. light microscope: (5 items

Answer 19

1. TEM larder
2. TEM uses electrons (not light)
3. Beam of electrons projected downwards (light upwards)
4. TEM uses electromagnetic coils to focus beam of electrons and to magnify image (light uses glass lenses)
5. TEM in vacuum (light operates in air).

Question 20

Optical path of TEM

Answer 20

1. Cathode (heated) to disrupt electrons
2. Electrons emitted to anode (electric potential of zero, drop in V causes electrons to accelerate towards it
3. Electromagnetic condenser lens focuses electrons onto specimen plane.
4. Specimen (thin and dead)
5. Electromagnetic objective lens picks up electrons passed thru specimen, magnifies the image in focal plane of it.
6. Electromagnetic projector lens (like ocular lens) picks up electrons from focal plane of obj lens. It then focuses and magnifies them onto specimen detector.

Question 21

Why must TEM specimens be stained?

Answer 21

Epoxy slices with elecrons passing through with low atomic number, so details need to be seen with staining of high atomic numbers. It will define parts of organelles, etc.

Question 22

Resolution (D) for TEM =

Answer 22

0.2nm; 1000x more resolution than proteins. Allows to see interactions of proteins.

Question 23

Fluorescent microscopy

Answer 23

Uses fluorescents of excitation (lower wavelength) and emission of higher wavelength. If a compound is illuminated at it’s excitation wavelength and viewed through a filter (for just excitation wavelength to pass), it is seen to glow against a dark background.

Question 24

Energy of a photon of light is inversely proportional to

Answer 24

its wavelength.

Question 25

Three steps of fluorescence

Answer 25

1. Excitation 2. Energy dissipation 3. Fluorescence emission

Question 26

Why can't we inject the fluorescent chemical into cell?

Answer 26

Because it would bind to all proteins. So attach it to antibody specific to that protein.

Question 27

Optical path in fluorescence microscope

Answer 27

1. Light source goes through first barrier filter 2. and 3. Fluorescent dye (fluorescein) in specimen excited to emit light (fluoresce) at a specific (longer) wavelength 4. Beam splitting mirror reflects light below 510 nm but transmits light above 510nm 5. Second barrier filter cuts out unwanted fluorescent signals, allowing through excitation wavelengths

Question 28

Confocal microscopy improves

Answer 28

Image of conventional fluorescence microscope...

Question 29

Confocal light microscopy optical path

Answer 29

1. Light from a laser passes through pinhole, 2. dichroic mirror and objective lens focuses the light at specific depth in the specimen. 3. Emitted light from specimen focused at second confocal pinhole, reaches detector 4. Emitted light from elsewhere of specimen largely excluded form this second confocal pinhole.

Question 30

Using antibodies to bind to specific proteins (how is it done)?

Answer 30

Purify protein X, secondary detects any (other animal) antibody so you don't have to tag your own every (other animal) antibody. Primary antibody recognizes antigen, wherever it is in cell. Secondary antibody binds to any other animal's primary antibody.

Question 31

Secondary antibody allows to

Answer 31

amplify the signal.

Question 32

Fixation of sample (locks) two main types

Answer 32

1. Cross-linking 2. Pecipitation (dehydrates sample)

Question 33

Different animals antibodies raised in useful in immunofluoroscopy because:

Answer 33

Can tag different parts/components with different colours (make sure different wavelengths).

Question 34

Immunogold electron microscopy

Answer 34

Put gold on the antibody because the atomic number is super high. You need to stain it with a high atomic number which will effectively deflect those electrons and show where that antibody is

Question 35

Live cell imaging via

Answer 35

green fluorescent protein linkage

Question 36

Subnuclear fractionation steps

Answer 36

1. Disrupt plasma membrane in isotonic solution, on ice to preserve proteins, organelles, etc... Then blend, sonicate or homogenize (grinding manually) but important to leave organelles intact. 2. Results in suspension of homogenate, extract, lysate. 3. Differential centrifugation forming pellet (sedimentation) which extracts at rate that depends on size and density. -Low speed start, takes bigger things out to fractions. Then speed it up -Only nuclei sediment at 10min for 1000g's -Results in post-nuclear supernatant 4. Next is mitochondria, peroxisomes, lysosomes, golgi (intermediate sizes and densities): sediment at 20min 20000g's.

Question 37

Differential centrifugation does NOT

Answer 37

yield totally pure organelles,

Question 38

Equilibrum density-gradient centrifugation

Answer 38

Pellet resuspended in buffer and differentiated organelles strictly via their density by subjecting pellet to a gradient of solute (sucrose) density (top tube lower density and lower in tube higher in density. Densities of the components of pellet separate depending on density at it's equilibrium position Then tube is punctured, and fractions are collected.

Question 39

Western blotting analysis steps

Answer 39

1. Separate proteins on a polyacrylamide gel 2. Transfer the proteins onto a thin membrane 3. Probe the membrane with a specific antibody 4. Detect the antibody Requires an antibody, separated based on molecular size.

Question 40

Gel electrophoresis (2 types)

Answer 40

1. Native gel electrophoresis (PAGE) is without denaturant 2. SDS PAGE

Question 41

PAGE

Answer 41

keeps protein complexes intact, migrating in electric field dependent upon: size, shape, charge.

Question 42

SDS binds

Answer 42

Uniformly to proteins

Question 43

SDS PAGE migration in electric field

Answer 43

Strictly dependent upon size. Larger proteins migrate through the polyacrylamide gel more slowly while smaller proteins migrate more quickly.

Question 44

A reducing agent (b-mercaptoethanol or dithiothreitol (DTT)) is used in _____ included to break _______

Answer 44

SDS PAGE, Disulfide bonds

Question 45

3 types column chromotography

Answer 45

1. Ion exchange excludes based on charge using salt gradient and beads: non charged pass first, charged will interact and elute slower and begin to increase salt gradient. 2. Size exclusion (gel filtration) excludes just on molecular size larger ones don't interact in beads 3. Affinity chromotography: Specific sequence within the protein due to presence of ligand that binds specific to protein of interest in the column.

Question 46

Three ways purified protein interactions can be studied:

Answer 46

1. Immunoprecipitation 2. Yeast two-hybrid 3. FRET (Fluorescence resonance energy transfer)

Question 47

FRET

Answer 47

Asking whether Z and Y interact (proteins). So we drag YFP and CFP together and then suggest interaction if the protein is close enough together and energy is only absorbed if it is close enough together. If they are close enough, CFP close enough to YFP will transfer energy and the excitation wavelength will be yellow, implying an interaction.

Question 48

How do you assess the function of the protein? Gene silencing!

Answer 48

facilitated by examining the effects of removing that protein from the cell. One such method is called RNA interference (RNAi).