exam 1 visualizing cells

Question 1

what are model eukaryotes (+ 4 features of them)

Answer 1

model organisms in which

• the genome has been sequenced
• site-directed and tissue specific mutagenesis is possible
• expression of multiple genes can be tracked across many cells simultaneously
• the developmental sequence is known

Question 2

what is the main issue with light microscopy

Answer 2

resolution is limited

Question 3

what is the issue with electron microscopy

Answer 3

it requires special preservation and staining techniques

Question 4

what does light microscopy image

Answer 4

large-scale cellular structures

Question 5

what, in light microscopy, provides contrast and sensitivity

Answer 5

chemical stains and fluorescent molecules

Question 6

which form of microscopy has a higher resolution than light microscopy

Answer 6

electron microscopy

Question 7

how does flow cytometry work

Answer 7

• uses fluorescent labels to measure levels of biomolecules and ions
• sorts cells into different tubes based on expression levels

Question 8

what is Cell Theory and who proposed it

Answer 8

that all organisms are made of cells - Schleiden and Schwann

Question 9

what is resolution

Answer 9

the ability to distinguish two objects that are close to each other

Question 10

what is the smallest organelle that can be seen clearly by light microscopy

Answer 10

mitochondria

Question 11

what is the issue with magnification ** on exam or final

Answer 11

we can magnify an image as much as we want, but it may be blurry (due to resolution)

Question 12

what is detection ** on exam or final

Answer 12

the ability to see something (an item smaller than 0.2 um can be detected if it emits light

Question 13

what is the limit of resolution for microscopy

Answer 13

the wavelength of the light and the size of the lens

- the bigger the lens, the bigger resolution you can get

Question 14

why are cells hard to visualize

Answer 14

they are colorless and translucent

Question 15

what do phase-contrast and differential-interference-contrast microscopy do and what is the result

Answer 15

they use the change in phase of light waves as they pass through a cell
- gets the visualization of live, un-manipulated cells, but have limited detail

Question 16

what is the pro and con of staining with chemical dyes

Answer 16

pro - chemical dyes can reveal details of cells

con - requires fixation (kills and preserves cells)

Question 17

how can tissue samples be visualized

Answer 17

they have to be cut into thin sections because they are so large

Question 18

how do chemical dyes work

Answer 18

by absorbing specific wavelengths of light so the intensity of light goes down
- fluorescent molecules absorb light at one wavelength and emit light of a longer wavelength

Question 19

what does fluorescence microscopy allow for

Answer 19

detection of very small numbers of fluorescent molecules since they are viewed against a dark background

Question 20

how can fluorescent stains be introduced

Answer 20

• chemical dyes
• fluorescent molecules attached to antibodies
• intrinsically fluorescent proteins expressed by cell itself (GFP - green fluorescent protein)

Question 21

how does fluorescent microscopy work

Answer 21

uses barrier filters and beam-splitting mirror to let in certain wavelengths, transmit light, then cut out unwanted fluorescent signals and be seen through eyepiece

Question 22

how is fluorescence microscopy limited

Answer 22

by fluorescence coming from out-of-focus parts of cells, which reduces practical resolution

Question 23

how does confocal microscopy work

Answer 23

uses a scanning laser and pinhole apertures to limit detection to the focal plane - creation of an optical section with much better resolution = no need to slice the cell

Question 24

what type of image of a sample can be reconstructed using confocal microscopy

Answer 24

3D images - formed by taking images from various focal planes (“z sections”)

Question 25

how does FRET work

Answer 25

fluorescence resonance energy transfer - uses 2 fluorescent proteins where the excitation energy of the 2nd matches the emission of the first. if 2 are very close together, exciting protein 1 will allow protein 2 to fluoresce
- can tell if the molecules are in contact with each other

Question 26

how does 2-photon microscopy work

Answer 26

2 separate long-wave photons of light excite the fluorophore. this allows for deeper penetration into a sample without sectioning (“vital microscopy”)

Question 27

what occurs with a long wavelength

Answer 27

the longer the wavelength, the further it can get into the sample before it’s absorbed

Question 28

what occurs with a shorter wavelength

Answer 28

the shorter the wavelength, the higher the energy

Question 29

how does electron microscopy work

Answer 29

a focused beam of electrons replaces light - more than 200x increase in resolution than light microscopy

Question 30

why is a vacuum required in electron microscopy

Answer 30

so electrons don’t bounce. cells must be fixed and desiccated and then sliced into ultra-thin sections

Question 31

when do you have to use light microscopy specifically

Answer 31

when using live samples

Question 32

how do scientists solve the issue of cells being mostly transparent to electrons

Answer 32

electron-dense materials are used to stain cells. can also use gold-tagged antibodies to mark specific proteins/structures

Question 33

what colors do electron microscopy show up in

Answer 33

black and white

Question 34

what is SEM (scanning electron microscopy)

Answer 34

3D - images the outside surface - uses staining but no sectioning

Question 35

what is TEM (transmission electron microscopy)

Answer 35

2D - images internal structures - sliced ultra thin

Question 36

what does an electron beam give more resolution than light

Answer 36

because the wavelength of an electron is much shorter than the wavelength of a photon

Question 37

what is a simple definition of flow cytometry

Answer 37

single cell analysis - live cells are analyzed in real-time in an aqueous stream as they pass through a laser and a computer collects fluorescence data and laser scatter