Topic 1: Studying Cells, Techniques

Question 1

What is the Schleiden and Schwann Cell Theory?

Answer 1

all organisms are composed of cells

the basic unit of life is a cell

all cells arise from pre-existing cells

Question 2

What are prokaryotic cells?

Answer 2

bacteria and archaea

does not include any membrane-bound organelle

no true nucleus

most are unicellular

Question 3

What are eukaryotic cells?

Answer 3

contains membrane-bound organelles

contains one true nucleus

can either be unicellular or multicellular

Question 4

What does unicellular mean?

Answer 4

one cell = one organism

eg. yeasts and protists

Question 5

What does multicellular mean?

Answer 5

often consist of differentiated cell types (specialized cell types)

eg. plants, fungi, animals

Question 6

How small are cells?

Answer 6

plant cells (20-30 um)

animal cells (20-30 um)

bacterium (1-2 um)

ribosome (25 nm)

microfilament (7 nm)

nm scale requires electron microscopy to resolve

Question 7

How do wavelengths impact microscopy?

Answer 7

light waves travel through objects and can change their path

when paths intersect of different waves of light they can reinforce or interfere with each other

two wavelengths of light can interact

two waves in the same place = reinforcement = appear brighter

if two waves are out of phase = interference = dimmer areas of a specimen

Question 8

What is resolution?

Answer 8

the minimum distance that two objects have to be in order to be distinguished as seperate

Question 9

What is light microscopy?

Answer 9

as light passes through the specimen, the phase and frequency of light can change

limit of resolution of any light microscope is 200 nm because of the visible spectrum of light cannot be used to prove structural details much smaller than it’s own wavelength

Question 10

What is the formula for resolution?

Answer 10

R = 0.61(wavelength)/NA

Low R = good microscope = better resolution

Question 11

What is interference microscopy?

Answer 11

increase contrast by manipulating the light source

phase contrast microscopy

differential interference contrast (DIC) microscopy

Question 12

What is phase contrast microscopy?

Answer 12

generates an image where contrast depends on refractive index of specimen

Question 13

What is differential interference contrast (DIC) microscopy?

Answer 13

use a prism to change phase of light passing through specimen

contrast is generated by differences in the index of refraction of the object and it’s surrounding medium

Question 14

What is fluorescence microscopy?

Answer 14

a type of light microscopy

very powerful technique for visualizing subcellular structures and even individuals proteins

fluorescent molecules are added to cell

see only the signal from the fluor

Question 15

What is fluorescence?

Answer 15

a molecule gets excited by one wavelength of light and emits another wavelength within visible spectrum

Question 16

What is immunofluorescence?

Answer 16

using fluorescently labelled antibodies

antibodies are immunoproteins made to detect a specific antigen

they can be used to detect specific proteins in cells

advantage: very specific and good resolution
disadvantage: requires fixed (dead specimens)

Question 17

What is antibody staining?

Answer 17

use 1o antibody = protein produced by immune system that is specific to antigen

first step: 1o binds antigen
second step: 1o antibody is detected by a 2o antibody, 2o antibody has been covalently linked to a fluor

Question 18

What is a green fluorescent protein (GFP)?

Answer 18

naturally occurring fluorescence protein

gene was cloned and added to DNA of other proteins

fusion protein retains normal protein function and glows

advantage: real-time localization, time-lapse images, many different GFP variants available in all sorts of colors, localization of known proteins

disadvantage: requires complicated genetic engineering and DNA reintroduction techniques, some proteins don’t like it

Question 19

What is confocal microscopy?

Answer 19

type of fluorescence microscopy

laser excites the specimen in a single plane of focus

get crisp images of only one plane of focus

can use it for live species, but time consuming

advantage: high resolution images with distinct focal planes

disadvantage: takes time to scan an entire specimen plane by plane

Question 20

What is electron microscopy (EM)?

Answer 20

much higher resolution because it is a beam of electrons, not wavelengths of light

requires fixed and sectioned cells (dead cells)

two types: scanning EM and transmission EM

approx. 40,000x better than light microscopy for theoretical limit of resolution

Question 21

What is transmission EM?

Answer 21

use e- beam instead of light

a very thin section of a fixed specimen encased in wax in a vacuum (this can distort specimen)

e- bounce off electron-dense structures

often use a gold-conjugate antibody to increase e- density of specific proteins

Question 22

What is scanning EM?

Answer 22

same concept as TEM but now it measures amount of electron scatter off the surface of a heavy-metal coated object

only visualize surfaces

can freeze fracture to visualize internal surfaces

Question 23

How do you study subcellular components?

Answer 23

many studies on cell structure and function require isolated organelles and cellular structure

a cell homogenate is first prepared by: sonication, chemical lysis, pressurized homogenates among others

need to disrupt the tissues/cells in a controlled way = called homogenate

Question 24

What is centifugation?

Answer 24

differential centrifugation allows separation of a filtered homogenate by sinning the suspension at increasingly higher speeds

separation based on density

Question 25

What is electrophoresis?

Answer 25

separation of proteins based on size

1: take sample of prep proteins
2: mercaptoethanol breaks S-S, denature proteins using SDS = detergent that denatures proteins and adds a net negative charge to unfolded protein
3: add proteins to gel

larger proteins stay near top
shorter proteins run further down gel

proteins migrate through the gel at a rate that reflects their size and number of amino acids

Question 26

What is western analysis?

Answer 26

method to find a specific protein from a mixture on a gel

“blot”: transfer proteins out of gel onto a filter paper where pattern from gel is maintained

use 1o and 2o antibodies to detect protein of interest

Why use?: detect presence of proteins, size, amount (thicker the band the more protein)