Intro + Microscopy

Question 1

what are the 3 divisions of the living world

Answer 1

bacteria, archaea} (prokaryotes), eukaryotes

Question 2

what divides the 3 branches?

Answer 2

the distance between them is based on the differences in their nucleotide sequences as there is vast variability

Question 3

what are the characteristics of eukaryotes?

Answer 3

• have a nucleus
• 1000x larger by volume, - a phospholipid bilayer membrane (have an inner and outer membrane)
• intercellular communciation
• high degree of organization and compartmentalization
• made of 70% H2O, ATP required
• chemical comp: sugars, fatty acids, amino acids, nucleotides

Question 4

what is the average size of a eukaryote

Answer 4

10-100 microm

Question 5

what about water’s structure makes it important?

Answer 5

• covalent bonds that make it weak enough to be a universal solvent
• weak hydrogen bonds ensure different H2O molecules
• can be a solid liquid or gas
• can support a lot of reactions
• hydrophobic effect that causes the cell membrane to function

Question 6

What are some characteristics of the plasma membrane?

Answer 6

• amphipathic (hydrophobic/philic)
• arranged in oil and water
• phospholipids arrange in a bilayer
• permeable, has certain membrane proteins that allow for exchange of important ions, proteins, etc that are not permeable

Question 7

list the types of microscopy

Answer 7

light and electron microscopy

Question 8

name the different types of light microscopy?

Answer 8

conventional, fluorescence, confocal, two-photon

Question 9

what is the range of electron microscopy?

Answer 9

0.1nm - 1.1mm

Question 10

what is the range of light microscopy?

Answer 10

10nm - 1.1mm

Question 11

what is the range of naked eye?

Answer 11

100 microm - 1cm

Question 12

how do light microscopes work and what are they used for?

Answer 12

utilizes: basic light path
used for: live or fixed cells and tissue

Question 13

what is the use of a upright vs inverted microscope?

Answer 13

upright microscope: tissues
inverted microscope: isolated cells

Question 14

what is the structure of a light microscope?

Answer 14

eye > eyepiece > tube lens > objective > specimen > condenser > iris diaphragm > light source

Question 15

name the type of conventional light microscopy?

Answer 15

bright field, phase contrast, differential interference contrast, dark field

Question 16

how does bright field microscopy work?

Answer 16

Light passes through the sample, and the image is formed based on light absorption, scattering, or transmission.

Question 17

how does phase contrast microscopy work?

Answer 17

It converts differences in the refractive index aka phase differences (light-bending properties) of transparent samples into differences in light intensity.

wave properties of light can be exploited. in unstained cells, a phase shift will occur as light travels through the cell. phase alignment is related to increased brightness; if not aligned, decreased brightness. observable with phase contrast

Question 18

what is differential interference contrast microscopy?

Answer 18

• similar principles as with phase contrast
• polarized light is separated and recombined
• more definition

Question 19

what is dark field microscopy?

Answer 19

lateral light source shows
only scattered light.

Question 20

what’s the structure of phase contrast microscopy?

Answer 20

light source > annular diaphragm > condenser lens > direct light (phase unaltered by specimen) > phase plate > image plane

Question 21

what’s the structure of dic?

Answer 21

light source > polarizer > wollaston prism > conenser lens > 2 beams of plane-polarized light separated by the prism below > stage > specimen > objective lens > wollaston prism > analyzer (rotated 90* with respect to polarizer) > image plane

Question 22

list some facts about fluorescence microscopy

Answer 22

• detects specific molecules and ions
• works as it atomically abrobs a photon, then emits it at a specific, longer wavelength so a light signal is detected
• fluorescent molecules are used to emit certain lights; each has special characteristics from each dye and molecule

Question 23

What kinds of dyes and molecules are used for fluorescence microscopy?

Answer 23

DAPI, GFP (green fluorescent protein), FITC (dye)

Question 24

what’s the structure of a fluorescence microscope?

Answer 24

looks similar to a light microscope
light source (high energy lamp) > first barrier filter (reduces unwanted wavelengths) > beam-splitting mirror uses chromophores to excite wavelengths > second barrier filter (reduces unwanted wavelengths, transmitting wavelengths

Question 25

how are GFPs used?

Answer 25

permit selective labelling and imaging of cells in live specimen

Question 25

how are tissues prepared for microscopy?

Answer 25

1. fixed 2. embedded 3. sectioned 4. stained

Question 25

what does the fixation stage of tissue prep entail?

Answer 25

exposures to chemical reagents to preserve and stabilize a tissue (may produce unwanted effects/reactions)

Question 25

what does the embedding stage of tissue prep entail?

Answer 25

plastic or polyethylene glycol to cover and embed tissue

Question 25

what does the sectioning stage of tissue prep entail?

Answer 25

cutting thin (1-10 microm) tissue sections with a microtome

Question 25

what does the staining stage of tissue prep entail?

Answer 25

only applicable sometimes, involves exposures to dyes

Question 26

what is immunofluorescence?

Answer 26

technique that allows detection and localization of biomolecules within cells and tissue

Question 26

what is the process of immunofluorescence?

Answer 26

1. antibodies produced and collected in host animal 2. tissues are fixed, permeabilized () and treated with primary antibodies direct to target a specific molecule/antigen 3. antibodies bind to antigen on or within cell 4. secondary antibodies are conjugated with a fluorescent marker to bind to the primary antibodies 5. allows for the labelling of cell structures based on where these antibodies bind

Question 27

what the advantages and disadvantages of confocal microscopy?

Answer 27

advantages: - technique provides clear images that reduce background signal - useful for applications involving thick sections disadvtanges: expensive

Question 28

what is confocal microscopy?

Answer 28

confocal meaning there is an equal distance between a light source and object as the object to the detector - uses fluorescence and high-energy lasers with a light source focusing on a specimen through a pinhole of light - 3D reconstructions can be possible (xyz axes)

Question 29

what are some advantages and disadvantages of two-photon microscopy?

Answer 29

advantages: - use higher-order light-matter interactions from multiple photons to generate contrast (instead of single photon like fluorescence) - allows deep tissue imaging up to a mm in depth - absorption occurs in the infared region while near infared light penetrates deep into tissues disadvantage: very expensive

Question 30

how does two-photon microscopy work?

Answer 30

excites molecules rapidly - protons excited then emitted - signal is dependent on photon density and sample thickness so absorption is confined by space (excitation only happens at the focal point) - high-ernergy laser pulses emit photons, no pinholes

Question 31

what are the two main types of electron microscopy?

Answer 31

transmission electron and scanning electron

Question 32

explain how electron microscopes work, their advantages and disadvantages

Answer 32

uses a bombardment of electrons instead of light advtange: resolution is significantl better, 0.1 nm=1.0 Ångströms (Å), lower resolution in some preperations like 2 nm disvtange: time consuming preparation

Question 33

what is the structure of a transmission electron microscope

Answer 33

electron gun > condneser > specimen > objective lens > projector lens > viewing screen

Question 34

how do transmission microscopes work?

Answer 34

- electrons are emitted at filament (wire) or cathode (negatively charged electron) - accelerated by the high voltage in a vacuum and magnetic coils focus electron beams like a lens - samples can be stained to produce electron-dense images

Question 35

how are samples prepared for tem

Answer 35

can take days-weeks - tissues fixed in glutaraldehyde - OsO4 is added to increase electron density - sample is dehydrated and infiltrated with plastic resin giving support - ultrathin sections (50-100 nm) cut with diamond knife - sections cant be handled directly and are placed on copper grids

Question 36

what is immunogold electron micropsocpy?

Answer 36

uses antibodies to bind to a specific cell and locates through a secondary antibody conjugated with gold nanoparticles. shows up as dark dots

Question 37

what is the structure of a scanning electron microscope?

Answer 37

electron gun > condenser lens > beam difiner (goes through a scan generator and video screen so a detector can produce images as the electrons hit the specimen) > objective lens > electrons hit the specimen and force it to give off its own electrons

Question 38

how does scanning electron microscopy work?

Answer 38

- produces 3d images of surface structures to study whole cells and tissues - prepped similarly to TEM - cells are coated with a heavy metal and scattered electrons from the specimen's surface are collected to produce an image

Question 39

how does ion changing work?

Answer 39

since changes in intracellular ion concentrations are important, ion-selective indicators emit light depending on said concentrations to reveal rapid intracellular dynamics

Question 40

what is the process of calcium imaging?

Answer 40

low Ca2+ can be tracked during fertilization with bioluminescent proteins like aequorin. fluorescent dyes can be used to emit wavelengths and decipher info about binding/concentration (dye delivered through injection or membrane-permeable AM (acetoxymethyl) esters

Question 41

how does x-ray crystallography work?

Answer 41

assesses 3D structures of macromolecules in atomic resolution through x-rays (01.nm) to bombard and diffract pre-crystallized proteins. the way they diffract shows macromolecule's arrangement

Question 42

what are the downsides to xray crystallography?

Answer 42

very time-consuming (especially when first founded), needs a large amount of materials and it is hard to crystallize proteins

Question 43

what did x-ray crystallography help discover?

Answer 43

the structure of DNA, K+ ion channels