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BIO 3153 > Intro + Microscopy > Flashcards

Intro + Microscopy Flashcards

1
Q

what are the 3 divisions of the living world

A

bacteria, archaea} (prokaryotes), eukaryotes

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2
Q

what divides the 3 branches?

A

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

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3
Q

what are the characteristics of eukaryotes?

A
  • 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
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4
Q

what is the average size of a eukaryote

A

10-100 microm

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5
Q

what about water’s structure makes it important?

A
  • 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
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6
Q

What are some characteristics of the plasma membrane?

A
  • 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
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7
Q

list the types of microscopy

A

light and electron microscopy

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8
Q

name the different types of light microscopy?

A

conventional, fluorescence, confocal, two-photon

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9
Q

what is the range of electron microscopy?

A

0.1nm - 1.1mm

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10
Q

what is the range of light microscopy?

A

10nm - 1.1mm

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11
Q

what is the range of naked eye?

A

100 microm - 1cm

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12
Q

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

A

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

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13
Q

what is the use of a upright vs inverted microscope?

A

upright microscope: tissues
inverted microscope: isolated cells

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14
Q

what is the structure of a light microscope?

A

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

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15
Q

name the type of conventional light microscopy?

A

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

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16
Q

how does bright field microscopy work?

A

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

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17
Q

how does phase contrast microscopy work?

A

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

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18
Q

what is differential interference contrast microscopy?

A
  • similar principles as with phase contrast
  • polarized light is separated and recombined
  • more definition
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19
Q

what is dark field microscopy?

A

lateral light source shows
only scattered light.

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20
Q

what’s the structure of phase contrast microscopy?

A

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

21
Q

what’s the structure of dic?

A

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

22
Q

list some facts about fluorescence microscopy

A
  • 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
23
Q

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

A

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

24
Q

what’s the structure of a fluorescence microscope?

A

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

25
Q

how are GFPs used?

A

permit selective labelling and imaging of cells in live specimen

25
Q

how are tissues prepared for microscopy?

A
  1. fixed
  2. embedded
  3. sectioned
  4. stained
25
Q

what does the fixation stage of tissue prep entail?

A

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

25
Q

what does the embedding stage of tissue prep entail?

A

plastic or polyethylene glycol to cover and embed tissue

25
Q

what does the sectioning stage of tissue prep entail?

A

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

25
Q

what does the staining stage of tissue prep entail?

A

only applicable sometimes, involves exposures to dyes

26
Q

what is immunofluorescence?

A

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

26
Q

what is the process of immunofluorescence?

A
  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
27
Q

what the advantages and disadvantages of confocal microscopy?

A

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

disadvtanges: expensive

28
Q

what is confocal microscopy?

A

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)

29
Q

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

A

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

30
Q

how does two-photon microscopy work?

A

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

31
Q

what are the two main types of electron microscopy?

A

transmission electron and scanning electron

32
Q

explain how electron microscopes work, their advantages and disadvantages

A

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

33
Q

what is the structure of a transmission electron microscope

A

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

34
Q

how do transmission microscopes work?

A
  • 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
35
Q

how are samples prepared for tem

A

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

36
Q

what is immunogold electron micropsocpy?

A

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

37
Q

what is the structure of a scanning electron microscope?

A

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

38
Q

how does scanning electron microscopy work?

A
  • 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
39
Q

how does ion changing work?

A

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

40
Q

what is the process of calcium imaging?

A

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

41
Q

how does x-ray crystallography work?

A

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

42
Q

what are the downsides to xray crystallography?

A

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

43
Q

what did x-ray crystallography help discover?

A

the structure of DNA, K+ ion channels

BIO 3153 (5 decks)

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