Intro + Microscopy Flashcards
what are the 3 divisions of the living world
bacteria, archaea} (prokaryotes), eukaryotes
what divides the 3 branches?
the distance between them is based on the differences in their nucleotide sequences as there is vast variability
what are the characteristics of eukaryotes?
- 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
what is the average size of a eukaryote
10-100 microm
what about water’s structure makes it important?
- 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
What are some characteristics of the plasma membrane?
- 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
list the types of microscopy
light and electron microscopy
name the different types of light microscopy?
conventional, fluorescence, confocal, two-photon
what is the range of electron microscopy?
0.1nm - 1.1mm
what is the range of light microscopy?
10nm - 1.1mm
what is the range of naked eye?
100 microm - 1cm
how do light microscopes work and what are they used for?
utilizes: basic light path
used for: live or fixed cells and tissue
what is the use of a upright vs inverted microscope?
upright microscope: tissues
inverted microscope: isolated cells
what is the structure of a light microscope?
eye > eyepiece > tube lens > objective > specimen > condenser > iris diaphragm > light source
name the type of conventional light microscopy?
bright field, phase contrast, differential interference contrast, dark field
how does bright field microscopy work?
Light passes through the sample, and the image is formed based on light absorption, scattering, or transmission.
how does phase contrast microscopy work?
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
what is differential interference contrast microscopy?
- similar principles as with phase contrast
- polarized light is separated and recombined
- more definition
what is dark field microscopy?
lateral light source shows
only scattered light.
what’s the structure of phase contrast microscopy?
light source > annular diaphragm > condenser lens > direct light (phase unaltered by specimen) > phase plate > image plane
what’s the structure of dic?
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
list some facts about fluorescence microscopy
- 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
What kinds of dyes and molecules are used for fluorescence microscopy?
DAPI, GFP (green fluorescent protein), FITC (dye)
what’s the structure of a fluorescence microscope?
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
how are GFPs used?
permit selective labelling and imaging of cells in live specimen
how are tissues prepared for microscopy?
- fixed
- embedded
- sectioned
- stained
what does the fixation stage of tissue prep entail?
exposures to chemical reagents to preserve and stabilize a tissue (may produce unwanted effects/reactions)
what does the embedding stage of tissue prep entail?
plastic or polyethylene glycol to cover and embed tissue
what does the sectioning stage of tissue prep entail?
cutting thin (1-10 microm) tissue sections with a microtome
what does the staining stage of tissue prep entail?
only applicable sometimes, involves exposures to dyes
what is immunofluorescence?
technique that allows detection and localization of biomolecules within cells and tissue
what is the process of immunofluorescence?
- antibodies produced and collected in host animal
- tissues are fixed, permeabilized () and treated with primary antibodies direct to target a specific molecule/antigen
- antibodies bind to antigen on or within cell
- secondary antibodies are conjugated with a fluorescent marker to bind to the primary antibodies
- allows for the labelling of cell structures based on where these antibodies bind
what the advantages and disadvantages of confocal microscopy?
advantages:
- technique provides clear images that reduce background signal
- useful for applications involving thick sections
disadvtanges: expensive
what is confocal microscopy?
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)
what are some advantages and disadvantages of two-photon microscopy?
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
how does two-photon microscopy work?
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
what are the two main types of electron microscopy?
transmission electron and scanning electron
explain how electron microscopes work, their advantages and disadvantages
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
what is the structure of a transmission electron microscope
electron gun > condneser > specimen > objective lens > projector lens > viewing screen
how do transmission microscopes work?
- 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
how are samples prepared for tem
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
what is immunogold electron micropsocpy?
uses antibodies to bind to a specific cell and locates through a secondary antibody conjugated with gold nanoparticles. shows up as dark dots
what is the structure of a scanning electron microscope?
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
how does scanning electron microscopy work?
- 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
how does ion changing work?
since changes in intracellular ion concentrations are important, ion-selective indicators emit light depending on said concentrations to reveal rapid intracellular dynamics
what is the process of calcium imaging?
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
how does x-ray crystallography work?
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
what are the downsides to xray crystallography?
very time-consuming (especially when first founded), needs a large amount of materials and it is hard to crystallize proteins
what did x-ray crystallography help discover?
the structure of DNA, K+ ion channels
