Lab 4: Microscopy Flashcards
condenser lens
gathers the diffuse rays from the light source and illuminates the specimen with a small cone of bright light.
Objective lens
collects light rays focused on specimen by condenser lens
Magnification
ocular x objective magnification
Empty magnification
no further detail produced by an increase in magnification because there is no further detail to enhance
Resolving power
defined in terms of the ability to see two neighbouring points in the visual field as two distinct entities—limited by the wavelength of the illumination.
Limit of resolution for light microscope
0.2 um (200 nm) —sufficient to resolve larger cellular organelles (nuclei or mitochondria)
Best ways to make thin, translucent specimen visible? Downsides?
- staining with dye (specific dyes bind with certain biological molecules)
- usually kills cell
Bright-Field Microscopy
cone of light that illuminates the specimen is seen as bright background and specimen is dark (good for specimens of high contrast)
Whole mount
intact object (living or dead)
section
very thin slice of specimen (cells must be killed by immersing tissue in fixative which essentially immobilizes cell in as close to its live state as possible. Then embedded in paraffin wax so it can be sliced, then slide with wax is immersed in toluene to dissolve wax).
Common fixatives for light microscopy
formaldehyde, alcohol, acetic acid
Phase-Contrast Microscopy
Makes highly transparent objects more visible. Extremely useful in teaching situation (observing live cells). Haloes around objects.
Differential Interference Contrast Microscopy
DIC or Nomarski
- very similar to phase contrast microscopy; delivers image with apparent 3D quality
Fluorescence Microscopy
Allows viewers to observe location of certain molecules. Can be used to study dynamic processes in cell like tagging DNA or RNA with specific sequences.
Fluorophores
absorb photons of a specific wavelength and re emit portion of energy in longer wavelength (fluorescence).
GFP
Green fluorescent protein
- doesn’t require an additional cofactor to absorb and emit light; instead the light absorbing/emitting chromophore is formed by self modification of three of the amino acids that make up primary structure of the GFP polypeptide.
- coding region of GFP is joined to coding region of gene for protein under study
FRET
Fluorescence Resonance Energy Transfer
- can measure distances between fluorophores in nanoscale range. Usually used to measure changes in distance between two parts of a protein.
- uses idea that excitation energy can be transferred from one fluorescent group to another as long as bodies are close enough (1 to 10 nm)—efficiency of the transfer depends on distance
WHat can FRET be used for?
can follow protein folding or association and dissociation of components within a membrane
FRAP
Fluorescence recovery after photobleaching
- used to study rate of protein turnover in living cells
Confocal Microscopy
Produces image of thin plan situated within much thicker specimen. Laser beam rapidly scans specimen at single depth thus illuminating only a thin plane (optical section)
Light Sheet Fluorescence Microscopy
Noninvasive imaging of large, three-dimensional samples (whole embryos or organisms)
TEM
Transmission Electron Microscopy
- forms images using electrons transmitted through specimen
- much higher res images that any light microscope because wavelengths are that much smaller
-
SEM
Scanning EM
- uses electrons that have bounced off surface of specimen
Limit of resolution for TEM
practical limit is 3 to 5 angstroms (tenth of nm) but actual limit when observing cellular structures is more typically 10 to 15 angstroms
The scattering of electrons by a part of the specimen is proportional to
the size of the nuclei of the atoms that make up the specimen.
Specimen preparation for TEM
Heavy metals used to increase the electron scattering capabilities of biological tissue. Fixatives must stop life of cell without altering the structure of the cell.
Artifact
Structure that isn’t present in living cell, was a result of fixative chemicals changing cell. Must prove structure isn’t an artifact by other imaging methods (live cell imaging ideally)
Common fixatives for EM
glutaraldehyde
- has aldehydes which react with amino groups in proteins and cross link proteins into insoluble networks
osmium textroxide
- heavy metal that reacts with fatty acids to preserve cellular membranes
Thickness of wax sections for EM
0.1 um (equiv to thickness of four ribosomes)
Stains for EM samples
heavy metal solutions
- uranyl acetate and lead citrate
Cyrofixation
rapid freezing of a sample
- less likely to lead to formation of artifacts because it doesn’t alter macromolecules but formation of ice crystals is an issue
- much faster prep
After fixative, samples must be…
dehydrated
Electron microscopy is well suited to study
very small particulate materials like viruses, ribosomes, multi subunit enzymes, cytoskeletal elements, and protein complexes.
Goal of specimen prep. for SEM
To produce object that has the same shape and surface properties as living state, but is devoid of fluid (so it can be observed in vacuum). Air drying samples results in destruction largely from surface tension at air-water interfaces.
Critical point drying
Used to prepare samples for SEM
- takes advantage of the fact that every solvent has a critical point where gas and liquid density are equal thus no surface tension exists between the two states which can alter 3D structure
- solvent of specimen replace with liquid CO2 which is vaporized under pressure
Difference between electron beams in SEM and TEM
TEM - electron beam is focused by condenser lenses to simultaneously illuminate entire viewing field (image = electrons that pass through specimen)
SEM - electrons are accelerated as fine beam that scans the specimen (image = electrons that are scattered back aka indirect image formation)
Resolving power of SEM
related to diameter of electron beam - resolutions less than 5 nm
What can you observe with SEM?
Structure of outer cell surface and all various processes, extensions, and extracellular materials.
