microscopy Flashcards
what are the three principles of cell theory?
1- basic unit of life is the cell
2- all animals and plants are made of cells
3-all cells are derived from pre-existing cells
what are the uses of microscopy?
- study cell structure
- study the intracellular localisation, dynamics and interactions of specific cellular components in living cells
how was mitosis first observed in a living cell?
drosophila embryos do not immediately undergo cytokinesis. first few divisions are nuclear. can observe e synchronous mitosis of nuclei in one cell
what is meant by amplitude?
‘height’ of the light wave, determines intensity of the light
why is wavelength important?
limits the size of the objects that can be seen
what happens when light interacts with an object?
there is a change in the phase relationship of the light waves
in what ways can the phase relationship of light waves be changed?
-if the light waves are in phase after passing through an object, this is constructive interference.
amplitude increases and object appears brighter
–light waves become out of phase - destructive interference. amplitude is smaller and the object appears lighter
what are the principle requirements of a light microscope?
1- magnification
2- resolution
3- contrast formation
4- illumination
what does the condenser of light microscope do?
focuses light from the light source onto the specimen
what is magnification determined by?
objective lens and eyepiece lens
total mag = eyepiece mag x objective mag
what is resolution defined as?
the ability to see two very small and closely spaced objects as separate entities
what is Abbe’s law used for?
for determination of the resolving power of the lens
what does numerical aperture of the lens measure?
light collecting ability
how does placing oil between the sample and the objective increase resolution?
increases the amount of light collected by the objective lens
in which two ways can contrast be increased?
1- biological stains
2- using light microscopes with special optical systems
give an example of a biological stain used to increase contrast
hematoxylin - has affinity for negatively charged molecules and will stain DNA, RNA and acidic proteins
how do biological stains increase contrast?
by reducing amplitude of light waves of a certain wavelength
what is meant by fluorescence?
a three stage process that occurs in molecules called fluorophores
how does fluorescence occur?
fluorophores are excited to a higher energy state, in this state, there are conformational changes and collisions which cause it to lose energy. when the molecule returns to the ground state, it has a much longer wavelength
which property of fluorescent dyes permit them to be used in microscopy?
each fluorescent dye has a characteristic excitation wavelength and emission spectrum
how are fluorophores excited in a fluorescent microscope?
using a halogen lamp
what is the function of the first barrier filter in a fluorescent microscope?
lets through only blue light of wavelength 450-490nm
what is the purpose of the dichroic mirror in a fluorescent microscope?
reflects light below 510nm but transmits light above 510nm
what is the purpose of the second barrier filter in a fluorescent microscope?
cuts off unwanted fluorescent signals, passing the emission to the eyepiece
what is immunofluorescent microscopy used for?
detecting specific molecules inside fixed cells
what is the difference between a monoclonal and polyclonal antibody?
polyclonal antibodies bind to and recognise several epitopes on one protein, whereas monoclonal antibodies bind and recognise only a single epitope
what is a hybridoma?
refers to the process of fusing a B-lymphocyte to a cancer cell, generating a single type of antibody
what is direct immunofluorescence microscopy used for?
high abundance proteins
why is BSA used as part of immunofluorescence microscopy?
prevents proteins from binding non-specifically via hydrogen bonding, hydrophobic effect etc.
what is different about indirect immunofluorescence microscopy?
you use a primary (unlabelled) antibody and aa secondary antibody to amplify the signal from the first antibody
what is useful about GFP?
can be used to study the intracellular location of a protein in living cells
what is the structure of GFP?
monomeric form is made up of 11 antiparallel B-strands in a beta barrel with a single a-helix in the centre. the a-helix contains 3 residues which make up the GFP fluorophore
what 3 amino acids make up the GFP fluorophore?
Gly-67, Ser-65 and Tyr-66
why are confocal laser scanning microscopes useful?
complex three dimensional objects are difficult to image using a conventional light microscope
how is a sample prepared for use in a transmission electron microscope?
1 - specimen is fixed
2- specimen is dried
3- specimen is embedded in a plastic resin
4- the stained sample is placed on a copper grid in a microscope
what is the resolution of an electron microscope?
0.2nm
