visualisation Flashcards
(19 cards)
how does fluorescence occur?
1) fluorescent compound is excited to a higher energy level
2) the molecule spends a finite length of time in this excited state (5-10ns)
3) fluorescent molecule undergoes a conformational change and collisions, resulting in a loss of energy
4) the electrons transition to their ground state, they emit photons at a much longer wavelength
what is the characteristic difference between emission wavelength and excitation wavelength that permits fluorescence microscopy?
emission wavelength is always longer
what is FITC and what is its structure?
Fluorescin Isothiocyanate; contains a polyaromatic ring structure with many double bonds
how does FITC join to proteins?
reacts with the amino groups of amino acids
at which range does FITC absorb and emit light?
absorbs: 495-529 wl
emits: 520-560 wl
what is the use of fluorescent dye-conjugated antibodies?
can be used to measure the cellular distribution of a particular protein
why might alexafluor dyes be preferred over FITC?
they are brighter and more photostable
they emit light in all parts of the visible spectrum and some infrared
how does a fluorescence microscope work?
by allowing us to selectively observe the emitted light by reflecting the excitation light onto a dichroic mirror into the sample
outline the series of barriers that permit emitted light to be isolated in a fluorescence microscope
1) first barrier filter: only lets through blue light in the wavelength 450-490nm
2) beam splitting (dichroic) mirror: reflects light below 510nm but transmits light above 510nm
3) second barrier filter: cuts off unwanted fluorescent signals, passing the specific green fluorescin emission between 520-560nm
what is a requirement for fluorescence microscopy?
a light source to excite the molecules of FITC
what is the use of immunofluorescence microscopy?
detecting specific molecules inside fixed cells
outline the structure of an antibody
two heavy chains, two light chains, held together by disulphide bonds. hinge region allows flexibility
where is the antigen binding site located on the antibody?
at the amino terminus
what is the difference between monoclonal and polyclonal antibodies?
monoclonal antibodies bind only a single epitope and do not cross react with other proteins whereas polyclonal antibodies bind several different epitopes
list the advantages and disadvantages of polyclonal antibodies
- inexpensive to produce
- low skills required for production
- quick to produce
- generate large amounts of non-specific antibodies
- recognise many epitopes on one antigen
list the advantages and disadvantages of monoclonal antibodies
- more expensive to produce
- more difficult technique
- hybridomas take a relatively long time to produce
- generate large amounts of specific antibodies
what is a hybridoma?
when a B-lymphocyte is fused to a cancer cell to produce a single, specific antibody
what is direct immunofluorescence microscopy ideal for?
high abundance proteins
what is indirect immunofluorescence microscopy ideal for?
low abundance proteins
