Methods Flashcards
How does TEM work?
cathode placed on top of cylindrical column
E-s accelerated by anodes & focussed by magnetic coils
spec in vacuum
electrons pass through spec stained with electron dense material –> scattered.
dense region is dark
how does a fluorescent microscope work?
illuminating light through 2 filters
exciting wavelengths pass through
emitted by dye fluorescing pass through
Outline confocal microscopy.
pinpoint illuminate a single point in specimen (depth)
fluorescence emitted, collected at detector with pinhole aperture (confocal to illuminating source).
what is a condenser used for?
lens system focussing light from source onto bright spot of specimen.
what combination produces the greatest resolution and what is the final magnification a combination of?
a higher OBJECTIVE LENS aperture
a shorter wavelength
objective (100) x eyepiece (10-20).
what are downsides to TEM?
What are electrons?
needs very strong fixation not to burn in electron beam thin sections (100nm) for electrons to get through.
particles that can act as waves but have amplitudes smaller than light.
which small compounds can be used when conjugated to fluorophores?
DAP1 - binds DNA
Phalloidin - binds MTs.
how does mitochondrial protein synthesis resemble that of prokaryotes?
fist amino acid is fMet
Antibiotics (streptomycin) doesn’t affect eukaryotes
Rifampicin (doesn’t inhibit RNA polymerase in eukaryotes).
How can microscopy be used to visualise cell trafficking?
fuse DNA with putative SS to DNA for CYTOSOLIC protein
identify fusion protein by immunostaining
what is Brefeldin A?
fungal antibiotic (macrocyclic lactone) inhibiting anterograde transport
(blocks ARF GTP binding protein - controls vesicle budding from ER)
Golgi disassembles but not remade
Briefly outline the modern cell theory?
all living organisms made of cells
cell is functional and structural unit and all metabolic reactions within cells.
cells arise from preexisting cells by diffusion.
cells contain hereditary info (DNA).
Why are yeast used as model organisms?
Eukaryotic
Easy to manipulate
Haploid
Small genome
