True or False:
fixation is easier than LM
If false, explain why.
- fixation more critical than LM
Why is fixation more critical than LM?
i. must stop life of cell without altering ità use very small pieces
- i. chemicals that denature and precipitate cellular macromolecules
- 1. chemicals having such action may cause the coagulation or precipitation of materials that had no structure in the living cell, leading to the formation of an artifact
True or False:
its not an artifact if its existence in cells fixed in a certain way or not at all
to view cells that haven’t been fixed, the tissue is rapidly __, and special techniques are used to __
- reveal the structure
Once fixed, what occurs?
i. tissues to be sectioned are usually embedded in __ and then bringing a plastic block down across a sharp cutting edge made of __ or __-->float up from __--> __
a. once fixed, water is removed by dehydration in alcohol and tissue spaes are filled with material for sectioning (thin)
- epoxy resins
- diamond or glass
- trough of water
can also stain with?
- floating the grids on drops of heavy metal solutions, which bind to macromolecules and provide atomic density required for electron scattering
- metal-tagged antibodies or others
a. cells don’t have to be __with __and embedded to be seen with __; they can be __ called __
What doesn't cryofixation do?
(doesn’t alter macromolecules and less likely to form artifacts)
Difficulty of cryofixing?
- a. : formation of ice crystals, which grow outward from sites where nucleation occurs
- i. as it grows, it destroys contents of the cell
How to avoid formation of ice crystals?
- i. avoid it by freezing quickly
- 1. as if water is frozen in liquid state= vitrified
- 2. small= immersed/ large= liquid nitrogen and hydrostatic pressure
a. __ can be prepared under a light or an electron microscope and useful for studying __
b. frozen cells don’t have to be __to reveal __
- Frozen sections
- internal structure
a. to get 3D image, __(3D, computational reconstruction) [putting pictures of different angles on top of each other is __ created by __]
- cryoelectron tomography
- Wolfgang Baumeister
a. shapes of proteins and nucleic acids can be resolved as long as contrast is __ --> __
- different from surroundings
- negative staining
What is negative staining?
heavy-metal deposits are collected everywhere on a specimen grid except where the particles are present
Shadow Casting for small particles:
a. __containing specimens are placed in a __, which is then evacuated by a __
i. chamber has __ (usually __) with __
- sealed chamber
- vacuum pump
- heavy metal
ii. __heated to high temps --> evaporate and deposit __ over surfaces within chamber
1. __ in EM are dark; __are bright
- metallic coat
- metal coated regions
a. using __ due to __, the particle appears __by a bright, white light with a dark shadow cast by the particle
b. great __and __ effect
- negative images
- photographic plates
What is viewed with freeze fracture replication and freeze etching?
small pieces of tissue put on metal disk and frozenà mounted on cool stage in vacuum chamber and the frozen tissue block is struck by knifeà 2 pieces
i. deviations in __ with __, __, and __ that reflect the contours of the __ traversed.
1. __gives info about contents of cell
- fracture plane
a. to make visibleà __
using fractured surface as template on which a heavy-metal layer is deposited to provide shadows that accentuate local topography