cours 3 - Microscopy XRay Flashcards
Describe a confocal microscope
- Only light focused at the pinhole will enter the detector.**
- This allows the confocal to provide clear images a few μm into tissue.
- In addition to the x and y axes, imaging may occur in the z axis.
- 3D reconstructions possible.
what are the advantages and disadvantages of two-photon microscopy?
Advantages:
• Non-linear technique (takes more than 1 photon to exite)
• Allows deep tissue imaging (up to 1 mm depth possible).
• In this process, absorption occurs in the near IR region,
Disadvantage:
• Very costly.
Describe the process of two photon microscopy
you need 2 photon arriving at the same time to exite 1 photon.
allows for deeper imaging
focus on a specific point (compare to fluo or confocal where everything is exited)
differs from confocal by exitation and detection pathway
uses rapid high energy laser pulses (100fs & 100Mhz)
no pinhole needed compare to confocal
What are the 2 types of electron microscope?
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
What are the advantages and disadvantages of electron microscopy?
resolution 2000X better than light microscopy
disadvantages: time consuming preparation
desctibe electron microscopy
- TEM: cut sections and look at organelles of cells
- SEM:see surfaces features like topology
- uses bombardment of electron rather than light
- configuration of the microscope is similar to light microscope (but condenser and objective lens are a magnets)
- electrons emitted at the cathode
- they are accelerated by a high voltage in a vaccuum
- a magnetic coil focus the electrons (like a lense)
- dark area: electron dense
- light area: electron nuisant
Descripe the tissus preparation necessary for transmission electron microscopy.
- Lengthy procedure that takes days to weeks.
- Tissue must be fixed in glutaraldehyde.
- Addition of OsO4 increases electron density.
- Dehydration and infiltration with a plastic resin gives extra support.
- Ultrathin sections (50 – 100 nm) must be cut with a diamond knife.
- Sections cannot be handled directly.
- Placed on copper grids.
describe TEM
- General configuration analogous to LM.
- Electrons emitted at cathode.
- Accelerated by high voltage (105 V) in a vacuum.
- Magnetic coils focus electron beam like a lens.
- Sample may be stained, producing “electron dense” images.
define immunogold EM
element like gold reflect electron bc it’s dense. so it dosent reflect light. so you can attach gold to protein to identify them
Describe scanning electron microscope.
- Produces 3D images of surface structures.
- Used to study whole cells and tissues, rather than intracellular structures.
- Principles of preparation and operation similar to TEM
- Cells/tissues coated with heavy metal.
- Scattered electrons from the specimen surface are collected.
Describe ion imaging?
- Changes in intracellular ion concentrations (e.g.Ca2+ and H+) are physiologically important.
- Ion-selective indicators emit light depending on local ion concentrations.
- These reveal rapid intracellular dynamics.
Describe Ca2+ imaging.
- Intracellular Ca2+ is low.
- Bioluminescent aequorin injected into a fish egg reveals Ca2+ wave propagated during fertilization. (to prevent fertilisation from other sperm)
- Other synthesized indicator molecules produce signal
- Ratiometric imaging exploits differential wavelengths associated with ionic
binding. - Dyes can be injected, or “AM” analogues can be used to cross cell membrane. = acetoxymethyl ester
What can x-ray crystalography detect?
- Structure - function
- Macromolecules
- Atomic resolution
- Crystallized proteins
- Bombardment and diffraction.
- e.g. Interference patterns
- e.g. structure of DNA
- e.g. Structure of K+ ion channel can be seen by X-ray crystallography. with Monoclonal antibodies
to “hold” protein.
What are the problems with x-ray crystallography?
- Years ago was very time-consuming (first structure…22 yrs!).
- Large amount of material required.
- Insoluble protein crystallization (e.g. membranes)?
describe x-ray crystallography
What microscope / type of microscopy is this image taken with?
bright field (light microscope)
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phase contrast (light microscope)
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Differential Interference Contrast (DIC) (light microscope)
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Dark field (light microscope)
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fluorescent microscope
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fluorescent microscope
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confocal
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2 photon
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TEM micrograph (Electron Microscopy)
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Immunogold EM (Electron Microscopy)
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SEM Micrographs (Electron Microscopy)
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SEM Micrographs (Electron Microscopy)
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DIC Micrographs (Electron Microscopy)
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TEM Micrographs (Electron Microscopy)
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X-ray Crystallography
