Flashcards in Microscopy Deck (27)
What happens at the limit of a microscope's resolution
Separate objects appear to merge
What does optical 'resolution' refer to?
The capacity of an optical system to reveal detail in a specimen
What is the resolution from a conventional light microscope
What is the resolution available from an electron microscope
What is the maximum available magnification from a student light microscope
What is the maximum available magnification from a electron microscope
Given the higher resolution and magnification, electron microscopes are said to display what aspect of cells and tissues
What are the two types of electron microscopy
Scanning and transmission EM
What are the features of scanning electron microscopy (2)
- 3D images
- Surface of object visible only
What are the features of transmission electron microscopy (4)
- 2D images
- Requires electrons to pass through the specimen to produce the image
- Requires ultrathin sections (50-100nm)
- More informative of tissue ultrastructure
How thin must sections be for transmission electron microscopy
What are the features of light microscopy (as compared to electron microscopy)? (3)
- Able to observe large areas of a specimen, (multiple cm2)
- Wide range of staining methods permitting identification of cell and tissue features (polychromatic)
- Can use slightly thicker sections to identify 3D features in certain specimens
What are the features of electron microscopy (as compared to light microscopy)? (4)
- Superior resolution and magnification - allows for identification of tissue ultrastructure
- Much smaller area available (<1mm2)
- Few staining methods, producing only monochromatic images
- Costly and time-consuming
What range of magnifications are available for EM
x500 - 190,000
How does an electron-dense area of an EM image appear?
How does an electron-lucent area of an EM image appear?
EM sections are almost featureless without which process?
Staining with heavy metals - uranium and lead salts
How does an area of an EM section come to appear electron-dense?
Heavy binding of the heavy metal staining, leading to impeded transmission of electrons through the specimen at this point (too dense to allow passage of electrons)
How does an area of an EM section come to appear electron-lucent?
Poor binding of the heavy metal staining, leading to greater transmission of electrons through the specimen at this point (allows easy passage of electrons)
Plasma and organelle membranes have what width?
What are histochemical techniques?
- Staining employing reagents known to react with defined cellular constituents (e.g. lipids, glycogen or DNA), thereby producing selective colourisation recognisable by LM
What is enzyme histochemistry?
A subset of histochemistry where staining is of enzyme substrates or end products, allowing demonstration of enzyme activity
What is immunohistochemistry?
Antibodies against specific cellular components (the antigen) is conjugated with an appropriate visual marker (for LM or EM) e.g. dyes, enzymes, particles of colloidal gold.
What problem with specimens is common to both LM and EM?
The need to prevent autolytic degeneration and preserve cellular ultrastructure
What do fixatives do?
Prevent autolytic degeneration by causing cross-linking of macromolecules, reducing and often arresting biological activity. Also renders cells more amenable to staining
What are the most common fixatives?