2.1.1 Microscopy Flashcards
How does the resolution differ between a light, electron and laser microscope?
TEM - 0.5nm
SEM - 3-10nm
Laser scanning confocal - 15-20nm
Light microscope - 200nm
How does the magnification differ between a light and electron microscope?
Electron (TEM and SEM) - x500,000
Light - x1,500
What are the differences between a scanning electron microscope and a transmission electron microscope?
SEM: beams sent across surface of specimen - produces 3D images, lower resolution
TEM: higher resolution, electrons sent through specimen
Explain the structure of a compound light microscope.
Two lenses - eyepiece at top to look through, objective lens that produces a further magnified image
Light comes from below the stage to illuminate the specimen
How does a laser scanning confocal microscope work?
A laser beam is moved along a specimen and reflected by a dichroic mirror and through an objective lens back onto the specimen. The light emitted from the specimen is filtered through to the detector. Light that is reflected close to the focal plane produces a clear image.
Why do electron microscopes produce greater detail than light microscopes?
Electrons have a much smaller wavelength (less than 1nm) than light waves
What is chromatic abberation?
The effect of refraction of different wavelengths of light through slightly different angles, resulting in a lack of focus.
What are artefacts?
Something that isn’t typical of the cell’s structure, but is the result of a process such as staining
Explain dry mount preparation.
Sample placed on slide, cover slip over the sample
e.g. hair sample
Explain wet mount preparation.
Sample suspended in liquid, cover slip over the sample
e.g. aquatic organisms
Explain squash slide preparation.
Wet mount prepared, then cover slip is pressed down over sample
e.g. root tips to look at cell division
Explain smear slide preparation.
Sample smeared thinly along slide, then cover slip placed on top
e.g. blood sample
What is positive staining?
Positive stain attracted to negative ions within cytoplasm of cells that absorb the stain
e.g. crystal violet, methylene blue
What is negative staining?
Negative stain repels negative ions within cytoplasm, so they stand out when all else is stained
e.g. congo red, nigrosin, safranin
What does acid fast stain do?
Helps differentiate acid-fast bacteria (Mycobacterium) from other bacteria. A lipid solvent carries carbolfuchsin dye into the cells, then it’s washed with acid-alcohol solution. It retains the dye while other bacteria lose the stain and are exposed to methylene blue stain.
What is the magnification formula?
Actual size = Image size / Magnification