Unit 2: Microscopy Flashcards
Specification reference 2.1.1
Define magnification
How much bigger the image is than the sample (specimen)
Define Resolution
How detailed the image is, it is the ability of a microscope to distinguish between to points that are close together
Equation for magnification
Image size = Actual size x Magnification
Two types of microscope
Electron
Light
Two types of electron microscope
Scanning (SEM)
Transmission (TEM)
Advantages/Disadvantages of TEM
Advantages - Provide high resolution images
Disadvantages- Specimen need to be sliced thinly.
Max Resolutions
Light microscope - 0.2 μm
TEM - 0.0005 μm
SEM - 0.003 - 0.01 μm
Lower resolution = better the image quality
Max Magnification
Light microscope - x1500
TEM - more than x1,000,000
SEM - less than x1,000,000 but stunning 3-D images
higher magnification the better
How does staining enable cell components to become visible?
Resolution is limited by wavelength of light and the diffraction of light as it passes through a sample. As most cell structures are normally transparent, images have low contrast as they do not absorb light. Stains increase the contrast as different components within the cells take up the stain to varying degrees, enabling components to become visible.
Give two examples of stains.
Methylene blue - positively charged dye, attracted to negatively charged materials in the cytoplasm - stains RNA/DNA
Congo red - negatively charged dye, that repels negatively charged cytosol. Stains outside of cell, leaving inside unstrained.
Eosin - The most common dye to stain used in histology, stains pink/orange. It is a negatively charged, acidic dye, that binds to basic components of a cell, mainly proteins located in the cytoplasm.
Iodine - Stain commonly used to observe plant cells.
What are the rules for producing good scientific drawings?
- Include a title
- State magnification
- Use a sharp pencil for drawings and labels
- Use white, unlined paper
- Use as much paper as possible for the drawing
- Draw smooth, continuous lines
- Do not shade
- Draw clearly defined structures
- Ensure proportions are correct
- Label lines must be parallel to the top of the page and drawn with a ruler. They must not cross or have arrow heads.
Describe electron micrograph images
They are always black and white (but sometimes colour is added artificially to images don’t confuse this with staining).
What is the Gram stain technique?
A technique used to separate bacteria into two groups, Gram-positive bacteria (e.g Streptococcus pneumonae - causes pnuemonia) and Gram-negative bacteria (e.g. Yersinia pestis - causes bubonic plague).
Crystal violet is first applied to a bacterial specimen, then iodine, which fixes the dye. Slide then washed with alcohol. Gram-positive bacteria retain the dye where as Gram-negative bacteria have much thinner walls and lose the stain.
Gram-negative bacteria are stained with the counterstain safranin, which makes them appear red.
What are the stages involved in preparing stained slides?
- Fixing - Chemicals like formaldehyde are used to preserve specimens.
- Sectioning - specimens are dehydrated with alcohols and then placed in a mould with wax/resin to form a hard block that can be thinly sliced.
- Staining - treatment of specimen with (often multiple) stains to show different structures.
- Mounting - specimens are then secured to a microscope slide an a cover slip placed on top.
What is an eyepiece graticule?
A glass disc marked with a fine scale of 1 to 100. The scale has no unit and remains unchanged whichever object lens is in place. The relative size of the dimensions, however, increases with each increases in magnification. The scale on the graticule at each magnification is calibrated using a stage micrometer.
