A2.2 - microscopy theory (3c) Flashcards
how do you calculate magnification?
magnification = eyepiece lens x objective lens
how do you calculate image size?
image size = actual size x magnification
how do you measure sizes using an eyepiece graticule, including producing a scale bar?
a graticule is located in the eyepiece and is engraved with equal units
- these units are arbitary and the size they measure depends on the particular microscope and the total magnification being used to view the specimen, based on the choice of objective lens
- to calibrate a graticule a stage micrometer is used
a stage micrometer is a slide containing a scale of known size (similar to a tiny ruler)
- by comparing the units of the graticule to the known unit size of the stage micrometer you can determine the size of the object being examined
scale bars are drawn proportionally to a structure of known size, as determined using a graticule and stage micrometer, but with a round-valued length
how do you calculate a graticule unit?
comparing the units of the graticule to the known unit size of the stage micrometer allows you to determine the size of the object being examined
how do you measure the actual size and image size?
actual size - scale bar
image size - ruler
how do you convert between units?
1cm = 10mm = 10000µm = 10000000nm
what is the difference between magnification and resolution?
magnification is the increase in an object’s image size compared to its actual size
resolution is the minimal distance between two points at which they have be distinguished as two points
how can you identify bacterial cells in a light micrograph?
rod-shaped
stain purple with crystal violet
small (1-10micrometres)
how can you identify animal cells in a light micrograph?
nucleus
irregular shape and position
larger (10-50micrometres)
how can you identify plant cells in a light micrograph?
nucleus and cell walls
cells are closely packed
chloroplasts
larger (10-100micrometres)
how can you identify bacterial cells in electron micrographs?
rod-shaped
flagella may be visible
smaller than eukaryotic cells (1-10μm)
how can you identify plant cells in an electron micrograph?
sap vacuole is visible
chloroplast and mitochondria are visible
larger than prokaryotic cells (10-100μm)
how can you identify animal cells with an electron micrograph?
mitochondria, but not chloroplasts are visible
larger than prokaryotic cells (10-50μm)
what is electron microscopy?
electron microscopes use the interaction of electrons with the specimen to produce an image
what are the 2 types of electron microscopy?
scanning electron microscopes (SEM) produce images of the surface of an object
transmission electron microscopes (TEM) use electrons passing through a thin section of a specimen to form an image
what are the pros and cons of electron microscopy?
pros:
- higher resolution
- higher magnification
cons:
- specimen must be dead and fixed in plastic
- structural features are produced during preparation which can hinder image accuracy
what is light microscopy?
light microscopes use light to form an image with a maximum 2000x magnification, and can use dead or living organisms
how are fluorescent stains used in light microscopy?
fluorescent stains bind to specific cellular components and fluoresce when the samples are irradiated with ultraviolet light
- different combinations of dye and cellular component cause different colours of fluoresence
how is immunofluorescence used in light microscopy?
immunofluorescence using antibodies with attached fluorescent dyes, so when the antibodies bind to a particular molecule, the dye is located to that area of the cell
- this allows the fluoresence technology to be targeted more precisely that with the standard fluoresence stains
- images fluorescing at different wavelengths can be colorised and merged to study relative locations of proteins within a cell
what is freeze fracture?
a way to prepare a sample for observation with an electron microscope; a biological specimen is rapidly frozen, before being physically broken apart (fracturing)
- this process reveals a plane through the sample that can be examined
what are the advantages of freeze fracture?
improves our understanding of cell membranes
- the fluid-mosaic model was developed due to micrographs showing proteins in the membrane on both this cytoplasmic and extracellular side, leading to a proposal of two layers of protein on either side of a lipid bilayer
what is cryogenic electron microscopy?
allows imaging of single-protein molecules and their interactions with other molecules; one 3D image is formed using computer enhancement of images of many copies of the molecule, which have been frozen in ice
what are the advantages of cyrogenic electron microscopy?
enhancing our understanding of virus structure, cell membrane component arrangement, protein synthesis and even gene expression
