2.1.1 Cell Structure Flashcards
Light / Optical Microscopes
Uses visible light to observe whole cells and form an image
Can visualise living samples and therefore see movement
Colour image is obtained
Poor magnification (x1500)
Poor resolution (200nm) (due to wavelength of light)
Can be used to observe larger organelles ( nuclei, mitochondria and chloroplasts),but cannot be used to observe smaller organelles such as ribosomes, the endoplasmic reticulum or lysosomes
How do optical microscopes work
- Light is directed through the thin layer of biological material that is supported on a glass slide
- This light is focused through several lenses so that an image is visible through the eyepiece
- The magnifying power of the microscope can be increased by rotating the higher power objective lens into place
When using an optical microscope always start with the low power objective lens and try using the coarse focus to get a clearer image
If blurry, consider whether the specimen sample is thin enough for light to pass through to see the structures clearly, or if there could be cross-contamination with foreign cells
Laser Scanning Confocal Microscope
(LCSM)
A high light intensity is used to illuminate a specimen treated with a fluorescent chemical
Moves a single spot of focused light across a specimen – causes the components labelled with a dye to fluoresce, and laser beam is reflected back –– which is focused through a pinhole aperture and detected by computer
Both 2d or 3d image can be produced by creating images at different focal planes
An image produced by a laser scanning confocal microscope has a lower resolution than an electron microscope, but is greater than a light micrscope.
Can show movement as can be used on living cells
Allows us to view different depths in specimens, and so we can view different layers
Electron Microscopes
Electrons have a greater resolution and magnification than Light and LSC microscopes
* A beam of electrons has a much smaller wavelength than light, so an electron microscope can resolve (distinguish between) two objects that are closer together
* Can be used to observe small organelles such as ribosomes, the endoplasmic reticulum or lysosomes
* Can’t be moved and produce a black and white image
* Dead or dehydrated specimens used, called artefacts, they prevent air bubble obscuring image through vacuum.
Transmission Electron Microscope
(TEM)
Uses a beam of electrons.
A thin sample is stained and then placed in a vacuum
Electron gun produces a beam of electrons that pass through the specimen
The electrons are focused on the sample by the lenses
The beam passes through the sample which modifies it and imprint its image.
The denser areas absorb more electrons, and so appear darker.
Beam is then magnified and detected, creating a 2D image of internal organs
Resolution = 0.2nm
Magnification = x500,000
Can only be used on very thin specimens and produces a 2d image
Greater resolution than SEM
Scanning Electron Microscope
(SEM)
Specimen does not have to be thin because electrons are not transmitting through (can be thick)
They scan a beam of electrons across the specimen covered in metal ions (platinum and gold)
Specimen must be dead, dehydrated and stained (artefact). This is so that the beam of electrons bounces off the surface of the specimen and the electrons are detected, forming an image.
SEMs can produce 3D images. Its then condensed and put onto a screen.
Lower res than TEM = 10nm
Magnification = x100,000.
Difference between the different types of micrscopes
Greatest magnification = TEM, SEM, LCSM, then Light Microscopes
Lowest Resolution = TEM, SEM, LCSM then light
Light microscopes = 2D and in coloud
LCSM - 2D or 3D and in colour
TEM - 2D, black and white
SEM - 3D, black and white
The preparation and examination of microscope slides for use in light microscopy
LIQUID SPECIMEN:
1) Add a few drops of the sample to the slide using a pipette
2) Cover the liquid with a coverslip and gently press down to remove air bubbles
3) Wear gloves to ensure there is no cross contamination with foreign cells
SOLID SPECIMEN
1. Use scissors to cut a small sample of the tissue - peel a very thin layer of cells to be placed on the slide (using a scalpel or forceps)
2. The tissue needs to be thin so that the light from the microscope can pass through
3. Apply a stain and place a coverslip on top at an angle and press down to remove any air bubbles
Wet Mount - To prevent the dehydration of tissue:
-The thin layers of material placed on slides can dry up rapidly, so adding a drop of water can prevent the cells from being damaged by dehydration (cover slip must be placed on from an angle)
Squash slides – A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip to squash the sample to ensure you have a thin layer and enable light to pass through
Dry mounts – Solid specimens are viewed whole or cut into very thin slices (sectioning) – cover slip is placed over the sample
Smear slides – The edge of a slide is used to smear the sample, creating a thin, even coating on another slide. A coverslip is placed on top after smearing.
Artefacts
Preparation of slides
Fixing – Chemicals like formaldehyde are used to preserve the specimen in as near natural a state as possible
Sectioning – Specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. This can then be sliced thinly with a knife called a microtome
Staining – Specimens are often treated with multiple stains to show different structures
Mounting – The specimens are then secured to a microscope slide and a cover slip is placed on top
Eye Piece Graticule
Inside light microscopes, there is a scale on a glass disc which is called the eyepiece graticule
* A graticule is a small disc that has an engraved ruler - It can be placed into the eyepiece of a microscope to act as a ruler in the field of view
* As a graticule has no fixed units it must be calibrated for the objective lens that is in use. This is done by using a scale engraved on a microscope slide (a stage micrometre)
* Line up the stage micrometre and eyepiece graticule whilst looking through the eyepiece.
* Then count how many divisions on the eyepiece graticule fit into one division on the micrometre scale
* Each division on the micrometre is 10micrometres, so this can be used to calculate what one division on the eyepiece graticule is at that current magnification
Stage Micrometer
A stage graticule / micrometer is a microscope slide with an accurate measuring scale – this is used to calibrate the value of the eyepiece divisions at different magnifications.
Staining in light microscopy
Many cells are naturally transparent (with the same refractive index) and let both light and electrons pass through - difficult to see detail - stains are used to increase contrast (make the tissue coloured) and therefore more visible and easier to identify organelles
Coloured dyes are used when staining specimens – the dyes absorb specific colours of light while reflecting others –
Certain tissues absorb certain dyes - which dye they absorb depends on their chemical nature
Differential Staining - Specimens or sections are sometimes stained with multiple dyes to ensure the different tissues within the specimen show up
Gram staining
Technique used to separate bacteria into two group:
Gram positive, which possess a thicker peptidoglycan cell wall AND
Gram-negative, which have a thinner cell walls (less peptidoglycan)
- Crystal violet (purple dye) is added, then iodine to fix the dye – helps to stick purple dye onto bacteria
- Alcohol is then used to wash away any unbound stain - The gram positive will retain the crystal violet due to thick peptidoglycan cell wall absorbing the dye, HOWEVER, the gram negative will not absorb the crystal violet, due to the thinner cell wall (less peptidoglycan)
- Counterstain with safranin (red stain) is used and stains the gram-negative bacteria red (purple has been washed off) – Doesn’t stain the gram-positive pink – gram positive is purple so you don’t see the pink
- Being able to distinguish between the two types of bacteria helps medics to prescribe an appropriate antibiotic to patients with bacterial infections
Guidelines for microscope drawings
- Draw in pencil – no colouring or shading – Lines should be clear, single lines (no overlapping)
- The drawing must have a title - indicating what specimen it is
- The magnification must be recorded with proper proportions
- Annotate cell components, cells and sections of tissue visible
Magnification equations
Magnification =
Image Size / Actual Size
eyepiece lens magnification x objective lens magnification = total magnification
