2.1.1 Cell ultrastructure Flashcards
Light microscopes
- Uses light to form an image
- Resolving power 200nm
- Useful magnification of optical microscopes is about x2000
- Living or dead specimens
- Natural colour
Laser confocal microscopes
- Uses laser beams to scan a specimen, which is tagged with a fluorescent dye.
- The laser causes the dye to fluoresce - give off light. This light is then focused through a pinhole onto a detector. The detector is hooked up to a computer, which generates an image. Image can be 3D.
- Pinhole → any out-of-focus light is blocked, so produces a much clearer image.
- Can be used to look at objects a different depths in thick specimen.
- 2D images of higher resolution can also be produced.
- Non-invasive
Transmission electron microscope (TEM)
- Uses electromagnets to focus a beam of electrons, which is then transmitted through the specimen. Denser parts of the specimen absorb more electrons, making them look darker on the image.
- Magnification x1,000,000
- High resolution 0.5 nm
- Non-living
- Internal structures
- 2D
Scanning electron microscope (SEM)
- Pass a beam of electrons across the surface of a specimen and then detect the rate at which electrons bounce back.
- Magnification to x1,000,000
- Resolution to 3-10nm
- 3D surface of specimen
- Non-living
How to prepare a microscope slide?
- Fixing - chemicals like formaldehyde used to preserve specimen in as near-natural state as possible.
- Sectioning - dehydrated with alcohols & placed in a mould with wax or resin → form a hard block. This can then be sliced thinly with a microtome.
- Staining
- Mounting - secured to a microscope slide and a coverslip is placed on top.
Dry mount
- Solid specimens are viewed whole or cut into very thin slices with a sharp blade – sectioning.
- Specimen is placed on the centre of the slide and a coverslip is placed over the sample.
Wet mount
- Specimens are suspended in a liquid such as water or an immersion oil.
- Coverslip is placed on from an angle.
Squash slide
- A wet mount is first prepared
- then a lens tissue is used to gently press down the cover slip
3
How to calibrate a microscope?
- Line up the stage micrometer and eyepiece graticule whilst looking through the eyepiece
- Count up how many divisions on the eye piece graticule fit into one division on the micrometer.
- Each division on the micrometer is 10 micrometers, so this can be used to calculate what one division on the eye piece graticule is at current magnification.
Smear slide
- The edge of a slide is used to smear the sample, creating a thin, even coating on another slide
- Cover slip is then placed over then sample.
6
How to use a light microscope?
- Start by clipping the slide containing the specimen you want to look at onto the stage.
- Select the lowest-powered objective lens.
- Use the coarse adjustment knob to bring the stage up to just below the objective lens.
- Use the coarse adjustment knob to move the stage downwards, away from the objective lens until the image is roughly in focus.
- Adjust the focus witht eh fine adjustment knob, until you get a clear image of what’s on the slide.
- If you need to see the slide with a greater magnification, switch to a higher-powered objective lens and refocus.
Why is staining used?
Stains are used to make cells** more visible**, **increase contrast **and identify different cell components.
Crystal violet/methylene blue
Crystal violet/methylene blue are positively charged → attracted to negatively charged materials in the cytoplasm leading to staining of cell components.
Nigrosin or congo red
Nigrosin or congo red are negatively charged → repelled by the negatively charged cytosol, these dyes remain outside the cells leaving the cells unstained and the background stained.
What is the use of differential staining?
Differential staining can distinguish between two types or organisms. It can also differentiate between different organelles of a single organism.
Gram-staining technique
- Used to separate bacteria into two groups
- Crystal violet is first applied to a **bacterial specimen **on a slide, then iodine, which fixes the dye, slide is washed with alcohol.
- Gram positive bacteria → retain the crystal violet stain and will appear blue or purple.
- Gram negative bacteria → have thinner cell walls and loose the stain. They’re stained with safranin dye – counterstain. These bacteria will appear red.
How to draw a scientific drawing?
- Includes a title
- State magnification
- Use a sharp pencil for drawings and labels
- More than half the space
- No shading
- Continuous lines
- Label lines no arrow heads, parallel to the top of page and draw with ruler.
Magnification formula
Magnification = size of image/actual size
What is magnification?
The number of time larger an image is than the actual object
What is resolution?
The ability to distinguish separate points on an image as two separate object
