Microscopes Flashcards
Define magnification?
The degree to which the size of an image is larger than the object itself
Define resolution?
The minimum distance apart that two objects can be in order for them to appear as two separate items
What is used to measure the size of cells (in a microscope)?
Eyepiece graticule
What scale are cells/objects measured by using a microscope?
Eye piece units (epu)
What are the rules when completing a biological drawing?
- Use a sharp pencil
- Should take up at least half a page
- Line need to be clear and continuous (no shading/sketching/colouring)
- Ensure proportions are correct
- Label all features you have shown
- Rule label lines
- Don’t cross label lines or use arrows
- Don’t write on label lines
- Ensure label lines touch the part you are labelling
What is the equation for magnification?
IAM
Image size/actual size
Describe how you could make a temporary mount of a piece if plan tissue to observe the position of starch granules?
- Add a drop of water to the glass slide
- Obtain a THIN section of plant tissue (potato) to ensure light can pass through
- Stain with iodine in potassium iodide solution
- Lower cover slip on slide using a mounted needle (ensures no air bubbles)
How do you calibrate a microscope to work out the mean diameter of starch grains?
- Measure the diameter of the grain using the eye piece graticule
- Calibrate the eye piece graticule using a stage micrometer at the same magnification
- Randomly sample 10+ grains and calculate the mean
What is cell fractionation?
The process in which cells are broken up (lysis) and different organelles within the cell are separated out so they can be studied in detail
Before cell fractionation can begin what type of solution must the tissue be placed in?
A cold, buffered, isotonic (same water potential) solution
What does isotonic mean?
The solutions have the same water potential
Why should the solution be cold?
To reduce enzyme activity to prevent organelles being broken down (by digestion)
Why should the solution be buffered?
So the pH doesn’t fluctuate so proteins don’t denature
Why should the solution be isotonic (have the same water potential)?
To prevent organelles shrinking or bursting as a result of osmotic gain/loss of water
Describe the process of cell fractionation?
- Homogenisation- break open cells by vibrating or grinding them up (in a homogeniser - blender) to release organelles from cells
- Filtration - the resultant fluid (homogenate) is filtered to remove any debris (whole cells/bits of tissue’)
- Ultracentrifugation - the fragments in the filtered homogenate are separated in a centrifuge at increasing speeds
Describe the process of ultracentrifugation?
- The first centrifuge spin is low-speed and separates the heaviest organelles (nuclei) out first. They are forced to the bottom of the tube where a thin sediment/pellet forms whilst the less dense remain in suspension above (supernatant)
- The supernatant ( is removed leaving just the sediment) is then transferred to the next tube and spun at a faster speed ( this next pellet contains the next most heavy/dense organelle -mitochondria)
What are the three types of microscopes?
- Light/Optical
- Transmission Electron Microscope (TEMs)
- Scanning Electron Microscopes (SEMs)
What type of electromagnetic radiation do optical microscopes use?
Beams of visible light (has a long wavelength which reduces resolving power)
What type of electomagnetic radiation do TEMs and SEMs use?
Beams of high energy electrons (which have a shorter wavelength leading to a higher resolving power)
Compare the size of magnification between optical microscopes and TEMs/SEMs?
Optical microscopes have a lower magnification (x400)
SEMs and TEMs have a higher magnification (x 1,000,000)
Which microscope has the highest resolving power ?
TEMs
Compare the resolving power for all three microscopes?
- Optical = lowest (0.2 ųm)
- SEMs = Higher (5 - 20 nm)
- TEMs = Highest (0.2 nm)
Which is the only microscope to produce a 3D image?
SEMs
Scanning Electron Microscope
How do you do focus an optical microscopes?
Use pairs of lenses
How do you do focus an electron microscopes
Use electromagnets
Which microscope enables you to view a specimen in colour?
Optical microscope
The TEMs and SEMs only view specimens in black and white
Which microscope can view live specimens?
Optical microscope
The electron microscopes take place in a vacuum so only dead/dehydrated specimens can be viewed
Which microscope require a thin specimen?
Light/optical microscopes and TEMs
SEMs can view thicker specimens as they produce a 3D image
Which is the only portable microscope?
Optical microscopes
Compare the value of microscopes?
- Optical microscopes are cheap
- Electron microscopes are expensive
Which microscope requires the most complex staining process?
TEMs (transmission electron microscopes)
- requires use of heavy metals
SEMs and optical microscopes don’t need staining
What is an artefact?
Something you see under a microscope (when looking at a prepared sample) that isn’t actually part of the specimen (eg, dust and bubbles, fingerprints)
Which microscope suffers mostly from presence of artefacts?
TEMs due to more complex staining process
What are examples of artefacts?
Dust
Bubbles
Fingerprints
What stain is used to make the cytoplasm show up?
Eosin
