Methods of studying cells Flashcards
What are the principles of an optical microscope?
- use light to form a 2-D image
- lower magnification 1500x
What are the limitations of an optical microscope?
- 2D image
- Only used on thin specimens
- Low resolution → can’t see internal structures of organelles
-low magnification
What is an advantage of using an optical microscope?
Can see living organisms
What are the principles of a scanning electron microscope?
- use electrons to form a 2D image
- beams of electron scan surface knocking of electrons from the specimen, which are gathered in a cathode ray tube to form an image
-electrons, shorter wavelength → higher resolution
-high magnification
What are the limitations of a scanning, electron microscope?
- vacuum → can’t see living organisms
- lower resolution than TEM
What are the advantages of using a scanning, electron microscope?
- 3D image
- high resolution → see internal structures
- higher magnification
- used on thick specimens
What are the principles of a transmission electron microscope?
- use electrons to form a 3D image
- electromagnets, focus beam of electrons onto specimen, more dense → more absorbed → darker appearance
- shorter wavelengths → higher resolution
- high magnification
What are the advantages of transmission electron microscopes?
- high resolution → see internal structures
- high magnification
What are the limitations of the transmission electron microscope?
- 2D image
- only used on thin specimens
- vacuum → can’t be alive
Define magnification
How much bigger the image of a sample is compared to the real size
Define resolution
How well distinguished an image is between two points
Describe the process of cell fractionation and ultracentrifugation as used to separate cell components
1) homogenised tissue using a blender → breaks down cell membrane → release organelles
2) place in a cold, isotonic, buffered solution → cold reduced enzyme activity (organelles won’t break down) → isotonic (water won’t move in and out by osmosis so won’t burst/shrivel) → buffer (keeps pH constant so enzymes don’t denature)
3) filter homogenate → remove large debris
4) ultracentrifugation
a. Centrifuge homogenates in a tube at a low speed
b. Remove pallet of heaviest organ now and spin supernatant at a high speed
c. repeat at higher speeds until organelles separated out each time pellet is made of lighter organelles
What’s the order of density for organelles?
Nuclei → chloroplast → mitochondria → lysosomes → endoplasmic reticulum → ribosomes
Describe the process of preparing a temporary mount of a specimen on a slide
-use tweezers to place it in section of thin specimen, on a water droplet on a microscope slide
-add a drop of stain, e.g. iodine in potassium iodide dilution (stains starch grains)
-add coverslip carefully tilt and lower and no air bubbles to make it thin as possible
