Methods Of Studying From Cells Flashcards
What are the three key types of microscopes?
- Optical (light)
- Transmission electron
- Scanning electron
Magnification
- The number of times larger an object is compared with the real size of the object
Resolution
- Ability to distinguish between two points
What is the resolution of an optical microscope determined by?
- wavelength of light
What is the resolution of an electron microscope determined by?
- wavelength of the beam of electrons
Describe properties of optical (light) microscopes
- a beam of light is condensed to create image
- poorer resolution due to light having a longer wavelength
- lower magnification
- colour images
- can view living samples
Why do optical (light) microscopes have poorer resolution?
- light has longer wavelength
Describe the properties of electron microscope
- a beam of electrons is condensed to create the image
- electromagnets are used to condense the beam
- higher resolving power as electrons have short wavelength
- higher magnification
- black and white images
- sample must be in vacuum and therefore non living
What is used to condense the beam of electrons used in electron microscope?
- electromagnets
What is the disadvantage of optical microscopes having poor resolution?
- small organelles in a cell are not visible
Why must samples be in a vacuum in electron microscope?
- electrons are absorbed by air
Why can only non living specimens be examined in an electron microscope?
- sample must be in a vacuum
Why is the image black and white in an electron microscope?
- samples must be stained
How to transmission electron microscopes work?
- extremely thin specimens are stained and placed in a vacuum
- electron gun produces a beam of electrons that pass through specimen and some electrons are absorbed by specimen and appear dark
- produces 2D image
- shows detailed image on the internal structure of cells
Why may some parts of the image in transmission electron microscope be dark?
- some parts of the specimen absorb the electrons
- the darker it is, the more electrons have been absorbed
How do scanning electron microscopes work?
- specimens not need to be thin as the electrons are not transmitting through
- electrons are beamed onto the surface and electrons are scattered in different ways depending on the contours
- produces 3D image
Why do specimens not need to be thin in scanning electron microscopes?
- electrons are not transmitting through they are beamed onto the surface and electrons are scattered in different ways spending on the contours
What is the formula for image size?
- image size= actual size / magnification
How to convert metre (m) to millimetre (mm)
- multiply by 1000
How to convert millimetre (mm) to micrometer (um)
- multiply by 1000
How to convert micrometer (um) to nanometre (nm)
- multiply by 1000
How to convert nanometre (nm) to micrometer (um)
- divide by 1000
How to convert micrometer (um) to millimetre (mm)
- divide by 1000
How to convert millimetre (mm) to metre (m)
- divide by 1000
What should you record image size in?
- millimetres (mm)
What is an eyepiece graticule?
- scale on the glass disc found on the inside of optical microscopes
What is an eyepiece graticule used for?
- measure the size of objects you’re viewing under microscope
What is an eyepiece graticule used for?
- measure the size of objects you’re viewing under microscope
What is the limitation of eyepiece graticule?
- each time objective lens is changed and therefore the magnification, it has to be calibrated to work out what the distance between each division represents at that magnification
What is used to calibrate the eyepiece graticule?
- stage micrometer
What is a stage micrometer?
- glass slide with a scale on it which you place on the stage
- typically 2mm long and subdivisions are 10um apart
How to calibrate an eyepiece graticule?
- Line up stag micrometer and eyepiece graticule whilst looking through eyepiece
- Count how many divisions on the eyepiece graticule fit into one division on the micrometer scale
- Each division on the micrometer is 10um so this can be used to calculate what one division on the eyepiece graticule is at that current magnification
What is cell fractionation used for?
- isolate different organelles so they can be studied
What does cell fractionation enable?
- individual organelle structures an functions to be studied
Why must the cells be prepared in cold solution?
- reduce enzyme activity
- when cell is broken open, enzymes are released which could damage the organelles
Why must the cells be prepared in an isotonic solution?
- organelles must be the same water potential as the solution to prevent osmosis as this could cause organelles to shrivel or burst
Why must the cells be prepared in a buffered solution?
- solution has a pH buffer to prevent damage to the organelles
What solution must the cells in cell fractionation be prepared in?
- cold, isotonic and buffered solution
What is the first step of cell fractionation?
- Homogenisation- cells broken open using blender in the cold, isotonic and buffered solution. Solution is then filtered to remove large cell debris
What is the second step of cell fractionation?
- Ultra centrifugation- filtered solution is spun at high speed in a centrifuge at increasing speeds, separating organelles according to their density
Describe differential centrifugation
- centrifuge spins at high speed and centrifugal forces cause pellets of most dense organelle to form at bottom of the tube
- process repeated at increasing speeds, removing the supernatant each time and leaving behind the pellet
- supernatant is spun again in the centrifuge and process is repeated
Order of organelle fractionation
- Nuclei
- Chloroplasts And Mitochondria
- Lysosomes and endoplasmic reticulum
- Ribosomes
Compare TEM and SEM
- in TEM a beam of electrons pass through the specimen whereas in SEM a beam of electrons scan the surface. Reflected beam observed
- in TEM it has very good resolution (2000 greater than light microscope) whereas in SEM resolution is poorer than when using TEM
- in TEM able to observe the ultrastructure (greater detail) of cell organelle whereas in SEM Surface of organelle/structure able to be seen, great depth of field
TEM wavelengths of electron steps
- Electron gun
- Specimen
- Electromagnet
- Objective lens
- Viewer
Light microscope wavelength of light steps
- Light source
- Condenser
- Specimen
- Objective lens
- Ocular lens
- Viewer
Advantages and disadvantages of light compared to TEM
- light shows colour whereas TEM does not show colour
How to find 1 eye piece unit (epu)
- 1 epu = stage micrometer (smu) / eye piece units
Explain how you would calibrate a microscope and calculate an actual size of a specimen using microscope
- Place the micrometer eyepiece into the eyepiece of the microscope
- Place the stage micrometer slide on the stage of the microscope at the desired objective lens
- Line up the epu and stage micrometer to calculate 1 epu at that objective lens
- Remove the stage micrometer from the stage of microscope and add the specimen slide that you want to
measure to the stage. - Measure epu of the specimen and calculate actual size by your multiplying the number of epu of the specimen
by your calculated actual valve of 1 epu.
