2.1.3 Microscopy Flashcards
Define magnification
How many times bigger an object appears
Define resolution
The ability to distinguish two points next to each other
If the resolution of a microscopic image is too low, what can be said about the light/electrons used
The wavelength is too long
Magnification equation
Magnification = image size / actual size
1cm = ?mm
10
1mm = ?µm
1000
1µm = ?nm
1000
Measurement unit conversion image
How to measure an object using a microscope and graticule
- Line up graticule with object
- Count how many epu for the measurement
- Calibrate graticule using stage micrometer - how many µm per epu
- Multiply the number of epu by the calibration factor
Resolution of optical microscope
Low
Resolution of SEM
Medium
Resolution of TEM and ADVANTAGE
- High
- Allows organelles to be seen in greater detail
Magnification of optical microscope
Low
Magnification of TEM
High
Magnification of SEM
Medium
How does a optical microscope work
Light ray focused onto object then onto eye using glass lenses
How does a SEM work
- Electrons deflected/bounce off specimen
- Foccused by electromagnets
How does a TEM work
- Electrons transmitted/pass through specimen
- Foccused by electromagnets
Primary use of optical microscope
Study living cells
Primary use of SEM
View 3D image
Primary use of TEM
View cell ultrastructure
Are optical microscope images in colour
Yes
Are TEM images in colour
No
Are SEM images in colour
No
Non-limitations to optical microscopes
- Can be dead or alive
- No vacuum
- Not too thin
- Stained with dye
- No artefacts
- Doesn’t take too long
Limitations to SEM
- Must be dead
- In vacuum
- Very thin specimin
- Stained with metal
- Artefacts may occur
- Time consuming
Limitations to TEM
- Must be dead
- Must be in vacuum
- Specimin must be very thin
- Stained with metal
- Artefacts may occur
- Time consuming
In microscope images, if the resolution is high/low what can be said about the light or electrons wavelength
- Low resolution = long wavelength
- High resolution = short wavelength
Describe how to extract chloroplast from a cell sample
- Homogenise cells in homogeniser
- Filter to remove unbroken cells/debris
- Place homogenate in test tube and spin in a centrifuge at low speed
- Remove nuclei pellet at bottom
- Respin supernatant in new test tube at higher speed
- Remove chloroplast pellet
When extracting organelles from cells during ultracentrifugation, what is the order of heaviest organelles, that must be removed
- Nuclei
- Chloroplast - only if plant cell
- Mitochondria
When extracting organelles from cells, why must the solution be cold
Reduce enzyme activity, so organelles aren’t digested
When extracting organelles from cells, why must the solution be isotonic
Prevent water movement into organelles by osmosis - preventing lysis
When extracting organelles from cells, why must the solution be pH buffered
- Maintain pH
- Prevent denaturations of proteins and enzymes
