Methods of Studying Cells Flashcards
Magnification definition
How many times greater the image is when compared to the real object.
Magnification = size of image / size of real object
Resolution definition
- The minimum distance apart that 2 objects can be in order to be distinguished as separate objects in an image
- Measure of clarity of an image
- eg if the maximum resolution of a light microscope is 0.2 micrometers, objects that are closer together than that will be viewed as a single item
Artefacts definition
- Something you can see when looking at the specimen which isn’t part of the specimen itself
- Forms when the slide is prepared badly
- Eg air bubbles, fingerprints on the slide, dust trapped in the slide
When were light microscopes first developed and by whom?
Robert Hooks - 1665
Compare light microscopes (LM), TEMs and SEMs
1) LM uses a beam of light; TEM and SEM both use a beam of electrons
2) LM maximum magnification = x2000, whereas TEM = x50,000,000 and SEM = x1,000,000
3) LM maximum resolving power = 200 nm, whereas TEM = 0.1 nm and SEM = 20 nm
4) LM can analyse living samples; TEM and SEM can’t
5) LM and TEM can only view 2D images; SEM can view 3D images
6) Staining process is more complex for TEM and SEM than LM
7) LM can only be used to view large subcellular structures, TEM can view everything and SEM views the cell exterior only
8) LM can see a coloured image; TEM and SEM can’t even when they are stained
How does a light (optical) microscope work?
- There are 2 convex lenses - the objective lens and the eyepiece lens
- Objective lens (usually x10) produces a magnified image which is then magnified and directed into the eye by the eyepiece lens (typically 4x, 10x, 40x or 100x)
- The specimen is illuminated from underneath (from the stage) to give a clearer view
- Magnification of a light microscope = objective lens magnification x eyepiece lens magnification
- Light source —> condenser lens —> object —> objective lens —> (intermediate image)—>eyepiece lens —> eye
Limitations of the Light microscope
- Nuclei, Mitochondria, LPV, Chloroplasts, Centrioles, Cell walls and the CSM are the only organelles that can be viewed by a light microscope - and even then it isn’t in good detail - ribosomes, Golgi, ER and other important organelles cant be seen.
- Maximum magnification is x2000 (in fact only x1000 most of the time) resolving power is 200 nm - this is very limiting
When was an electron microscope first developed?
1930s
How does a TEM work?
- An electron gun produces a beam of electrons from BELOW the specimen
- Electron beam is focused onto the specimen (which must be very thin) by a condenser electromagnet
- Therefore the beam only passes through a thin section of the specimen
- Parts of the specimen absorb electrons so appear dark
- Other parts don’t absorb electrons so appear lighter
- A flat 2D image (micrograph) is produced on a screen
- To build a 3D image, we can take a series of sections through a specimen, then build a 3D image by looking at the series of photomicrographs produced
- However, this is long and complex so we use an SEM to build a 3D image instead
Why do electron microscopes have a higher resolution than light microscopes?
- A beam of electrons has a shorter wavelength than a beam of light
- Therefore we see a clearer, more detailed image
Why can the maximum resolution (0.1nm) be rarely achieved in practice?
- Difficulties preparing the specimen limit the resolution
- A very high energy beam of electrons is required which could damage (ionise) the specimen
How does an SEM work?
- An electron gun produces a beam of electrons from ABOVE the specimen
- But they aren’t focused onto the specimen by a condenser electromagnet as the electrons don’t need to penetrate
- Instead the electron beam is directed onto the surface of the specimen
- The beam is then passed back and forth across the specimen in a regular pattern
- The electrons are scattered by the specimen; the pattern of this scattering determines the contours of the specimen surface
- A 3D photomicrograph is built up by computer analysis of the pattern of scattered electrons and secondary electrons produced by the specimen
Limitations of the electron microscope
- The whole system must be in a vacuum therefore living specimens can’t be analysed
- A complex staining process is required and even then the image is not in colour
- The staining process can also introduce artefacts onto the image, and if artefacts are small it can be hard to actually tell what’s part of the cell and what isn’t
- The sample must be extremely thin (especially for TEM)
- Electrons can ionise the specimen, damaging it and therefore we might not get a 100% accurate view
How do you set up a slide on a light microscope?
- Place the sample on a clean glass slide
- Stain the sample with a drop of a stain (eg iodine in potassium iodide solution)
- Use forceps to place a cover slip over the sample, at an angle to prevent the entry of air bubbles
- Place the slide on the stage and look through using the lowest magnification eyepiece lens
- Turn the coarse adjustment knob until the sample is visible under the eyepiece lens
- Then turn the fine adjustment know until you see an image with a clear resolution
What are the different stains you can use? What colour are they? What do they stain?
- Methylene Blue and Toluidine Blue both stain DNA Blue
- Haematoxylin and Eosin (H&E) stains RNA/DNA Purple/Blue and cytoplasm pink
- Giemsa stains RBCs red and nuclei present in other blood cells are stained purple
- Iodine in potassium iodide solution stains starch blue-black and everything else orange-brown
How does a laser scanning confocal microscope work?
- Uses laser beams to scan a specimen tagged with fluorescent dye
- A laser beam is focused through a lens which is aimed at a beam splitted.
- This splits the beam and some of the light is directed to the specum
- When the laser hits the dye, it reflects fluorescent light
- The light is focused through a pinhole onto a detector
- The detector is connected to a computer to generate the image
What is an eyepiece graticule?
- A glass disc that fits inside the eyepiece lens
- There is a fine scale of arbitrary divisions etched onto it
- Once calibrated, this scale is used to measure the size of objects under a light microscope
What is a stage micrometer?
- A microscope slide with a coverslip with a scale fitted onto it
- This scale has known divisions and is used to calibrate the eyepiece graticulel
How to calibrate the eyepiece graticule
1) Align the eyepiece graticule with the stage micrometer by turning the eyepiece lens
2) Count how many divisions on the eyepiece graticule corresponds to a set number of stage micrometer divisions (eg 10 SM divisions = 21 EG divisions)
3) Calculate how big one EG division is - but you need to know how long each small division on the SM is (eg if one small SM division is 0.01 mm then ten divisions is 0.1mm, which is 21 EG divisions. Therefore one EG division = 0.1mm/21=0.00476 mm or 4.76 micrometers
4) Then we can measure the length of the sample using the eyepiece graticule (just multiply the number of eyepiece graticule divisions by 4.76 to find the length of the actual sample in micrometers
Why does the eyepiece graticule have to be re-calibrated for a) every different magnification used and b) every different microscope used?
Because if you make the magnification bigger, one EG division becomes smaller
What is cell fractionation and why is it done?
- Cells are broken up and the different organelles they contain are separated out into (preferably) pure samples
- It is done because in order to study the structure and function of organelles in detail, we need to obtain a large number of isolated organelles - looking at their appearance under a microscope isn’t enough.
- Therefore we need to do this to conduct research on them
Before cell fractionation can begin, the sample (a tissue) is placed in an ice-cold, buffered, isotonic solution. Why?
- Ice-cold to minimise enzyme activity so that any hydrolytic enzymes don’t destroy the organelles
- Buffered (same pH) to prevent pH fluctuations which could denature organelle proteins (inc. enzymes and membrane-bound proteins and histones) and so alter the structure of the organelles
Stage 2 - Homogenisation. What happens?
- The tissue-containing solution is homogenised using a homogenised
- This is a blender-like plunger that grinds up the cells by pushing them up and down to disrupt the tissue and break open the CSM
- This released the organelles from the cells into a resultant liquid - the cell homogenate
