Microscopy Flashcards
Describe what a microscope does and name 4 different types of microscope.
A microscope is an instrument which enables you to magnify an object a lot.
- compound light microscope
- laser scanning confocal microscope
- scanning electron microscope
- transmission electron microscope
Label a diagram of a light microscope with the names of the component parts
look at textbook
name and write the functions of the parts of a compound light microscope
- Eye piece- is what you look through and it magnifies the object 10x
- Scanning lens- It is used in preliminary observation and magnifies 4x.
- Low power objective lens- magnifies 10x and gives the middle amount of detail.
- High power objective lens- magnifies 40x and gives the most amount of detail.
- Stage clips- used to secure the slide and hold it in place
- Stage- stand above the the light source for the glass slide.
- Arm- where you hold a microscope
- Condenser- used to vary the intensity of the light projected
- Coarse- roughly puts things into focus and should only be used with the scanning lens.
- Fine adjustment- Puts things into full focus and should only be used with the low and high power objectives lenses.
- Illumination- projects light onto the slide.
Outline how an SEM works
A beam of electrons is fired at the surface of a specimen and the reflected electrons are collected to create a 3D image.
Outline how a TEM works
A beam of electrons is transmitted through the specimen and focused to produce an image.
What is flourescence
It is the absorption and re radiation of light.
Outline how a laser scanning lens works
It moves a single spot of focused light across a specimen (point of illumination). this causes fluorescence with the components labelled with a dye.
The emitted light from the specimen is filtered through a pin hole aperture. Only light radiated from very close to the focal plane (the distance that gives the sharpest distance) is detected.
Draw a table comparing the use and properties of light, SEM, TEM and laser scanning confocal microscope.
look at book
State the features of the images produced from light, SEM, TEM and laser scanning confocal microscopes.
look at book
Know a bit about the history…
look at book
Explain how to use a light microscope to view a specimen at low and high powers.
- When using low power lenses ( scanning or low power objective lens) use the coarse to roughly bring it to focus.
- When using a high power use the fine adjustment knob.
Describe how to produce a temporary wet mount of living tissue.
Specimens are suspended in liquid such as water or an immersion oil.
A cover slip is placed on from an angle, aquatic samples and other living organisms can be viewed this way.
Describe and explain the characteristics of a good slide preparation.
- no air bubble- artefact
- staining if needed
- glass and transparent
- coverslip - protection
- thin specimen- good quality and a good size
- good liquid levels.
- clean
Explain why slide preparations need to be thin.
- transmitted light is going through so it needs to be transparent or you won’t see anything
- you want to look at a single cell so you can measure it which you can’t do if they overlap.
Explain how to use a stage micrometer to work out the distance represented by the small divisions in an eyepiece graticule under 3 different objective lenses.
- Line up the eye piece graticule with the stage micrometer (the fixed scale on the slide).
- get the scale on the micrometer slide in focus.
- align the micrometer scale with the scale in the eyepiece take a reading from both of them.
- See what the equivalent value of stag units is to eyepiece units.
- work out the stage units in mm by dividing by ten ( 1 stage unit = 0.1 mm)
- Divide the stage units in mm by the number of eyepiece units.
- then put this in the right unit (micrometre) by multiplying by 1000 (magnification factor)
Explain how to use a stage micrometer and eye-piece graticule to add a scale bar to a drawing.
- measure the specimen with the eyepiece graticule.
- then multiply it by the magnification factor
- this will get the size of the specimen in micrometres
- draw specimen and put a bar underneath.
Explain how to use a stage micrometer and eye-piece graticule to calculate the
size of a specimen.
- measure the specimen with the eyepiece graticule.
- then multiply it by the magnification factor
- this will get the size of the specimen in micrometres
Describe how to choose an appropriate number of significant figures, or decimal places to present data.
Use the highest number of decimal places in the question for your answer.
Explain how an adjustment to the “plane of focus” can alter what is viewed within a cell.
If you change the focus while looking through a microscope you will notice different parts of the image will come into focus and other parts will go out of focus.
The plane of focus is the horizontal plane where what you are looking at is in focus.
It is usually not very thick so it is hard to focus on things that are not flat.
When you focus the microscope you move the plane of focus up and down, which is why different parts of what you are looking at come in and out of focus.
Explain how a tissue slice might be misleading due to the very thin nature of the slice.
- you can only see a cross-section of a cell not it’s overall shape- you might not see the actual shape of the cell.
- there are different organelles at different parts of the cell- you might miss structures that are inside the cell.
- Its not actually the diameter of the cell just the diameter of the cross section.
Explain why staining is useful for light microscopy
- a lot of specimens are transparent and colourless so difficult to see.
- staining provides colour- contrast with the background
- differential staining- you can distinguish between two types of organisms or two types of organelles of a single organisms.
Describe the properties a stain needs to have to be useful for light microscopy.
- Bind to other molecules- differential staining - bind to certain parts of the cell
- liquid- and be able to get through cell membrane.
- shouldn’t damage the cell
- very intense colour- to be able to colour something in small amounts.
Describe how to prepare a stained specimen for viewing under a light microscope.
- place sample on a slide an allow it to air dry
- this is then heat-fixed by passing through a flame.
- the specimen will adhere to the microscope slide and will take up stains.
Name two common stains and the molecules they bind to.
- methylene blue- DNA
- Giemsa- used to differentiate between different types of blood cell.
- Eosin- cytoplasm turn pink
