Microscopy and studying cells Flashcards
What is magnification?
Magnification is how much bigger the image produced is compared to the size of the actual image. It can be described as: size of image / actual size of specimen.
What is resolution?
Resolution describes how well defined features of an image produced by the microscope is and can be defined as the smallest distance two objects can be together while still being distinguishable as 2 distinct objects.
What is the maximum magnification of a microscope?
The largest magnification that can be produced by the microscope while still maintaining a good image quality.
How is an image produced by a Compound Light Microscope (CLM)?
- Light is produced by a light source directly under the specimen or is reflected onto the specimen by a mirror.
- A condenser lens focuses the light onto the specimen which is put onto a transparent glass slide and mounted onto a stage.
- Light then travels through the objective lens where the image is magnified. A CLM usually has several objective lenses to create different magnifications.
- Light then travels through the turret and reaches the eyepiece lens the image is further magnified and formed onto the retina.
What is the maximum magnification and resolution of a CLM and why?
- A CLM has a maximum magnification of x1500 which is limited by the resolution of the CLM.
- The resolution of the CLM is 200nm as it is limited by the wavelength of visible light. If two objects are closer together than 200nm, light would not be able to pass between them, and so the two objects cannot be defined.
How are specimens prepared for a CLM?
- Fixation: The specimen is treated with a chemical that preserves it in a life-like state and prevents decomposition.
- Dehydration: The water is removed by putting into a dehydrating agent (an alcohol is usually used).
- Clearing: Dehydrating agent is cleared out of the specimen by a clearing agent.
- Embedding: The specimen is put into something hard enough to support it during sectioning but soft enough to cut through (e.g. wax).
- Sectioning: The specimen is sliced into pieces thin enough for light to easily pass through it with a machine called a microtome.
- Staining: Staining is used to define organic tissue that are otherwise transparent so they can be seen.
Why is staining used?
- To define transparent features of the specimen.
- To distinguish one feature from another with the use of different coloured stains.
- To test the presence of specific features or chemicals.
What is the principle behind staining?
Staining is based on the principle that if you want to see certain features, you need a coloured chemical that would bind onto the substance the feature is made out of or primarily consists of. For example, to see cell walls, you need a chemical that stains cellulose. To see the nucleus, you need a substance that stains DNA.
How is an image produced by a Transmission Electron Microscope (TEM)?
- An electron gun, consisting of a cathode and anode is used to produce electron beam. The cathode emits electrons in all directions. The electrons are pulled towards the anode where a thin beam of electrons are let through a slit.
- The beam of electrons are focused onto specimen by a condenser lens. An electromagnetic lens is used as electrons have bad penetrating power and would be stopped by glass.
- Electrons pass through specimen and is magnified by the electromagnetic objective lens and projector lens.
- Electron beam is directed onto a swing-out screen coated in electron sensitive fluorescent chemicals so the image can be seen as the human eye cannot detect electrons directly.
- Photographic paper can be put beneath the screen to keep a permanent record of the image.
What is the maximum magnification and resolution of a TEM?
- A TEM has a maximum magnification of x500,000 as it has a much higher resolution compared to the CLM.
- The resolution of the TEM is 0.1 nm as electrons have much shorter wavelengths compared to light.
How is the CLM different from a TEM?
- TEM lenses made from electromagnetic fields whereas CLM lenses are glass.
- TEM turret is a vacuum whereas a CLM turret is filled with air.
- TEMs are large and fixed whereas CLMs are small and portable.
- TEM specimens are much thinner than CLM specimens.
- TEM images are in greyscale whereas CLM images are coloured.
How is the specimen preparation process for a TEM different from a CLM?
- The CLM specimen is embedded in wax whereas the TEM specimen is embedded in polymer resin as wax isn’t hard enough to allow for specimen to be sectioned to the thickness required.
- TEM specimens are nanometers thick whereas CLM specimens are micrometers thick.
- Microtome used to cut CLM sections (metal blades) whereas ultra microtomes (glass/diamond blades) are used to cut TEM sections.
- CLM stains are coloured chemicals whereas TEM stains are heavy metal salts that do not allow electrons to pass through.
- CLM specimens are mounted on glass slide whereas TEM specimens are mounted on copper grids.
How does a Scanning Electron Microscope (SEM) work?
- The structure of the SEM is identical to the TEM, except everything beneath the stage in a TEM is missing.
- An electron beam is shone onto a whole (unsectioned) specimen coated in heavy metals.
- Electrons are reflected off the surface of the specimen differently according to the surface contour.
- Reflected electrons are detected by detectors surrounding the specimen.
- Electron beam is scanned across surface until enough data is generated and 3 dimensional model is created by a computer.
- Zooming in simply involves making image on screen bigger, no physical adjustments are made to lenses.
What is the maximum magnification and resolution of an SEM?
- An SEM has a maximum magnification of x100,000; not as large as the TEM.
- The resolution of an SEM is around 5 nm.
What are the advantages of EMs compared to LMs?
- EMs can be used to create images with magnifications much larger than can be produced by LMs. This allows us to see features of cellular ultrastructure which we would have otherwise not seen with LMs.
- SEMs are able to produce 3 dimensional images of specimens. This allows us to study in detail the structure of the specimen.
