Microscopy and Electron Microscopy Lecture Notes

These flashcards cover the fundamentals of microscopy, including the electromagnetic spectrum, resolution limits, differences between light and electron microscopes, and the components and functions of TEM and SEM.

What is defined as the electromagnetic spectrum?

The range of different wavelengths.

What is the relationship between wavelength and frequency?

Longer waves have a lower frequency.

Do all waves in the electromagnetic spectrum travel at the same speed?

Yes, all waves travel at the same speed.

How does energy influence wavelength?

Greater energy results in shorter wavelengths.

How does molecule size affect the interference of visible light?

Larger molecules can interfere with visible light, while smaller ones typically do not.

What is the general rule for the limit of resolution in microscopy?

The limit of resolution is approximately one half the wavelength of the radiation used to view a specimen.

What happens when an object is smaller than half the wavelength of the radiation used to observe it?

The object cannot be seen separately from nearby objects.

What is the shortest wavelength of visible light mentioned in the notes?

$$400\,nm$$

What is the maximum resolution limit of a standard light microscope?

$$200\,nm$$

What is the approximate diameter of a ribosome?

$$25\,nm$$

What is the approximate diameter of a mitochondrion?

$$1000\,nm$$

Why can ribosomes not be seen using a standard light microscope?

They are approximately $25\,nm$ in diameter, which is below the maximum resolution limit of $200\,nm$, and they do not interfere with light waves.

Why can a stained mitochondrion be seen under a light microscope while a ribosome cannot?

A mitochondrion has a diameter of $1000\,nm$ and interferes with light waves, whereas a ribosome is only $25\,nm$ and does not interfere.

What happens when light waves encounter a transparent object?

The object allows the light waves to pass through it, meaning it does not interfere with them.

Why did biologists seek radiation with shorter wavelengths for microscopy?

To visualize biological structures smaller than $200\,nm$.

Why were X-rays rejected as a radiation source for microscopy?

Because of the difficulty in focusing X-rays.

What are two physical properties of electrons that make them ideal for microscopy?

Short wavelengths and negative electrical charge.

How are electron beams focused within an electron microscope?

Using electromagnets.

What is the relationship between the energy and wavelength of electrons used in microscopy?

Greater energy results in shorter wavelengths.

What is the typical resolution obtainable with an electron microscope?

$$0.5\,nm$$

Why do electrons possess wavelengths?

They exhibit both particle and wave properties, behaving like electromagnetic radiation.

Compared to a light microscope, how much better can the resolution of an electron microscope be?

It is $400$ to $2000$ times better.

Why is it impossible to view living specimens in an electron microscope?

The interior of an electron microscope must be a vacuum.

What are the specific specimen requirements for electron microscopy?

Specimens must be thin, dehydrated, and require very careful staining.

What causes artefacts in electron microscopy images?

They are caused by staining or the conditions within the microscope.

What are the two primary types of electron microscopes?

Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM).

How does a Transmission Electron Microscope (TEM) generate an image?

It passes electrons through the specimen and detects only those that are transmitted.

What kind of images are produced by a Transmission Electron Microscope (TEM)?

Flat 2-D images.

What is a primary benefit of using a Transmission Electron Microscope (TEM) in biological studies?

It allows us to see inside cells.

How does a Scanning Electron Microscope (SEM) operate?

Electrons are scattered from the surface of the specimen and then detected to form an image.

What type of images does a Scanning Electron Microscope (SEM) produce?

SEM produces 3-D images.

What is the typical resolution range of a Scanning Electron Microscope (SEM)?

Between $3 - 20\,nm$.

Why are specimens often coated in metals like gold for SEM?

To assist in scattering electrons for detection, though this can lead to artefacts (false images).

How are invisible electron beams visualized in electron microscopy?

Fluorescent screens are used to project them.

Why are specimens stained with metal ions in electron microscopy?

To prevent electrons from passing through, resulting in clearer images.

Why must an electron microscope be maintained in a vacuum?

To prevent electrons from colliding with air particles, which would cause scattering and result in the loss of a sharp image.

Why is it necessary to dehydrate specimens for electron microscopy?

In a vacuum, water boils at room temperature ($25^\circ\text{C}$), which would destroy fresh biological samples.

What is the primary function of the electron gun and anode?

They generate the beam of electrons required for imaging.

What is the role of the condenser electromagnetic lens in an electron microscope?

It directs the electron beam toward the specimen.

What is the function of the objective electromagnetic lenses in an electron microscope?

They are responsible for producing the image of the specimen.

What is the role of the projector electromagnetic lenses?

They focus the magnified image of the specimen onto the final viewing screen.

What is the primary function of the coarse adjustment knob on a light microscope?

It is used for initial focusing by facilitating large movements on the stage.

What is the purpose of the fine adjustment knob on a light microscope?

It is used for precise or final focusing by producing small movements to create a sharper image.

Which components of a light microscope regulate the amount of light entering the instrument?

The diaphragm and the condenser.