Introduction to microscopy

Question 1

What is microscopy?

Answer 1

Using microscopes to view objects/specimens that are not visible to the naked eye.

Question 2

What determines the resolution of an image?

Answer 2

→ The aperture of the objective determines the resolution.
→ The higher the numerical aperture the better the resolution power of the objective.

→ Resolution does NOT equal Magnification

Question 3

What does light microscopy aim to do?

Answer 3

→ Light and Lenses
→ To illuminate and magnify
→ Fundamental setup always the same
→ Brightfield, DIC, Phase

Question 4

What are the 2 types of electron microscopy?

Answer 4

Transmission EM
Scanning EM - allows for 3D images

Question 5

What makes a microscope a fluorescent microscope?(how is it different)

Answer 5

→ Firstly the source of light (emitted by a laser beam), has a fluorescence filter cube

→ Fluorescence involves absorption and emission, molecule receives light, is excited then loses energy and emits light

→ Excitation is always a higher wavelength than emission

→ This process is known as STOKES SHIFT- due to energy loss, the emitted light is shifted to longer wavelengths relative to the excitation light

Question 6

Where are fluorescent proteins , eg Green fluorescent protein found?

Answer 6

→ These proteins are naturally found in light producing cells of cnidarians

→ Fluorescent proteins can be fused with other proteins and introduced in cells via transfection. This allows live study of fluorescent tags in living cells/organisms.

Question 7

How can you use fluorescence?

Answer 7

→ Antibodies vs protein fusion (tag the gene)

Question 8

Compare widefield and confocal microscopic viewing

Answer 8

→ Higher z resolution and reduced out of focus blur make confocal pictures crisper and clearer.

→ However, only a small volume can be visualised by confocal microscopes at once- bigger volumes need time consuming sampling and image reassembling.