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.