Fluorescence Microscopy

Question 1

Draw a diagram to show the resolving power of different microscopes.

Answer 1

Question 2

What limits the resolution of light microscopes?

Answer 2

• Abbe’s diffraction limit
• This is around 0.2 micrometers
• Two points cannot be differentiated if they are less than this difference apart

Question 3

What are 3 main types of fluorescence microscopy?

Answer 3

• Widefield
• Confocal
• Super-resolution

Question 4

What is the difference between widefield and confocal microscopy?

Answer 4

• Widefield microscopes gather emitted light from many focal planes -> Both in-focus and out-of-focus planes.
• Confocal microscopes blocks most of the out-of-focus light and allows detection of light only in one focal plane.

Question 5

What can widefield microscopy be used to observe?

Answer 5

• Tissue culture cells
• Molecules in solution
• Very thin tissue sections

Question 6

How does confocal microscopy work?

Answer 6

• The in-focus light is passed through a pinhole, allowing it to be seen.
• The out of focus light does not pass through the pinhole.
• Hence, a HD image of one plane is created. Multiple planes can be reconstructed to produce a 3D image.

Question 7

What are the two main types of confocal microscope?

Answer 7

• Laser scanning confocal microscope -> Involves a laser that scans across the sample line by line. It takes produces very high resolution images but takes a long time.
• Spinning disc confocal microscope -> Involves imaging via multiple pinholes at once so that many images can be collected at once. This makes it good for live imaging.

Question 8

What is 2-photon microscopy? How does it work?

Answer 8

• It is a form of fluorescent microscopy that is different to confocal microscopy
• It does not require a pinhole because only a tiny part of the sample is excited and therefore all of it is in focus
• It works by exciting an electron twice -> This is highly unlikely to happen apart from at the area of interest

Advantages:

• Used longer wavelengths of light, which are scattered to a lesser degree than shorter ones, and penetrate deeper into the sample.
• These lower-energy photons are less likely to cause damage outside the focal volume.

Question 9

Describe how STORM super-resolution microscopy works.

Answer 9