Introduction to fluorescence microscopy

Question 1

What’s the difference between fluorescence and chemiluminescence?

Answer 1

Fluorescence requires energy from outside the molecular system (radiation).

Chemiluminescence is when a chemical reaction excites a molecule, facilitating a light emission.

Question 2

The larger the aromatic rings, the 1.____ delocalized electrons, the 2.____ a fluorophore is to excite.

Answer 2

1. more
   2.easier

Question 3

What’s the stokes’ shift?

Answer 3

It’s the shift in max emission and absorption for a fluorophore.

Emission is always less energetic than absorption.

Question 4

What’s the purpose of a barrier filter in a fluorescence microscope?

Answer 4

It ensures that the wavelength used for excitation isn’t reflected into the camera used for fluorescence detection.

Question 5

What does an excitation filter do in a fluorescence microscope?

Answer 5

A fluorescence filter ensures that the wavelength of interest is filtered away from the rest when exciting the fluorophores.

Question 6

What’s the purpose of a chromatic beam splitter in a fluorescence microscope?

Answer 6

It is placed downstream of the excitation filter at 90 degrees from the sample. It reflects short wavelengths, filter away long wavelengths.

Question 7

What’s the principle behind digital monochromatic cameras?

Answer 7

1. Picture is divided into an array.
2. Each pixel light intensity is quantified.
3. Photograph using RGB filters to make a coloured image.

Question 8

What’s pseeudocolouring?

Answer 8

1. Insert multiple fluorophores with different emission spectras.
2. Visualize each fluorophore separately.
3. Allocate different RGB-spectrums to each fluorophore.
4. Overlap the images.

Question 9

What’s the limiting factor for microscope resolution in light microscopes?

Answer 9

The diffraction of light when it passes through the objectives (causes interference patterns). The refraction pattern causes what’s known as an airy disc.

In practical terms, the best resolution possible is 0.2 micrometers.

Question 10

What’s a n airy disc? Why are airy discs important?

Answer 10

1. Airy disc: onceptualized infinietely small, very bright object which is captured in a microscope. The light is defracted in an interferaence pattern.
2. The overlap of two objects’ airy discs dictate whether you can tell them apart or not. The minnimum distinguishing length = The width of the airy disc.

Question 11

What does numerical apperture (NA) have to do with blurry images?

Answer 11

Numerical apperture (NA) dictates the focus plane of the microscope. If cells are thicker than the focal plane, the images will be blurred.

Question 12

How is out-of-focus light blocked out in confocal microscopy?

Answer 12

Confocal microscopy focuses on small areas of the cell at the time. There’s a pinhole in front of the fluorescence detector that blocks light deriving from other places thtan the illuminated area.

Question 13

Explain the principle behind image deconvolution.

Answer 13

1. Cell is divided into z-stacks (z-axis fragments).
2. Generate images of fluorescence at different planes. Each image has out-of-focus light.
3. Cancel the light which derives from a z-stack lower than the z-stack you’re looking at. This effectively relocates the light to its original sources.

Question 14

What’s photobleaching?

Answer 14

It’s when you burn out a fluorophore permanently by exciting it too long.

Question 15

Name the different types of fluorophores used in microscopy and what they bind to.

Answer 15

1. Organelle probes - organelles.
2. Coupled fluorophores - Any specific protein.
3. Environmental probes - Fluoresces depending on environmental conditions (ph, temp..)
4. Fluorescent proteins - generally fluorescent.

Question 16

Examplify how you could use GFP to study a protein of interest.

Answer 16

1. Fuse GFP to the protein by removing the transcription stop codon.
2. Fuse GFP gene next to promoter that allows transcription of the protein of interest.

Question 17

What problems can arise from fluorescence protein fusions?

Answer 17

1. Functional disturbance of the protein.
2. Poor protein localization.
3. Protein clustering (If the fluorescent protein has an affinity for other proteins).
4. GFP-protein fusion may not be folded the same natively.

Question 18

What does FRAP stand for? How is the method used?

Answer 18

1. FRAP = Fluorescence revovery after photobleaching.
2. You purposefully bleach fluorophores in certain spots.

Question 19

What’s split-GFP?

Answer 19

It’s GFP which can only fluoresce if two proteins interact. It’s a marker of interaction. The majority of GFP is fused to one of the proteins (the GFP lock). The remaining unit of GFP is fused to another protein (the GFP key). If the two units come together, functional GFP is generated.