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Medsci 733 > Lecture 10: Optics 3 > Flashcards

Lecture 10: Optics 3 Flashcards

1
Q

What is luminescence?

A
  • Luminescence –the emission of light at low temperatures(cf incandescence)
  • Luminescence is a light emission which represents an excess over the thermal radiation, and lasts longer than the period of electromagnetic oscilation.
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2
Q

What are the types of luminescence?

A
  • Chemiluminescence
    • Bioluminescence
  • Electroluminescence
  • Photoluminescence
    • Phosphorescence
    • Fluorescence
  • Radioluminescence
  • Thermoluminescence
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3
Q

What is chemiluminescence and how does it wrok?

A

Chemi-luminescence –the emission of light as a result of a chemical reaction.

The excited state created by the chemical reaction is transferred to a dye molecule, or fluorophore, and subsequently fluoresces back to the ground state.

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4
Q

Whats an example of bioluminescence?

A

Aequorin

A protein produced by the Aequor(ae) genus of jellyfish that produces a blue light on binding Ca2+.

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5
Q

What do creatures use bioluminescence for?

A
  • Bioluminescence occurs when living creaturesconvert chemical energy to light energy.
  • Used for communication, food location, prey attraction, camouflage, defense.
  • An estimated 90% of deep sea creatures produce bioluminescence.
  • Most emit blue or green light (440 -479 nm).
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6
Q

What is another bioluminescence tool used commonly?

A

The green fluorescent protein (GFP) of Aequoreavictoriais now widely used as an in vivoreporter for monitoring dynamic processes in cells or organisms.

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7
Q

Describe how aeqorin functions;

A

Ca binds
Chromophore becomes excited
Enzyme catalyzes the breakdown of luciferin, releasing light

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8
Q

What can aeqorin be used for then?

A

As a DYNAMIC Ca reported in isolated tissues

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9
Q

What is good about using Aequorin as a Ca reporter over fluorescent tags?

A
  • Bioluminescence does not require light excitation like fluorescent probes or proteins.
  • Therefore it does not induce autofluorescence, photobleaching, or biological degradation.

Non toxic
Doesnt bind other divalent cations
Doesnt interfere with the intracellular Ca2+ concentration buffer system even when microinjected at high concentrations

Has a low affinityfor Ca2+(Kd= 10 μM) & is therefore a good sensor in the range of biological [Ca2+]variations.

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10
Q

What are some of the specific features of Aeqorin as a dynamic Ca reporter?

A
  • Emits bioluminescence (L/Lmax) on binding Ca2+ .
  • Molecule has 3 Ca2+ binding sites (2/3 reqdto emit 1 photon light).
  • L/Lmaxincreases monotonically from 10-7to 10-4M [Ca2+ ].
  • Not effected by photo bleaching.
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11
Q

What are the problems with Aeqorin?

A
  • Not good for low [Ca2+ ].
  • Difficult to load into cells.
  • Needs to be evenly distributed

Aequorin signals are very difficult to detect because of aequorin’s low light quantum yield (i.e. the number of emitted photons per protein that bind Ca2+).

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12
Q

List fast and slow photoluminescence techniques;

A
  • Phosphorescence–slow photoluminescence

* Fluorescence-fast photoluminescence

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13
Q

What is the key to photoluminescence?

A

•Luminescence stimulated by light absorption in UV-Vis-NIR spectral region.

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14
Q

What does photoluminescence represent?

A

•Representsany process in which material absorbs electromagnetic energy at a certain wavelength and then emits part of it at a different (usually longer) wavelength. -usually an excitation source emits in UV & the photoluminescence occurs in Vis or NIR.

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15
Q

Describe the time scale of phosphorescence;

A

•Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs. •The slower time scales of the re-emission are associated with “forbidden” energy state transitions in quantum mechanics.

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16
Q

Whats the difference between bioluminescence and fluorescence?

A
  • In bioluminescence, two or more substances combine to generate light (i.e. chemiluminescence).
  • In fluorescence, a substance absorbs light of one color and emits light of another color.
17
Q

What are factors that affect fluorescence?

A 
Quantum Yield
Fluorescence structures
Transition Type
Quenching
Effect of dye concentration
Intracellular Ionic concentrations
pH
18
Q

What is quenching?

A

Quenching: occurs when there is a non-radiativetransfer between the excited and quenching agent species. (e.g. Mn2+)

19
Q

What is Quantum Yield;

A

Quantum yield: used to compare how well a molecule will fluoresce or phosphoresce. It is the ratio of the number of molecules that are emitting light to the total number of excited molecules.

20
Q

What is sort of the threshold for quantum transition?

A

Transition types: rarely occurs from absorption at wavelengths less than 250nm because there is insufficient energy.

21
Q

What is the most popular method of iCa measurement?

A

Fluorescent probes

22
Q

What are many fluorescent probes based on?

A

•Many of fluorescent probes based on non-fluorescent Ca2+chelator EGTA.

  • Non-ratiometricor ratiometric
  • Penta-K+salt or acetoxymethyl(AM) forms
23
Q

What alters fluorescent signal qualities?

A
  • Light source intensity
  • Concentration of dye loaded
  • Hardware (objective NA)
  • Exposure (100ms or 1s)
  • Cell depth in preparation
  • Focal plane
  • Photobleaching
  • Dye leakage
24
Q

What can vary between calcium sensitive dyes?

A

The wavelength emission and intensity per a given calcium concentration

25
Q

Whats an example of a ratiometric Ca fluorescent probe and how does it work?

A

Fura-2

  • Two excitation wavelengths
  • Emission at 510nm
  • As Calcium increases 340nm signal increases and 380nm signal decreases and their ratio of emission indicates the level of calcium in the cell.
26
Q

Whats another factor to consider when choosing a probe?

A

Ensure their are no spectral overlaps in excitation and overlap if using multiple probes

Kd of the probes is a major consideration as in high calcium concentrations or major fluxes then a low Kd will consequently lead to saturation and the incapacity to report further change.

27
Q

What are the advantages of using a ratiometric indicator?

A
  • Corrects for differences in dye loading (between and within recordings)
  • Signal strength (e.g. cell depth, light intensity, variable cell thickness)
  • Does not require ‘calibration’ per recording
  • Corrects for photobleaching and dye leakage
28
Q

How is low light emission detected?

A

Photomultiplier tube

29
Q

How does a photomultiplier work?

A
  • Photomultipliers are able to multiply the signal produced by the incident light by figures up to 100 million.
  • In addition to their very high levels of gain, photomultipliers also exhibit a low noise level, high frequency response and a large collection area.
30
Q

What are the potential loading methods of fura-2?

A

Iontophoretic loading (claimed to reduce compartmentalisation)
Membrane permeable
Direct Pipette?

31
Q

Describe membrane permeable fura 2?

A

Acetoxymethyl ester (AM) form:•

  • Taken up freely into the cells
  • Subsequently cleaved within the cell by esterases.
  • Becomes negatively charged ‘active’ form
32
Q

What is the The Grynkiewicz equation ?

A

An equation to calibrate fura 2 in preparations for accurate reporting

33
Q

Describe the Grynkiewicz equation;

A

[Ca2+]=Kd beta [(R-Rmin)/(Rmax-R)]

Beta is the ratio of the fluorescence of the Ca2+freeform to the Ca2+boundform at l2(380nm) excitation;

Kd is the dissociation constant;

Rmin is the limiting value of R at zero [Ca2+], i.e. Sf1/Sf2;

Rmax is the limiting value of R at saturating [Ca2+], i.e. Sb1/Sb2

34
Q

What affects intracellular dyes

A 
Intracellular behaviourof the dye affected by:
•Viscosity
•Intracellular binding & uptake
•pHi
•Ionic strengh
35
Q

What would you calibrate in cells?

A

Calibration in cells:

•Aim is to introduce different [Ca2+]i, but takes a long time & cells have different mechanisms for controlling [Ca2+]

Medsci 733 (14 decks)

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