Lecture 10: Optics 3 Flashcards
What is luminescence?
- 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.
What are the types of luminescence?
- Chemiluminescence
- Bioluminescence
- Electroluminescence
- Photoluminescence
- Phosphorescence
- Fluorescence
- Radioluminescence
- Thermoluminescence
What is chemiluminescence and how does it wrok?
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.
Whats an example of bioluminescence?
Aequorin
A protein produced by the Aequor(ae) genus of jellyfish that produces a blue light on binding Ca2+.
What do creatures use bioluminescence for?
- 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).
What is another bioluminescence tool used commonly?
The green fluorescent protein (GFP) of Aequoreavictoriais now widely used as an in vivoreporter for monitoring dynamic processes in cells or organisms.
Describe how aeqorin functions;
Ca binds
Chromophore becomes excited
Enzyme catalyzes the breakdown of luciferin, releasing light
What can aeqorin be used for then?
As a DYNAMIC Ca reported in isolated tissues
What is good about using Aequorin as a Ca reporter over fluorescent tags?
- 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.
What are some of the specific features of Aeqorin as a dynamic Ca reporter?
- 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.
What are the problems with Aeqorin?
- 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+).
List fast and slow photoluminescence techniques;
- Phosphorescence–slow photoluminescence
* Fluorescence-fast photoluminescence
What is the key to photoluminescence?
•Luminescence stimulated by light absorption in UV-Vis-NIR spectral region.
What does photoluminescence represent?
•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.
Describe the time scale of phosphorescence;
•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.
Whats the difference between bioluminescence and fluorescence?
- 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.
What are factors that affect fluorescence?
Quantum Yield Fluorescence structures Transition Type Quenching Effect of dye concentration Intracellular Ionic concentrations pH
What is quenching?
Quenching: occurs when there is a non-radiativetransfer between the excited and quenching agent species. (e.g. Mn2+)
What is Quantum Yield;
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.
What is sort of the threshold for quantum transition?
Transition types: rarely occurs from absorption at wavelengths less than 250nm because there is insufficient energy.
What is the most popular method of iCa measurement?
Fluorescent probes
What are many fluorescent probes based on?
•Many of fluorescent probes based on non-fluorescent Ca2+chelator EGTA.
- Non-ratiometricor ratiometric
- Penta-K+salt or acetoxymethyl(AM) forms
What alters fluorescent signal qualities?
- Light source intensity
- Concentration of dye loaded
- Hardware (objective NA)
- Exposure (100ms or 1s)
- Cell depth in preparation
- Focal plane
- Photobleaching
- Dye leakage
What can vary between calcium sensitive dyes?
The wavelength emission and intensity per a given calcium concentration
Whats an example of a ratiometric Ca fluorescent probe and how does it work?
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.
Whats another factor to consider when choosing a probe?
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.
What are the advantages of using a ratiometric indicator?
- 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
How is low light emission detected?
Photomultiplier tube
How does a photomultiplier work?
- 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.
What are the potential loading methods of fura-2?
Iontophoretic loading (claimed to reduce compartmentalisation)
Membrane permeable
Direct Pipette?
Describe membrane permeable fura 2?
Acetoxymethyl ester (AM) form:•
- Taken up freely into the cells
- Subsequently cleaved within the cell by esterases.
- Becomes negatively charged ‘active’ form
What is the The Grynkiewicz equation ?
An equation to calibrate fura 2 in preparations for accurate reporting
Describe the Grynkiewicz equation;
[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
What affects intracellular dyes
Intracellular behaviourof the dye affected by: •Viscosity •Intracellular binding & uptake •pHi •Ionic strengh
What would you calibrate in cells?
Calibration in cells:
•Aim is to introduce different [Ca2+]i, but takes a long time & cells have different mechanisms for controlling [Ca2+]
