Lecture 6: fluorescence microscopy Flashcards
What’s the definition of fluorescence?
Luminescence that is caused by the absorption of radiation at one wavelenght followed by nearly immediate reradiation usually at a different wavelenght and that ceases almost at once when the radiation source stops.
Electrons have three different energy states, the ground state (S0), first excited state (S1) and second excited state (S2). How do molecules go from ground state to one of the two excited states?
Molecules in ground state (S0) are irradiated by a specific wavelenght to reach the first or second excited state. E.g. λ1 wavelenght irradiation causes molecules to reach the first excited state, while λ2 wavelenght irradiation causes molecules to reach the second excited state.
What happens when molecules reach S2 and go from S2 to S1?
There’s fast relaxation, where molecules lose some of their energy without the emission of photons by their movement/vibrations.
How does fluorescence occur?
Fluorescence occurs when molecules reach (S2 and so through) S1 and slowly release their energy (through photon emission). This energy release is what causes the fluorescence.
Why is there more energy needed to excite a molecule to S2 (absorption) than the amount of energy released to cause fluorescence (emission) from the same molecule?
Because part of the energy is lost (without photon release) due to vibrations of the molecules, which happens when molecules go from S2 to S1. The energy that is left, will be emitted by photons, which will cause fluorescence.
What is Stokes shift?
Due to the difference in energy absorption and emission (caused by molecular vibrations), the emission wavelenght is longer compared to the absorption wavelenght. Stokes shift is the difference between positions of the band maxima of the absorption and emission spectra of the same electronic transition.
For what is Stokes shift important?
It is crucial for fluorescence in microscopes, since it allows spectral separation of excitation light from fluorescence light.
A fluorophor can re-emit light upon light excitation and so can emit fluorescence. What are important properties for dyes in fluorescence microscopy?
- Optical properties (color, low photobleaching, stable fluorescence etc.)
- Physical properties (not too large)
- (Bio)chemical properties (non-cytotoxic, specific, environmental probe (pH, Ca2+)
Synthethic dyes, and with this fluorescent dyes, are materials that both absorb and emit strongly in the visible region of the spectrum. What are characteristics of these dyes?
- They can covalently bind to proteins or DNA
- Pretty stable (also pH stable)
- Used in e.g. antibody labeling
YOYO-1 is a green fluorescent dye used in DNA staining. It’s an intercalating dye and hardly fluorescent in solution. What’s an intercalating dye and why is it hardly visible in solution?
An intercalating dye is a dye that is inserted in between base pairs of DNA during DNA amplification, so that fluorescence intensity increases with each amplification. This is also the reason why it is hardly visble in solution.
What kind of dye is Indo-1?
A dye that changes spectrum or intensity upon ion binding. Indo-1 is a calcium indicator and has a dual emission peak: the main emission peak is in calcium-free solution and in presence of calcium this emission peak narrows.
What are quantum dots?
Quantum dots are made out of semiconductors (somewhere between a conductor and insulator), like CdSe (cadmium selenide). They have broad absorption and sharp emission. The confinement of energy depends on the quantum dot’s size and so the emission wavelenght also depends on the size of the quantum dot.
What are advantages and disadvantages of quantum dots?
- Advantage: very bright and very photostable
- Disadvantage: blinking on and off, big, finding the right attachment complex, toxic
What are intrinsic and extrinsic fluorophores?
- Intrinsic: fluorophores that occur naturally, like aromatic amino acids, NADH and flavins.
- Extrinsic: synthetic or genetically encoded fluorophores, like GFP.
Most intrinsic fluorophores don’t work very well and therefore in most cases extrinsic fluorophores have to be added.
How is the fluorophore formed in Green Fluorescent Protein?
The fluorophore is a tripeptide consisting of serine, tyrosine and glycine residues. Autocatalytically and in the presence of oxygen the fluorophore is formed from these three residues. Here, residues undergo cyclization, dehydration and subsequently oxidation.
The excitation source can be a lamp or a laser. What lamp can be used and what are its advantages and disadvantages?
Arc-discharge (electrical breakdown of a gas that produces a prolonges electrical discharge). It is used because of its brightness.
- Advantage: relatively cheap, many colors
- Disadvantage: highly inefficient, temperature, no sharp lines, cannot be foccused to difraction limited spots.
There are three different filters:
- exciter/excitation filter
- dichroic mirror/beam splitter
- emission/barrier filter
What do these filters do?
- exciter/excitation filter → transmits wanted color and blocks the rest
- dichroic mirror/beam splitter → reflects the excitation colors and transmits fluorescence
- emission/barrier filter → transmits fluorescence and block the rest
With the use of filters & dichroics, it is determinded which wave lenghts can or cannot pass through the filters. Exluding and including certain wave lengths, have different names:
- Long-pass
- Short-pass
- Band-pass
- Dichroic long-pass
Explain what wave lenghts are included/excluded through these mechnisms.
- Long-pass → reflects short wavelenghts while transmitting/passing long wavelengths.
- Short-pass → reflects long wavelenghts while transmitting/passing short wavelenghts
- Band-pass → reflects all wavelenghts except certain wavelenghts between e.g. 500 and 550 nm, which are transmitted/passed on.
- Dichroic long-pass → reflects a certain wavelenghts (e.g. 450-500 nm) and transmits/passes all other wavelenghts.
Explain how a charged-coupled decive (CCD) camera can visualize fluorescence and what advantages and disadvantages there are.
CCD is a chip that converts light (photons) into electric charge (electrons). Through an array of detectors fluorescent light is separated and a whole image can be recorded.
- It’s very sensitive and has very low noise
- It’s rather slow (every pixel has to be read out to convert the image).
What is wide field epifluorescence?
Here, the whole sample is illuminated with light of a specific wavelength coming from a parallel beam of laser. Emitted light is visualized through eye pieces or detected by a camera (e.g. CCD camera).
What is a disadvantage of wide field epifluorescence?
The entire sample is illuminated with the same intensity. When working with e.g. thick samples, it can be difficult to tell how deep in the sample the fluorescence originated from. It also creates a blurred image.
So it’s not typically suited for 3D imaging.
What is confocal fluorescence microscopy?
Here, one spot in the sample is measured at a time with the help of a point detector. This provides true 3D optical resolution. One voxel is measured at a time, which is why a laser or sample scanner is needed.
Why is it possible to visualize thick samples or 3D structures with confocal fluorescence microscopy?
A pinhole can be used, which suppresses out of focus light. The light of an object in focus can pass through the pinhole, while the light of an out of focus object can not.
