Let there not be light! If you don’t see fluorescence you might be FRET-ing (and lots of information getting!) Fluorophores are molecules that take in high-energy light (often light with such high energy we can’t see it) and spit out light that we can see. Different ones take in and emit different frequencies of light which we can describe as their excitation and emission spectra). But what if we have a fluorophore, we give it light we know it likes (in the excitation range), and we still can’t see it? Its energy may be being “stolen” by another fluorophore via Forster Resonance Energy Transfer (FRET) (sometimes referred to as Fluorescence Resonance Energy Transfer). 

blog form: 

note: apologies if some stuff is repeated and this post kinda sucks, I mixed and mashed some old stuff (in addition to the new) because, well, I’m a full-time grad student, remember so patience please! (similarly, sorry if I can’t answer every question. I didn’t set out to have thousands of followers and have no interest in being an influencer, doing paid endorsements, any of that stuff. I just want to help people, but research is my “job” (and love) and it keeps me busy!)

FRET can only happen if they’re really close and they have good SPECTRAL OVERLAP (the emission range of the first overlaps with the excitation range of the second). It can get confusing because FRET doesn’t actually involve light transfer between the 2, just energy, so the key thing to remember is that light is really just traveling packets of energy, so we can think about energy amounts overlapping. 

Molecules are made up of atoms which are like basic “units” of elements. If you were to look at a periodic table “menu” and “order” 1 of an element, they’d bring you an atom. In biochemistry, some of the most popular “orders” are carbon (C ), hydrogen (H), oxygen (O), nitrogen (N), and phosphorus (P). Within atoms are smaller parts called subatomic particles. Positively-charged protons and no-charge neutrons hang out together in a dense central core called the nucleus, but negatively-charged electrons aren’t so keen to stay put, so they whizz around the nucleus in electron clouds.

Electrons move around a lot, but they still have to follow laws of physics, so their movements and orientations are somewhat restricted. We can visualize molecular orbitals that are like electron apartment buildings where the different floors are different energy states. It costs more “energy money” to live further from the nucleus so in order to take an elevator upstairs, you need to absorb energy. And to go back down, you give back energy.

Within the floors (energy states) are different “vibrational levels” which are kinda like standing on a bed or a table or a dresser versus the carpet. You’re still in the same floor (state) but you have slightly more or less energy. You can move around on that floor more easily than going between floors. You need an “elevator ride” for that and that costs “energy money” to go up and you have to give back energy money to go down. 

Just like you have to invest work to carry a bowling ball up a hill, you have to invest energy to move the electron further from the nucleus (remember the nucleus is plus-charged cuz all those protons and opposite charges attract -> we’re asking the electron to go the opposite direction it should “want” to go). This energy for taking the elevator upstairs (excitation) can come in different ways (heat, chemical reactions, etc.). In the case of fluorescence, it comes in the form of “light”

Light is ElectroMagnetic Radiation (EMR) – when people think of “light” they usually think of “visible light,” but that’s just a teeny little piece of the EMR spectrum squeezed in between infrared to the left (lower energy, longer wavelength) and ultraviolet to the right (higher energy, shorter wavelength).

Light can be thought of as little packets of energy (photons) traveling as waves through space. The only thing different between the light you see and the light you don’t see is the amount of energy in their photons. All light travels at the same speed (the speed of light), but the photons with more energy want to go “faster” but they can’t go faster forward so they just bounce more along the way – they have a higher frequency (think of a surfer getting hit by more waves in the same amount of time) and, to keep the overall forward speed at the speed of light, the peaks of their waves have to be closer together to compensate. So the more energy in a photon, the higher the frequency (f) and the shorter the wavelength (λ).

From the perspective of a molecule in light’s path, it’s like being bombarded with “coins” of “energy money” with different wavelengths corresponding to different coin denominations. Molecules can only grab the coins if they have the right size “coin slots” which are determined by where their electrons are housed and how much it costs for those electrons to take the elevator a “higher floor” house (higher energy orbital). Different molecules accept different coin denominations depending on their chemical makeup, so they absorb different wavelengths

If the coin and the coin acceptor match, the photon is absorbed and the energy is used to boost an electron to a higher, excited state. terminology note: a part of a molecule that absorbs light is called a chromophore (chromo for color because they “steal a slice of the rainbow” leading us to see color – if they give back light we call them a fluorophore. 

Sometimes the elevator drops an electron off on top of a table, but it can quickly roll off onto the carpet (vibrational relaxation) but, unless the top vibrational level of the floor below is super close, it can’t “go through the floor” through vibrational relaxation – if it does we call it internal conversion & it can happen in high-energy orbitals where the floors are closer together. Normally, it’s gonna have to give up more energy than simple vibrational relaxation in order to fall back down a floor(s). 

In FLUORESCENCE, it gives back the energy as another photon. But it used up some of the energy for vibrational relaxation (table->carpet), lost some as heat, etc. so the amount it has to give back is lower, so the photon given off will have less energy (like taking in a dime and giving back a nickel). Since energy, wavelength, & frequency are directly linked, the emitted light will have longer wavelength and lower frequency. So the emission wavelength is red-shifted compared to the excitation wavelength and the difference between the 2 is called the Stokes shift. http://bit.ly/2Or1J3q

But that’s not the only way to give back energy. In addition to the “boring” stuff like bumping into other molecules that act as sort of “shock absorbers,” if certain conditions are met something much cooler can occur. Something known as Forster Resonance Energy Transfer (FRET) (sometimes referred to as Fluorescence Resonance Energy Transfer). It’s like a direct money transfer. Energy exchanges hands, but not light. No photons are exchanged, just “vibes”

Basically, in FRET, we couple 2 fluorophores (think 2 side-by-side apartment buildings). Instead of receiving energy from light, the second one receives energy from the first – one electron’s “going down” in apartment building 1 at the same time another electron’s “going up” in apartment building 2. This can only happen if they’re really close together, so if we see fluorescence from the second fluorophore we can tell that molecules are interacting. 

This energy transferring from the 1st to the 2nd is NOT in the form of light (it’s NON-RADIATIVE). It’s transferred through something called Förster Resonance Energy Transfer. There aren’t any particles flowing from one to another (no photons or electrons), just energy “vibes.” 

If we go back to our analogy of energy as a sort of “money” and photons as “coins,” fluorescence would be like taking in a dime and giving back a nickel (some of the energy gets used for “wiggling” and stuff). 

FRET, on the other hand, is more like a sort of wire money transfer, and this sort of transfer can only happen if the molecules are really close. And by close, I mean REALLY close. Visible light has wavelengths of ~380-740 nm (there are 1 billion nm in a m). And FRET can only happen at distances of < 10nm. For some perspective, an “average” human cell is ~ 20,000 nm (20um), bond lengths are ~0.15nm and an “average” protein has an ~4-5nm diameter (check out the bionumbers website for some more cool factoids). Note: sometimes, you see values in Angstroms (A) – an Angstrom (A) is 0.1nM. 

quick jargon-y detail note: The ability for FRET to occur decreases rapidly with distance – FRET efficiency (E) varies by the inverse sixth power of the distance between them (r).  EFRET = 1/[1 + (r/R0)6]

R0 is the Förster radius and it’s the distance at which E is at 50% of its max. This distance is usually a few nm. When r is less than R0, FRET is very efficient, but once you pass R0 things go downhill fast thanks to that “to the sixth” part, with a useful range ~ 0.5-1.5 x R0

This closeness requirement can be really useful. You might have seen microscopy images where people stain cells with a dye that binds to one thing and another dye that binds to another thing and then they overlay the images? It might look like the molecules are really close – they “co-localize”, but they might not be directly interacting. But with FRET, you know they really are. 

In addition to closeness, the other major requirement for FRET is “spectral overlap.” This means that the wavelength emitted from the donor (emission spectrum) overlapping with the wavelength absorbed by the acceptor (absorption/excitation spectrum) but in FRET there’s not actually light being transferred from one to another.

remember: We often talk about light in terms of wavelength (e.g. 500nm is reddish and 700nm is blueish) but FRET can make more sense if you think of it in terms of energy. The key thing is to remember is that light is just packets of energy (photons) traveling through space as waves. I And the wavelength & frequency of light are directly related to the energy of the photons. More energy, higher frequency, shorter wavelength. 

Even though photons aren’t being transferred, energy still is. And, when an excited electron falls back down, it has to give back the same amount of energy no matter how it chooses to give it back. But since there are different choices for this post-absorption philanthropy, there are lots of different versions of FRET.

Sometimes you use it to make things visible when they’re close, but you can also use it to make things “invisible” when they’re close. It all depends on what the FRET acceptor does with the energy it receives. If it lets it off as light, you see light. But if it lets it off as heat, etc. you don’t see light – even if you would have seen light if that acceptor wasn’t nearby. This “quenched fluorescence” is the basis of things like the TaqMan reporter probes used in qPCR (including diagnostic tests for SARS-CoV-2). 

The “classic” FRET example is – take 2 fluorophores and let one act as a donor to give energy to the acceptor. If you shine a wavelength the donor can absorb the donor can absorb it, and if its emission spectrum overlaps with the acceptor’s absorption spectrum, the acceptor will absorb it. 

The acceptor offers an alternative path for giving off energy. So the acceptor, even if it still absorbs the same amount of light as before, will emit less light as before if a FRET partner’s nearby because it’s giving off the energy it absorbs in non-radiative (non-light) transfer to the FRET partner. The donor’s fluorescence is being quenched.

The acceptor on the other hand has a different absorption spectrum than the donor. So it won’t absorb the original light. The “only” way it can fluoresce is if it gets the energy directly from the donor. 

So the acceptor won’t emit light in the absence of the donor. Once it gets energy from the donor, it can emit light (we call this sensitized emission) and this light will be at a different wavelength than the light emitted by the donor (but the same as if it absorbed energy directly from a photon).

In quenched fluorescence, the acceptors take in the light but don’t give off light they just release the energy as heat, etc. So you can measure quenching – look at presence or absence of fluorescence versus amounts of fluorescence at 1 wavelength vs the other. 

A lot of times you measure the stopping of fluorescence quenching. For example, in undergrad, I researched a metallopeptidase (a peptide-cutter). To study its cutting activity I used peptides (short amino acid (protein letter) chains) with a donor fluorophore at one end and an acceptor at the other end. The acceptor was a quencher, so it didn’t give off light of its own – it absorbed energy from the 1st but gave that energy off non-radiatively. So if FRET occurred, you couldn’t see anything. And the only way FRET could occur is if the peptide got cut because then the fluorophore was freed from the quencher and could shine. By measuring an increase in fluorescence, I could track enzymatic activity.

A similar quenched fluorescence scheme can be used for nucleic acids (DNA and RNA), the classic example being TaqMan probes, which are used in qPCR. qPCR is a technique where you make lots of copies of a specific region of DNA and measure the number of copies as they’re made to see how many copies you started with. The traditional diagnostic coronavirus tests use it to look for SARS-CoV-2 genetic information – if present, it will get amplified, and you can see it thanks to the ending of quenched fluorescence. 

PCR happens in cycles – you use a strand of DNA as a template for making the complementary strand, then you unzip the strands and do it again. and again. and again. With TaqMan probes, after each unzipping you add a quenched fluorescence probe in the form of a short piece of DNA that binds to the copied sequence, blocking the DNA copier (DNA Pol)’s travel. The DNA Pol thus has to chew up the probe to get past it and when it does so it frees the fluorophore and the quencher so you see light. You measure the light to see how many PCR cycles it takes to get above the background noise – this number of values is called the Ct value, and the lower it is (the fewer cycles it took) the more there was to begin with. So a lower Ct value indicates a higher amount of viral RNA in the initial sample. http://bit.ly/coronavirustesting 

technical (but important) caveat: you don’t know if that viral RNA is actually part of intact viruses, but a lower Ct value is more indicative of contagiousness. High Ct values, on the other hand, might represent post-infection shedding of “dead” virus and viral RNA pieces which are harmless – so some people test “positive” with PCR tests for weeks after infection even if they aren’t contagious. More on this here: http://bit.ly/reallyrapidtests 

How ‘bout another non-coronavirus example for a little mind break? Let’s go back to the DNA sequencing example we looked at yesterday. I’m not going to go into it in detail here, but classical there are 4 DNA letters and “Sanger sequencing” involves using labeled dideoxy nucleotides which are “chain terminators” – they don’t have the 3’OH part of DNA needed for linking to another letter. So once they get added you can’t add more letters. By including a mix of normal and terminator and asking DNA Pol to copy DNA you get pieces of various lengths ending in various letters which you can read. http://bit.ly/DNAsequencingmethods 

Each DNA letter is labeled with a different fluorophore, so we need to label 4 different things (ddATP, ddTTP, ddCTP, and ddGTP) and be able to tell them apart, which offers additional challenges.

Their emission wavelengths have to be far enough apart that you can tell them apart (have good spectral resolution). And not just the “peak” – fluorophores have a maximum emission (and absorbance), but there’s also “fuzziness” around it – the absorbance and emission spectra are like bell curves. And we want the curves to be “sharp” so they don’t overlap – we don’t want to mistake a T from an A, etc.

To help with this we can use energy-transfer conjugated dyes. We label the ddNTPs with dyes in which a donor and an acceptor are physically linked. The laser light excites the donor electron, which hops up to a higher energy level. When the electron falls, it’s gonna give off energy. But instead of giving off that energy as light, it gives it off as “nonradiative energy” (not-light) that the acceptor molecule snatches up -> excites the acceptor molecule’s electron to a higher level -> now, when this electron falls it does give off its energy in light form (fluoresces)

We can use the same donor for each of the 4 letters so they absorb the same wavelength – so you can excite them all with a single laser as they run through the gel.But the donors are conjugated to different acceptors, which have different emission wavelengths, and we want their emission spectra to be well-separated.

Because the signal strength is proportional to the absorbance, you want the absorbance wavelength to be optimal, which is why these donor-acceptor dyes are great because they have the same optimum absorbance, so we don’t have to optimize for one at the expense of the others or use a wavelength that’s just “meh” for each of therm. And, ideally, the signal strengths for each of the letters should be comparable so that the quiet guys don’t get drowned out. 

The “classic” dyes used are “BigDye” terminators. They use 5-carboxy-dichlororhodamine dyes as acceptor dyes & 5- or 6-carboxy isomers of 4′-aminomethylfluorescein as donor dyes. The 5-carboxy-dichlororhodamine dyes are slightly different for each letter so they give off different colored light. Linking the linked donor-acceptor to the nucleotide is a propargylamino (PA) or propargyl ethoxyamino (EO) linker.

A couple more uses: 

  • you can see if molecules interact (e.g. tag 2 proteins with FRET partners), whether molecules change shape (e.g. tag 2 ends of the same protein with FRET partners), etc. 
  • For test-tube work you can also use small molecule FRET partners like Cy3 and Cy5, which are frequently used to label DNA or RNA.

more on FRET:  http://bit.ly/2UaXNqv

#365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 

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