We can take advantage of luminescence in the lab to make molecules glow & thus results show! “Luminescence” is the term we use to describe molecules giving off light, which some do when they have some “extra energy” to release. This energy can come from various sources including: light (in which case we call the process fluorescence) or a chemical reaction (in which case we call the process chemiluminescence – and, if it comes from a biological/biochemical reaction, we can call it bioluminescence). Normally this extra energy is held by the outer electrons. Electrons are one of 3 main types of subatomic particles – unlike the positively-charged protons & neutral neutrons, which stay glued together in a dense central atomic nucleus, the electrons are free to whizz around that nucleus (but you’re most likely to find them in certain areas called orbitals).

note: (text adapted from past posts, video new)

An electron’s “usual” position is its “ground state” – it’s basically as close as the electron can comfortably get to the nucleus – taking into account all the other electrons it has around it. An electron’s ground state is the “default” because it requires the least amount of energy – basically the positive pull of the protons in the nucleus is a bit like a leash on the electrons and the electrons need energy to tug back & stretch out that leash if they want to move further away. But, when electrons have extra energy, the can venture a little further out into an “excited state”

It’s kinda like a dog on a leash that sees a squirrel – it gets excited, tugs on the leash, maybe gets a little further from the walker – but eventually runs out of steam, starts suffocating itself, and falls back into the “normal” walking distance that doesn’t choke it. Since it’s easier to walk at the closer distance, the dog doesn’t have to spend as much energy, so it can use the energy it saves for doing things like daydreaming about squirrels. But electrons don’t have use for the extra energy they save, so they just release it.

A lot of times they release it as “non-radiative energy” – for example, they might just gradually wiggle around, bump into other things, etc. – letting off little bits of energy on their way back down. But sometimes, if the energy’s just right, they’ll release that extra energy in the form of a photon. A photon is a little packet of energy that – along with a lot of other photons – travels in a wave to produce ElectroMagnetic Radiation (EMR) – aka LIGHT! And we call this light-releasing process luminescence.

In order for the light to be released, energy first must be absorbed – so how do you get an electron “dog” to “see a squirrel?” A common way is with light. Parts of molecules called chromophores can, depending on their chemical structure, absorb photons of specific energies (and energy is inversely related to wavelength (higher energy, higher frequency, shorter wavelength). If you think of light as baseballs being thrown wiggly-wise (with higher-energy (higher-frequency, shorter wavelength) balls wiggling more), chromophores are like special mitts that can capture certain balls. 

In fluorescence, a molecule absorbs a photon of higher energy, exciting an electron. But then the electron “crashes” and falls back down, releasing the energy it no longer needs in the form of another photon (but of lower energy because some of it’s lost as heat, vibrating, etc.) (It’s like a molecule catching a really wiggly ball and then throwing a slightly less-wiggly one.  But that exciting energy doesn’t have to come from light – for example, in chemiluminescence the energy comes from chemical reactions. In bioluminescence, the energy comes from biological/biochemical reactions. Common examples are firefly luciferase, recilla lucifeyrase, and nanoluciferase. They’ll only give off light if you give them the chemical substrates they need. So you can get lower background signal, etc.

helpful resources:

Often the absorb – release energy cycle’s pretty fast. But what if the energy source only periodically (and, to make things trickier – only randomly and not that frequently) releases energy? (e.g., radioactive decay?) You’re left squirrel-waiting. And if the signal from each squirrel-sighting’s really small you might not see it at all. So what you want to do is basically trap all the excited dogs in an excited state and get them to all “crash back” at the same time. 

So to visualize radioactively-labeled products (e.g. labeled DNA or RNA) in gels I use digital radiography – I “collect the energy” given off from the individual decays on a storage phosphor screen and then when I scan the screen with a laser it’s like releasing a bunch of pent-up energy all at once – that energy gets converted to light which gets amplified and converted into an electronic signal.

Much more here, but here’s an overview: blog form: http://bit.ly/phosphorimaging 

I can label things with radioactive isotopes that’ll decay & let off a signal – but they do so randomly and at their own pace. So instead of watching for each individual decay, I capture the energy given off by the decay on a storage phosphor screen – I expose the white side of the screen to a saran-wrapped membrane or gel or wherever my labeled thing is. When those decays happen, they’ll excite electrons in the screen (and in the position right above them so we can see location) from a bored state to an excited but trapped state. I let the decay process carry out, with more and more phosphoruses decaying and thus more and more electrons getting excited and stuck. 

Depending on how “hot” (radioactive) the sample was, I expose it for a few hours to a few days. And then I excite them even more  (with a laser scanner) so they get unstuck to an excited but unstable state. And then they fall back down to the “bored” state, giving off the now-unneeded energy as light. 

much more on fluorescence: http://bit.ly/fluorescentstains & http://bit.ly/fretandfluorescence  

more about GFP: https://bit.ly/gfpfunscience

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