P.S. I Love PhotoStimulated Luminescence (PSL) because it lets me see invisible (radioactive) things! It’s what happens when I “expose a screen” to see where radioactively-labeled things end up in an experiment like a slot blot – where I label RNA and see if (and how eagerly) it binds protein. I can use radiolabeling as a sort of GPS tracker – if I swap out one of the phosphoruses at the beginning of an RNA with a radioactive version, that RNA will give off radiation when that radioactive phosphorus decays. But nuclear decay is a random process that happens over time and I want to see it all!
So 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.
One of the first times I used a storage phosphor screen, I stuck the screen on my gels upside-down – I ended up getting a really week signal – and a chewing-out from the postdoc I was working with. That was just one of several experiences early in my PhD journey that got me to doubt whether I was cut out for science – I decided that I wasn’t going to let my self-doubt get in my way – instead I’d just make sure that I helped make scientific training a more positive experience for others. I’ve gone on to use these screens A LOT so I want to tell you more about them.
“Luminescence” is the term we use to describe molecules giving off light, which some do when they have some “extra energy” to release. 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)
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
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? 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.
I said before that the nucleus is pretty “boring” and “stuck,” but different forms (isotopes) of the same element can have different #s of neutrons – and if the # of protons & # of neutrons is too uneven, the nucleus can actually become unstable. RADIOACTIVE DECAY is a way that unstable atomic nuclei can “rearrange” themselves to more stable groupings, usually accompanied by the release of energy.
Something you might have picked up on is that when things go from less stable to more stable, they usually release energy – the way I like to think about this is that when you’re more comfortable you don’t have to fidget around trying to find a more comfy position anymore. I use a radioisotope of phosphorus called 32P – when it decays it lets of energy in the form of a beta particle.
This decay process is random, and it varies for each radioactive isotope, but you can look up the probability that any one molecule will decay at any timepoint and then take into account the number of molecules to figure out something called the HALF-LIFE, which is the average time it takes for half of the molecules to decay (for 32P this is ~14 days, so 1/2 of it’s “used up” in 2 weeks).
Each decay lets off a really small signal – the more molecules there are, the higher the signal will be, but they’ll still come randomly and staggered over time. So you want a way to “tally it up” and then read out the score. A way that we can do this is with Storage Phosphor Screens, which involve 2 reactions – the “tallying up” of how many squirrels were spotted and the “squirrel score reporting”
The first reaction (triggered by radioactive decay) gets electrons to actually move to other atoms (a dog gets so much extra energy it breaks free of its leash) but then gets “trapped” in a “metastable state”. And then the second reaction (triggered by the laser) gets the electrons to fall down – first the dog returns to its owner, but in an excited state, and then it “gives up” trying to escape and falls back to its normal “ground state”, releasing the extra energy as light.
So how does it work? This inorganic chemistry stuff’s outside of my expertise – and there are multiple theories, but here’s what I’ve been able to figure out (hopefully correctly).
The screen has a few different layers. At the base is a support layer and at the “top” is a protective layer. And in the middle of this “sandwich” is the really important part – the phosphor layer, which is ~10-20mm thick and – despite the name – doesn’t contain any phosphorus.
Instead “phosphor” is a kinda generic term for a luminescent material. The phosphor layer in the screens I use is is made up of a crystal lattice of Barium fluorobromide (BaFBr) w/a little bit (trace amount) of europium (Eu2+) replacing some of the bariums. Eu2+ is used as a color center (aka F-center) – it acts as the source of luminescence (light-releasing).
Remember – light-releasing = energy-releasing so first you need to do some energy-gaining, and, instead of light energy, our initial energy source is nuclear radiation. The type of radiation given off by radioactive decay is often called “ionizing radiation” because it can turn neutral molecules into ions (charged particles) and alter the charge of ions – by giving or “knocking-out” negatively-charged electrons from them and skewing the proton/electron ratio.
For example, when Eu2+ gets hit by radiation given off from 32P decay it can lose an electron to become Eu3+ (it’s kinda like a dog gets really excited and breaks the leash all together) & can hang out with BaFBr, giving you BaFBr-:Eu3+.
And this FBr associated state is a higher-energy state than the ground state of the Eu (but not as high as the excited state that’s easy to fall from). And, unlike the high-energy states of normal fluorescent dyes and stuff, this state is “metastable” – it’s not comfy, but it won’t change unless something else changes (kinda like if you get a new job with a higher salary, but you hate the job but you like the $ so you won’t go back to your old job unless you can be persuaded by an outside source)
If it gets a little more energy, it can get “untrapped” and it joins back up with its original owner. The persuading outside source comes from a light source – it’s not as high in energy as the radiation given off by the P32 – so it’s not going to excite any Eu2+ ions to become Eu3+, but it is enough to excite the “trapped” electron to a different energy state that’s less stable & easier to fall all the way down from.
Our laser and PMT are in this scanner device (TMed the Typhoon). When I stick my screen in and tell it to scan, a red laser hits the BaFBr-:Eu3+ & gets the electron to return to the Eu (molecular Br-exit?) to give you back BaFBr + Eu2+. But this is different from the “original” BaFBr + Eu2+ because the Eu2+ has an electron in an excited state – not excited enough to break free all the way, but excited enough to be tugging on the leash. And this is one of those much less stable states, so it’s not just gonna wait around like that – it’ll fall back down.
And when it falls back down, it releases that extra energy it doesn’t need anymore as a photon. And the amount of energy in that photon produces light of a color we see as blue-violet (~400nm). One of the cool things about it is that this is actually higher energy light than the excitation light because the electron was already “partway there” – just trapped. The signal’s pretty weak, so it gets captured by something called a PMT (PhotoMultiplier Tube) which amplifies it and converts it to an electronic signal.
And since you’re exciting all of them at the same time, your signal will correspond to the total number of exciting events, which corresponds to how many 32P molecules decayed – which corresponds to how many there were there in the first place (multiplied by the PMT stuff).
These screens were a big advance over traditional film – they’re more sensitive so you can use shorter exposure times & lower levels of radiation. Also unlike film, they can be reused – you just have to “blank” them by sticking them on really bright white light for a while. That gives it plenty of energy for all of the stuck electrons to unstick and fall back down so the screen’s regenerated.
So, to summarize – 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.
Normally I “sneak an early look” – scan it after an hour or so (it’s hard to wait long enough!) – then put on a NEW screen for an overnight exposure.