Jessica Winter – Interview

Making Quantum Dots Blink: An Interview with Jessica Winter

Compiled and edited by Carol Lynn Alpert from phone transcripts and emails during October 2010.

Carol Lynn: Jessica, we understand your role on the QSTORM team is to produce
quantum dots that “blink” on command.  Can you help us understand why this is such an essential aspect of getting to the overall goal of imaging of subcellular structures in living organisms?

Jessica: Well, let’s start by explaining that quantum dots, or QD’s are tiny
collections of  roughly 10,000 atoms  or so.  They form a crystalline structure that’s on the order of one to ten nanometers in diameter.  They’re made from semiconductor materials, usually cadmium combined with one of the Group VI elements: selenium, telerium, or sulfur.   Quantum dots can glow with a very bright fluorescence when they are activated, and the color of the light they emit is determined by the size of the particle, because in the excited state, the electron “cloud”  — remember we are talking quantum mechanics here – the electron is a probability/wave function and not a discrete particle — wants to be larger than the size of the particle physically permits it to be. This is called “quantum confinement.”   The QD “electron cloud” size is constrained by the particle size, which determines how much energy (and what wavelength of energy) the particle can adsorb and therefore emit, which determines the color of its glow.

Carol Lynn: What color QDs are you using for the QSTORM experiments?

Jessica: We chose green.  Color choice depends on the application.  The QSTORM
biologists, Ge and Beth, are going to be microinjecting these QDots into living organisms.  For in vivo applications like this, most of the time we go for green,  because red is the same color as blood, and blue requires UV excitation, which can be dangerous to the organism.

Carol Lynn: What do you mean by “excitation?”  What makes them glow?

Jessica: Almost all structures can absorb and reflect light. Light has energy. If an
insulator (like, say, your desk) absorbs light, it will turn this energy into a small amount of heat via atomic vibration. If a QDot (which is a semiconductor) absorbs light. the energy can be sufficient to “excite” an electron from a ground state to a higher level shell in the atomic structure. This excited electron can react with another chemical molecule, be removed by electrical flow (current), or dissipated as heat through atomic vibration.  If none of those things happen, it will instead produce a fluorescence signal, a glow made up of photons.  Fluorescence is the inverse of the adsorption process. Light is adsorbed to the surface of the QD, exciting an electron, and then emitted as a photon when the electron “relaxes” back to its ground state.   Energy cannot be perfectly conserved when it converts between different forms (the second law of thermodynamics covers this – and it is why we can’t have a perpetual motion machine), so the emitted light has slightly less energy than the adsorbed light. This means that it is “red-shifted” in the visible spectrum. So a QD that adsorbs ultraviolet light may emit blue light, and a QD that adsorbs green light might red light.

Carol Lynn: What kind of light do you generally use to activate your Qdots?

Jessica: Actually, I usually use a broad spectrum light source, mercury lamps with filters. These are less expensive then lasers, which normally come tuned to only one particular wavelength.  The filtered mercury lamps throw a small range of wavelengths, but as long as those include the QD-tuned wavelength, it will work to activate them.  QDs can in fact respond to a small band of wavelengths to excite them, but, in turn, they only emit one wavelength.

Carol Lynn: OK, now let’s get back to what these QDots are for.  You’re going  to give the QDots you make to the QSTORM biologists, and they’re going to inject them into a cell inside a living organism… wait, how many QD’s are they going to put inside each cell?

Jessica: Normally it takes tens of thousands to illuminate what we want to see.

Carol Lynn: Wow.  OK, so are the QDots literally shining light on what is to be
Imaged? OR is it the QDots themselves that we are imaging?

Jessica: We image the dots.  We get them to attach to one particular molecular
structure inside the cell, and they light it up the way strings of Christmas lights can outline the structure of a bare-leaved tree at night.  The dots are so small that they completely outline the molecular structure we want to image.

Carol Lynn: How do you get the QDots to target a particular molecular structure
inside a cell?

Jessica: Well, the biologists help us pick a targeting molecule, something like an
antibody that  is attracted to and binds with a particular type of molecule in the cell.  We tether this targeting molecule to the QDots before we inject them, and they guide the QDot to the structure we want to image.

Carol Lynn: So are we literally imaging the QDots glowing right through the cell
and the organism?

Jessica: Yes.  The organism will be immobilized.  We expect to see a very faint glow
from a portion of the QDots sticking to the molecules they’re tethered to.

Carol Lynn: OK, now I think we’re ready to tackle the blinking question.  Why do we
need to be able to “turn” the QDots “on and off?”

Jessica: Here’s the issue:  The quantum dots in the cell are so tiny and so close to
one another that when we activate them with the mercury lamp, and they are all switched “on” at the same time, the light that each one emits will interfere with the light being emitted by their close neighbors.   Remember, that light is actually a wave function.  An interference pattern is produced when two wave functions overlay each other.  When you’re trying to focus the light source with a circular lens you get this circular diffraction pattern called an Airy disc.  It’s named after an English astronomer, George Airy, who was able to explain mathematically why telescope images of stars also can have these kinds of blurry distortions.   But, if we could selectively turn on and off some subset of the QDots, the probability is that some of them will be glowing without neighbors glowing right next to them, and thus we can image them in high resolution without diffraction.  And then the next time we image them, another set of QDots will be “on” and another set “off,” and so on.  So we hope to be able to construct a complete QSTORM image by superimposing multiple images in the same frame until we have a complete outline of a structure.  To see what I mean, check out the very cool YouTube video demonstration of what it would look like to image the Eiffel Tower through this kind of reconstructive imaging.  Its called QuickPALM/Storm over the Eiffel Tower.

Carol Lynn: That’s a pretty cool visual demonstration. I recommend this to all
readers:  http://www.youtube.com/watch?v=RE70GuMCzww&feature=related.

Now that we have an idea why you want to get the QDots to switch on and off, or blink, can you tell us how you’re going to manage to do it?

Jessica: Good question!  That is at the heart of our QSTORM research here.  We have
to figure out how to control the blinking from outside the cell.   One method that has been tried is to change the pH of the cell.  That worked, but it would be disastrous to try to do in living cells – they need to keep a very careful balance to their internal chemistry to function properly.   Instead, we’re going to try to work with a second external light source as a control signal, a laser beam this time.  While the mercury light excites the QDots, this second light source controls their blinking.  It’s like a string of Christmas lights.  They won’t work unless they’re plugged in, but their blinking is controlled by a separate electrical circuit.  The switch off mode we call “photocaging” or “quenching” the light emission of some of the dots, so that we can image others more clearly. We won’t be able to control exactly which subsets of QDots turn on and off – they’ll respond on a Bell curve and the probability is that we will eventually be able to image almost all subsets of them.  That’s one of the reasons we’re using QDots instead of traditional fluorescent dyes.  Those dyes can photobleach and fade.  QDots don’t fade, and they emit photons two orders of magnitude faster. The quicker we can collect each light emission, the better the image.

Carol Lynn: Has anyone tried to photocage quantum dots before?

Jessica: Not that we know of.   We’ve got a few ideas that we’re going to try.

Carol Lynn: You mentioned that quantum dots can sometimes blink on and off
without  an external  control signal.  Is that a problem?

Jessica: Yes, it can be. The energy of the excited electrons can be dissipated by heat
instead of by emitting photons.  So, while they all blink, they don’t all do it at the same time in response to a signal.  That’s one of the reasons we thought we might also see if we could use carbon nanoparticles instead of QDots, because they fluoresce without this random blinking.  But, they’re not well understood.  Just today I read an interesting paper about polymer nanoparticles (PDots) – and they might be worth a try too.  They’re even brighter.  People have been trying to make non-blinking QDots for long time.  We’re NOT trying to stop QDots from blinking.  We’re just trying to photocage and control the blinking.  Our first hope is that a laser beam combined with some kind of doping mechanism will work to photocage the QDs.  If we can do that, all the QDs in a particular sample might be photocaged, or switched off.  Then we would apply light at an energy level which would release only some of them at a time.  That would allow us to build up a collection of image frames with super resolution.

Carol Lynn: Jessica, you and your students have a lot of chemistry, physics, and
engineering to do in your lab over the next few months!  We’ll check back with you periodically to see where you’re at with the experiments. Thanks so much for taking the time to talk with us.

Jessica: Good luck writing up this interview.