Super Resolution Microscopy

I heard it said a lot when I lived on the west coast. When it comes to business, ideas are worth nothing— it’s the execution that matters. More than I ever expected, that theme is echoed in science. And elsewhere. Anywhere I look, the things most worth doing are hard enough to do that, while it takes some decent smarts to think of the idea, it takes real vision to actually try it.

I met Eric Betzig the day he came to give a talk at Harvard, shortly before he won the Nobel Prize. He and my advisor were old friends, so a group of us students got to have lunch with the guy. To be tapped for hosting a speaker is kind of an honor for a new student.  He was a successful scientist in my field; had invented some microscopy technique I’d never heard of. I wore nicer clothes than usual.  I tried to make small talk. Anyways, over salad I described to him my project— the set up, the design, the challenges I was facing, the goal— to measure the repulsive Casimir force, a quantum electrodynamical force, using frustrated total internal reflection.

“Why?” he said, after a few seconds.

I didn’t realize quickly enough that he was challenging me.   I said something automatic– about fundamental limits.  I repeated the words from the research proposal: new materials, friction-less systems, the challenge of… He was shaking his head.

My face burned.  He had a reputation of being eccentric and brilliant. In his eyes I could see my ordinariness, my fear.

I should try to do something of actual value, he said. That I believe in.

The idea behind PALM is very simple, really. It’s the difference between the error on a single measurement and the error on the average of a bunch of measurements. Resolution in imaging is the limit of distinguishability of two nearby sources of light. The photons from each source land in a distribution, with some uncertainty in position on the detector determined by the width of this distribution. So each source appears not as a point, but as a spread out blob of light.


Two sources emitting at the same time getting closer and closer together will at some point appear indistinguishable. This limit, related to the width of each blob, is normally set by the size of the light-focusing element—an objective, a dish, or a lens. Incidentally this is why we have things like this:


But, imagine if we covered up all the emitters but one. We’d still get a blob of light on our detector, sure, with the same width as before, but now we can find with ease the center of that spot.


The uncertainty on the peak, or mean, position is many times smaller than the width of the distribution. How many times smaller depends on how much light you have collected.

It turns out, you don’t need to do this one emitter at a time. As long as the data is sparse, that is, the bright points are separated by adequate dark space, a large number of fits can be done all at once.

Later that day, on the way out of Betzig’s talk, which had been packed (I sat on the stairs) someone in my group said he didn’t see what the big deal was. Someone else said, yeah, the ideas are basic, almost brute force, even.

Turning on and off emitters is not a trivial business. It’s not like we have little wires and little switches attached to each molecule, fluorophore, or each star, or whatever we’re imaging. We can imagine trying to use selective illumination to excite emitters, but our excitation light is subject to the same diffraction limits as the light that forms the image.

But some fluorescent molecules were discovered to be activatable. They can be switched from a “dark”, non-emitting state, to an “active” state by exposure to UV light. Then, they can be turned off again through the process of photo-bleaching. There is a rate associated with the activation and deactivation of fluorophores. It does not mean that each molecule goes gradually from being active to inactive, it means that first some molecules are activated, and then some more, until finally the whole batch is lit up.

So, send a relatively weak pulse of light, over the whole sample, enough to activate, in some randomly chosen manner, about 1% of the fluorophores. Image these until they go dark. Find the centers of this sparse set of emitters. Send another pulse. Image those. And again. Stack the resolved images together.


The first time they took an image with this technique, it took 12 hours. Seven years later, they’d moved on, were now showing videos of live cells at 100 frames per second. Cancer cells crawling through a collagen matrix. T-cells attacking their targets. Not a biologist, I didn’t know the technical significance of what I saw. Still, my hair stood on end. Still, it haunted me— like something I was not supposed to have seen.

I wasn’t sure that I’d gone to the same talk as my colleagues.



I wish I could tell you a happy ending to my story. That I followed my passions, worked hard on some closely held idea and saw success, however moderate; that I’m on my way to not just graduation but contentment. But that wasn’t the way, and maybe that’s not the nature of the thing. I got rid of that original project, at least, but I’m still looking for something truly original, of value. That I believe in.


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