Tracking RNA with a double-helix point spread function

I’m spending the first half of this week at the Biophysical Society’s annual meeting , which is being held in Baltimore. The meeting is large. More than 6500 biophysicists from around the world are attending. Rather than try to provide an overview, I’ll focus instead on a few talks that I find surprising and interesting. If I liked them, maybe you will, too.

In yesterday’s New and Notable symposium, I learned about an innovative optical technique for tracking the three-dimensional paths taken by biomolecules as they carry out their evolution-ordained tasks. In general, you can’t use an optical microscope to locate individual protein molecules because the wavelength of visible light (380–750 nm) is a hundred times larger than the molecules themselves.

However, if you know how your microscope blurs the light from a point source—that is, if you know your microscope’s point spread function (PSF)—you can circumvent that diffraction limit. The trick is to insert a fluorescent tag into the molecules you want to track. Most microscopes have axially symmetric PSFs. If yours does too, you’ll know that when you observe glowing disks of fluorescence hundreds of nanometers in diameter, the protein isn’t just anywhere in the disk; it’s dead center.

Superlocalization, as this principle is known, can track molecules as they move from side to side, but it has a tougher time following their up and down motion, even within the microscope’s shallow focal plane. That’s because the PSF’s z component resembles a vertically oriented sausage. You can’t tell whether a tag is in the middle or at one of the ends of the sausage.

In his talk yesterday, Stanford University’s W. E. Moerner explained how his group is able to apply superlocalization in 3D. Following a recipe devised in 2000 by Rafael Piestun, Yoav Schechner, and Joseph Shamir, Moerner and his group create focused beams of light whose PSF in the z direction resembles not one but two sausages.

What’s more, the sausages wind around each other in helical pattern. Consequently, a horizontal slice through the PSF consists of two spots whose rotation angle in the xy plane corresponds to depth in the z direction. The upshot, as Moerner and his team demonstrated in 2009, is that they can pinpoint the location of a fluorescent tag with 20-nm precision throughout their microscope’s 2-μm focal depth.

In the 2009 proof-of-principle experiment, fluorescent molecules were scattered about and fixed in a polymer substrate. In his talk yesterday describing recent 2010 work in collaboration with Stanford’s Patrick O. Brown, Moerner showed the results of tracking the progress of messenger RNA molecules as they emerge from transcription and participate in translation.

For me, the most amazing aspect of the technique is not the valuable biological information that it yields, but the way one generates the double-helix PSF: by simply inserting a suitably patterned mask in an otherwise standard microscopy system. Of course, you do need to know how to pattern the mask. See the 2008 paper by Sri Rama Prasanna Pavani and Rafael Piestun for details.