The_Scientist_-_December_2018

60 THE SCIENTIST | the-scientist.com

LAB TOOLS

NOZAKI ET AL.,

MOL CELL

, 67:2, 2017; VADIM BACKMAN, NORTHWESTERN UNIVERSITY

WIGGLES AND WAVES

RESEARCHER: Kazuhiro Maeshima, structural biologist,

National Institute of Genetics, Mishima, Japan

PROJECT: Super-resolution imaging of chromatin structure in

living cells

CHALLENGE: For electron microscopy and array tomography,

cells are treated with chemicals to fix their structures for imaging.

The result is crisp and detailed images—and dead cells. That pre-

vents observational studies of how chromatin packaging responds

to environmental changes inside and outside the cell.

SOLUTION: Advances in fluorescent probes and in strategies

for sample illumination have pushed the possible spatial resolu-

tion of fluorescent images down to fewer than 10 nanometers—a

scale small enough to reveal chromatin changes in living cells.

Maeshima combined a super-resolution imaging technique

called photoactivated localization microscopy with imaging soft-

ware that tracks the movement of a single DNA-wrapped his-

tone structure, called a nucleosome. The researchers found that

chromatin in more open configurations moved more than tightly

packed chromatin did. Maeshima suggests that these shifts can

make even more genes accessible to transcriptional machinery.

NEED TO KNOW: Since the development of super-resolution

microscopy nearly a decade ago, researchers have developed long-

lasting dyes that make these kind of tracking experiments possible,

says Maeshima. But some limitations remain: “We cannot see all

of the nucleosomes at once,” he says. “If we did, we couldn’t see the

individual movements—so we can only pick out a limited number

of nucleosomes to see at a time.”

USE IT: The team used the image-processing software Poly-Particle-

Tracker to trace the movements of their labeled nucleosomes.

NATURAL LIGHT

RESEARCHER: Vadim Backman, biomedical engineer,

Northwestern University, Evanston, Illinois

PROJECT: Imaging chromatin in its natural state within the cell

CHALLENGE:Most fluorescent dyes that bind to DNA are toxic. If

they don’t kill the cell right away, they can still produce undesirable

effects. “I always cringe, because I never know if what I look at is going

to be the reality of chromatin or if it is an artifact,” says Backman.

SOLUTION: If labels are toxic, why use them at all? Some biological

molecules give off their own natural fluorescence, but researchers

didn’t think DNA was one of them until Backman’s group saw DNA

“blink.” They discovered that DNA does give off natural fluores-

cence, but only sporadically. Most of the time it remains in a dark

state, but a patient eye can catch the flash as the DNA absorbs light,

gets excited, and emits its own fluorescence. Backman found that

even low levels of illumination are sufficient to produce the blink,

which means that the light exposure won’t damage the cells. “You

can run experiments for days, imaging the entire cell cycle,” he says.

NEED TO KNOW: Not all of the DNA strand glows at the same time,

however, so the compromise is that microscopists can’t get an image

of each and every chromatin molecule using this technique. Further-

more, the technique isn’t yet at the super-resolution stage, so while

changes that happen at the 20-nanometer scale can be detected,

they can only be localized to a 200-nanometer-wide region. Even so,

Backman says, “you can look at hundreds of cells over days.”

USE IT:Backman and his colleagues are currently developing a

resource core at Northwestern dedicated to label-free imaging of

DNA. They also plan to create a website next year to provide guid-

ance for other scientists who want to try their hand at the method. g

MOTION CAPTURE: Stem cells encouraged to start differentiating

(right) show more densely packed chromatin than stem cells with an

inhibitory chemical (left). Tracking the movement of fluorescent particles

on the genetic material also shows that chromatin in the differentiating

cells moves less (more blue in lower right panel).

LABEL FREE: A technique that looks for

small fluctuations in the light coming from

DNA molecules in living cells can show

changes in chromatin structure over time.