The_Scientist_-_December_2018

12.2018 | THE SCIENTIST 59

OU ET AL.,

SCIENCE

, 357:6349, 2017

matin’s higher-order structures, between 20 and 200 nanome-

ters, light microscopy struggles. The scale is too small to resolve

with conventional microscopes and too large for super-resolution

approaches. Electron microscopy can offer a clearer picture at that

scale, but most of the stains used in electron microscopy do not

react with DNA exclusively. As a result, scientists have generated

conflicting models of how chromatin fibers fold together.

SOLUTION: O’Shea’s team developed a system that labels chromatin

for electron microscopy using multiple chemical steps. First, a fluores-

cent dye called DRAQ5 binds exclusively to DNA. Then, the research-

ers excite that fluorescent dye, which in turn catalyzes a reaction that

dusts a thin layer of polymers on nearby DNA and its associated pro-

teins. Finally, the researchers paint that polymer dust with a metal that

can be visualized with electron microscopy, revealing the chromatin.

O’Shea and her colleagues combined this staining approach

with a version of electron microscopy called electron tomography

that images the sample at multiple angles. “You get the computa-

tional equivalent of cutting through the nucleus with a ham slicer,”

she says. The researchers were able to reconstruct those virtual

“slices” into a three-dimensional model of the nucleus.

NEED TO KNOW: Typically, electron tomography uses a specimen

holder that is adjusted by hand to image different angles. O’Shea

and her colleagues needed eight different angles to wrap around

the whole chromatin fiber and compensate for blind spots inher-

ent to electron tomography, so they built a sample holder that

could make those fine adjustments automatically. “Having that

piece of hardware was key,” she says.

USE IT: The group is working with Nikon to develop a roadmap

and parts list so that other labs can modify their microscopes and

acquire similar images.

THIN SLICES

RESEARCHERS: Gail Mandel, neuroscientist, and Michael Lin-

hoff, research assistant scientist, Oregon Health and Science Uni-

versity, Portland, Oregon

PROJECT: Study chromatin shape changes in cells deep within tissue

CHALLENGE: Mandel wanted to understand why chromatin is

denser in the neurons of mice with a mutation in a gene called

MeCP2. Mutations in human MeCP2 cause Rett syndrome, a dis-

order that impairs communication and motor skills. Mandel set

out to track the presence of chemical tags that can cause chromatin

to pack tightly. Fluorescent markers that highlight those chemical

tags exist, but Mandel ran into two big problems. First, it’s hard to

use more than three or four different fluorescent markers in the

same image, and she wanted to track five or more chemical tags.

Also, the light used to excite those markers doesn’t penetrate deep

into tissue, making it difficult to acquire images crisp enough to see

chromatin in detail inside the brain.

SOLUTION:Mandel turned to Linhoff, an imaging expert who sug-

gested an approach based on array tomography. In this technique,

researchers embed tissue into plastic or gelatin and shave it into sec-

tions 200 nanometers wide—about 100 times thinner than the diam-

eter of a human hair. Then they image each section and assemble the

series to create a detailed view of nuclei deep within the mouse brain.

Linhoff stained the sections with one fluorescent marker at a time,

imaged them, then stripped off that first marker and added the next.

The embedding makes the tissue stable enough to withstand this

relatively aggressive chemical treatment. They found that the loss of

MeCP2 in neurons resulted in 20 percent more chromatin, tightly

packed in the nucleus.

NEED TO KNOW: Linhoff warns that there is a steep learning

curve with array tomography. Fortunately, the tissue sectioning

can be outsourced. He sent his mouse tissues to the Cell Sciences

Imaging Facility at Stanford University, but also recommends the

services of a Stanford-based company called Aratome. This com-

pany coats their coverslips with carbon, which allows for even

more aggressive treatment of the tissue before imaging.

USE IT: Reconstructing a 3-D picture from the many slices of

array tomography can be a complicated process. Linhoff and his

colleagues suggest using a detailed manual available through

Stanford’s Neuroscience Microscopy Service to align images.

IN THE SPOTLIGHT: A light-catalyzed reaction highlights the structure of

chromatin. The six lower panels show zoomed-in views of four cells from

the top two panels, revealing the visual difference between chromatin

exposed to a specific light wavelength and chromatin outside

the illumination spot.