Cool Tools: Pushing the Limits of High-Resolution Microscopy

Cell biologists would love to shrink themselves down and actually see, touch and hear the inner workings of cells. Because that’s impossible, they have developed an ever-growing collection of microscopes to study cellular innards from the outside. Using these powerful tools, researchers can exhaustively inventory the molecular bits and pieces that make up cells, eavesdrop on cellular communication and spy on cells as they adapt to changing environments.

In recent years, scientists have developed new cellular imaging techniques that allow them to visualize samples in ways and at levels of detail never before possible. Many of these techniques build upon the power of electron microscopy (EM) to see ever smaller details.

Unlike traditional light microscopy, EM uses electrons, not light, to create an image. To do so, EM accelerates electrons in a vacuum, shoots them out of an electron gun and focuses them with doughnut-shaped magnets onto a sample. When electrons bombard the sample, some pass though without being absorbed while others are scattered. The transmitted electrons land on a detector and produce an image, just as light strikes a detector (or film) in a camera to create a photograph.

This image, showing a single protein molecule, is a montage. It was created to demonstrate how dramatically cryo-EM has improved in recent years. In the past, cryo-EM was only able to obtain a blobby approximation of a molecule’s shape, like that shown on the far left. Now, the technique yields exquisitely detailed images in which individual atoms are nearly visible (far right). Color is artificially applied. Credit: Veronica Falconieri, Subramaniam Lab, National Cancer Institute.

Transmission electron microscopes can magnify objects more than 10 million times, enabling scientists to see the outline and some details of cells, viruses and even some large molecules. A relatively new form of transmission electron microscopy called cryo-EM enables scientists to view specimens in their natural or near-natural state without the need for dyes or stains.

In cryo-EM—the prefix cry- means “cold” or “freezing”—scientists freeze a biological sample so rapidly that water molecules do not have time to form ice crystals, which could shove cellular materials out of their normal place. Cold samples are more stable and can be imaged many times over, allowing researchers to iteratively refine the image, remove artifacts and produce even sharper images than ever before. Continue reading

Cool Image: Adding Color to the Gray World of Electron Microscopy

Color electron micrograph of an endosome, a cell organelle. Credit: Ranjan Ramachandra, UCSD

As his Christmas gift to himself each year, the late biochemist Roger Tsien treated himself to two weeks of uninterrupted research in his lab. This image is a product of those annual sojourns. While it may look like a pine wreath dotted with crimson berries, it is in fact one of the world’s first color electron micrographs—and the method used to create it may dramatically advance cell imaging.

Electron microscopy (EM) is a time-honored technique for visualizing cell structures that uses beams of accelerated electrons to magnify objects up to 10 million times their actual size. Standard EM images are in grayscale and any color is added in with computer graphics programs after the image is made. With their new technique, Tsien, who received a Nobel Prize for his development of green fluorescent protein into a tool for visualizing details in living cells using light microscopes, and his colleagues have found a way to incorporate color labeling directly into EM. Continue reading

Visualizing Skin Regeneration in Real Time

Top: Colorful skin cells on a zebrafish . Bottom: Cells from the outer surface of the scale.
More than 70 Skinbow colors distinguish hundreds of live cells from a tiny bit (0.0003348 square inches) of skin on the tail fin of an adult zebrafish. The bottom image shows the cells on the outer surface of a scale. Credit: Chen-Hui Chen, Duke University.

Zebrafish, blue-and-white-striped fish that are about 1.5 inches long, can regrow injured or lost fins. This feature makes the small fish a useful model organism for scientists who study tissue regeneration.

To better understand how zebrafish skin recovers after a scrape or amputation, researchers led by Kenneth Poss of Duke University tracked thousands of skin cells in real time. They found that lifespans of individual skin cells on the surface were 8 to 9 days on average and that the entire skin surface turned over in 20 days.

The scientists used an imaging technique they developed called “Skinbow,” which essentially shows the fish’s outer layer of skin cells in a spectrum of colors when viewed under a microscope. Skinbow is based on a technique created to study nerve cells in mice, another model organism.

The research team’s color-coded experiments revealed several unexpected cellular responses during tissue repair and replacement. The scientists plan to incorporate additional imaging techniques to generate an even more detailed picture of the tissue regeneration process.

The NIH director showcased the Skinbow technique and these images on his blog, writing: “You can see more than 70 detectable Skinbow colors that make individual cells as visually distinct from one another as jellybeans in a jar.”

This work was funded in part by NIH under grant R01GM074057.

Another Piece to a Century-Old Evolutionary Puzzle

After mating about 55,000 pairs of fruit flies and sifting through 333,000 daughter flies, a research team found six sons that each had mutations in the same gene that helped make two fruit fly species unique from each other. Credit: Jim Woolace, Fred Hutch News Service.

Nitin Phadnis and Harmit Malik Exit icon set out to conduct an experiment that could solve a century-old evolutionary puzzle: How did two related fruit fly species arise from one? Years after they began their quest, they finally have an answer.

The existence of a gene that helps make each of these fruit fly species unique and separate from each other had been guessed at since 1940, following experiments decades earlier in which geneticists first noticed that the two types of flies, when mated, had only daughters—no sons.

Scientists had previously discovered two other genes involved in driving the fruit fly species apart, but they knew those two genes weren’t the full story. Continue reading

Recognition for CRISPR Gene-Editing Tool

The CRISPR gene-editing tool was recognized today by Science magazine as its “breakthrough of the year.” We support a number of researchers working in this exciting area and have featured it on this blog. To learn more about this exceptionally promising new method, see below for our illustrated explanation of the CRISPR system and its possible applications.

How the CRISPR System Works

Illustration of CRISPR system

The CRISPR system has two components joined together: a finely tuned targeting device (a small strand of RNA programmed to look for a specific DNA sequence) and a strong cutting device (an enzyme called Cas9 that can cut through a double strand of DNA).

Continue reading

Cracking a Ubiquitous Code

We asked the heads of our scientific divisions to tell us about some of the big questions in fundamental biomedical science that researchers are investigating with NIGMS support. This article is the third in an occasional series that explores these questions and explains how pursuing the answers could advance understanding of important biological processes.

Ubiquitin (Ub) molecules
Ubiquitin (Ub) molecules attached to proteins can form possibly hundreds of different shapes. Credit: NIGMS.

Researchers are on a quest to crack a code made by ubiquitin, a small protein that plays a big role in coordinating cellular function. By attaching to other proteins, ubiquitin determines what those proteins should do next.

Just as zip codes direct letters to specific towns, the ubiquitin code might direct one protein to help with DNA repair, another to assist in cell division, and a third to transport molecules into and out of cells. Continue reading

Cool Image: Tracing Proteins in Action

Bright amorphous loops

These bright, amorphous loops represent a never-before-seen glimpse at how proteins that play a key role in cell duplication are themselves duplicated. Credit: Sue Jaspersen, Zulin Yu and Jay Unruh, Stowers Institute for Medical Research.

Looking like necklaces stacked on a dresser, these bright, amorphous loops show the outlines of yeast proteins that make up the spindle pole, a cellular component found in organisms as diverse as yeast and humans. Each cell starts with a single spindle pole, which must somehow duplicate to form the pair that works together to pull matching chromosomes apart during cell division. Scientists don’t completely understand how this duplication occurs, but they do know that errors in spindle pole copying can lead to a number of health conditions, including cancer. Continue reading

Cool Image: DNA Origami

Computer-generated sketch of a DNA origami folded into a flower-and-bird structure.

A computer-generated sketch of a DNA origami folded into a flower-and-bird structure. Credit: Hao Yan, Arizona State University.

This image of flowers visited by a bird is made of DNA, the molecule that provides the genetic instructions for making living organisms. It shows the latest capability of a technique called DNA origami to precisely twist and fold DNA into complex arrangements, which might find future use in biomedical applications. Continue reading

Bacterial ‘Fight Clubs’ and the Search for New Medicines

Competition encourages bacteria to produce secondary metabolites with therapeutic potential that they would otherwise hold in reserve. Credit: Michael Smeltzer, Vanderbilt University.

Bacteria hold a vast reservoir of compounds with therapeutic potential. They use these compounds, known as secondary metabolites, to protect themselves against their enemies. We use them in many antibiotics, anti-inflammatories and other treatments.

Scientists interested in developing new medicines have no shortage of places to look for secondary metabolites. There are an estimated 120,000 to 150,000 bacterial species on Earth. Each species is capable of producing hundreds of secondary metabolites, but often only under specific ecological conditions. The challenge for researchers is figuring out how to coax the bacteria to produce these compounds.

Now, Brian Bachmann Exit icon and John McLean Exit icon of Vanderbilt University and their teams have shown that by creating “fight clubs” where bacteria compete with one another, they can trigger the bacteria to make a wide diversity of molecules, including secondary metabolites. Continue reading

Mapping Our Skin’s Microbes and Molecules

Last month, we shared some facts about the microbes that inhabit us. Here’s another: From head to toe, our skin bacteria coexist with chemicals in hygiene products, fibers from clothes and proteins shed by dead or dying skin cells.

These images highlight the complex composition of our body’s largest organ. They show the association between microbial diversity (top images) and skin chemistry (middle images). The different colors note the abundance of a certain bacterium or molecule—red is high, and blue is low. The skin maps remind NIH Director Francis Collins of a 60’s rock album cover. Continue reading