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View to a cell

Subject: New optics shatter the diffraction barrier, illuminating life within us

Imagine if your best knowledge of human anatomy came from viewing the body through binoculars from a mile away. You might make out the shape of a hand, but knuckles and fingernails would elude you. Experiments could tell you there’s a pumping heart inside, but to see that heart with any clarity you would have to fix it in formaldehyde or liquid nitrogen, blast it with electrons and add dyes to impart contrast. For a long time, that’s what it’s been like for biologists trying to observe cells.

English scientist Robert Hooke coined the term cell in 1665 after examining a slice of cork through a light microscope. The plant parts he saw reminded him of the cells of a monastery. In the centuries since, we’ve learned that our bodies comprise some 200 different cell types and a total of several trillion cells, not counting microbes, at any given moment. (As you read this sentence, about 50 million have died and been replaced.) Within a single cell there may be 10,000 different proteins; thousands of the energy factories called mitochondria; and half a billion actin molecules, which provide scaffolding to support the cell and help it move and change shape.

Making twins

Mitosis, the cellular division that produces two genetically identical daughter cells, is perhaps the most fundamental process in biology. Without it, multicellular life wouldn’t exist, a broken bone would never heal, tissues would disintegrate. Cancer — essentially mitosis gone rogue — also wouldn’t exist. Scientists have been watching mitosis through a microscope for about 150 years, but new views are fleshing out the less-detailed pictures of the past (bottom, right). After DNA replicates, the nuclear envelope surrounding it dissolves. Spindle fibers (gold above, red at right) align pairs of chromosomes (blue) and then separate the genetic material into two daughter cells (shown forming, above).

Top: L. Schermelleh/Univ. of Oxford; Bottom: J. Stout/Indiana Univ.; Bottom, right: Andrew Syred/Science Source

The structures and activities within our cells are a major force in determining the stuff we’re made of, even though each of us begins with the humble fusion of just two. Following genetic instructions and taking cues from its environment, each cell realizes its fate — a fate that is inextricably linked to our own.

Until now this internal machinery has remained largely hidden from sight. Viewed through microscopes similar to Hooke’s, most cells are see-through and colorless; it’s hard to discern fine features. Due to diffraction, the bending of light, objects smaller than about 250 nanometers — the size of the smallest bacteria — are fuzzy when viewed through an optical microscope, if they can be seen at all. (Consider that most proteins are merely a few nanometers across.) This diffraction barrier, explicitly defined by German physicist Ernst Abbe in 1873, makes a smeared blur of much that happens in and on a cell.

That’s all changed in the last few decades. Scientists have developed a suite of new optical techniques that circumvent the diffraction barrier and show us a cell’s full guts and glory. With new fluorescent tags that light up structures in the dense darkness inside a cell these new optical approaches produce detailed images of what was once invisible. In the pages that follow, some of the most striking images are highlighted, all from animal cells that scientists use to understand basic cellular processes and disease.

By capturing the inner workings of a cell and interactions between cellular neighbors, scientists can now connect knowledge gleaned from genetic experiments to actual structures and activities they can see. Discoveries will lead to new hypotheses and experiments that will further our understanding of animal development and function — and how both can go awry.

Some of these super-resolution techniques are still so new and challenging that many scientists think the major breakthroughs they will yield are yet to come. But for now, we can all enjoy stunning, unprecedented views of the cellular world.

Click thumbnails for larger versions of images

POWERHOUSES Scientists are expanding their view of mitochondria, long known for their energy-producing role and quintessential kidney bean shape. It turns out that mitochondria are at the center of numerous biological processes, including regulating cell death. Mitochondria are also highly dynamic, traveling through cells on tracks, elongating into slender tubes, condensing, dividing and fusing (becoming, for example, one giant network to power energy-sucking DNA replication). Blurred by conventional techniques (left), mitochondria’s details are revealed through super-resolution microscopy (shown color-coded by depth, center, and in cross section at right). Credit: Zhuang Lab/Harvard/HHMI

HEALTHY DAUGHTERS Protein structures called kinetochores (red) are largely responsible for a crucial task: ensuring that each daughter cell produced when a cell divides in two ends up with a complete set of chromosomes. These protein structures assemble on chromosomes (not visible). Kinetochores attach to hollow rods called microtubules (green) and pull the chromosomes into each daughter cell. If kinetochores don’t attach properly, daughter cells can end up with extra or missing chromosomes, a condition called aneuploidy that can cause miscarriages and birth defects, including Down syndrome. Credit: K. deLuca/Colorado State Univ.

E. coli, for example, have tricks that induce cell ruffling, allowing entry of toxins or the microbes themselves. Scientists also have a growing interest in ruffling as a vehicle for drug delivery. Credit: Betzig Lab/Janelia Farm Research Campus/HHMI" style="border: 0px; height: auto !important; max-width: 100%; float: left;">

PACKING IT IN The ruffles decorating this monkey kidney cell aren’t as dainty as they look. They will fold in on the cell, enabling it to gulp a considerable amount of fluid and solid cargo. Vigorous cell ruffling is a defining feature of macropinocytosis, one of several ways that cells bring in dissolved molecules and fluid from the environment. At times the process also allows entry of viruses and bacteria; salmonella and <em>E. coli</em>, for example, have tricks that induce cell ruffling, allowing entry of toxins or the microbes themselves. Scientists also have a growing interest in ruffling as a vehicle for drug delivery. Credit: Betzig Lab/Janelia Farm Research Campus/HHMI

A FLY IS BORN Around two hours after fertilization, a fruit fly embryo has grown from one cell to about 3,500 (left). This nascent fly is still a single layer of cells, sitting atop the egg’s yolk within a shell. Using an approach that looks at the embryo from four different perspectives at once, scientists can witness a wave of cell division (purple) pass through (left to right), nearly doubling the number of cells to about 6,000. The cells do not yet have membranes; they exist as tiny islands of cytoplasm and nuclei. A few minutes after this wave of cell division, the cell membranes will form, and within 22 hours the fly larva will hatch. Credit: Keller Lab/Janelia Farm Research Campus/HHMI

BRANCHES IN THE BRAIN Better views of dendritic spines, the leaflike structures that protrude from nerve cells (seen here in live mouse-brain tissue), can elucidate how the human brain functions. Dendritic spines are essential for nerve-to-nerve communication; throughout life they continue to morph, grow and perish. A recent study reveals that dendritic spines in a live adult mouse can undergo shape changes on a time scale of minutes. The spines play a pivotal role in memory formation and learning, and specific changes in dendritic spine size and shape have been implicated in brain disorders including schizophrenia and Alzheimer’s disease. Credit: J. Tonnesen and U.V. Nägerl/CNRS and Univ. of Bordeaux

GETTING A MOVE ON Cells would go nowhere without actin, the most abundant protein in eukaryotic cells (the sperm cells of some roundworms are thought to be the only ones with absolutely no actin). Actin filaments (color-coded by depth, blue farther away) act as the muscles of cells and are especially prominent in the cellular skeleton, where they constantly re-form and dissolve into stiff networks that are crucial for cell locomotion. “Stress fibers” that enable contraction are made from a combination of actin and motors made of myosin. The fibers are visible in this bottom layer of two cancer cells. Actin can also contort into treadlike spikes (visible on the lower and right edges). The cell can use these as battering rams to push and pull itself into a tissue. When a tumor cell crawls along a blood vessel or an immune system cell rushes to a site of injury or infection, it’s actin that’s making them move. Credit: D. Burnette/NICHD/NIH

MAKING MUSCLE Skeletal muscle is unusual: Its cells form from the fusion of precursor cells called myoblasts, and the resulting megacells may have tens to hundreds of nuclei each. In addition to making muscle, myoblasts can also differentiate into bone precursor cells or even be coaxed into becoming fat. This mouse myoblast cell already has two nuclei, but they are preparing for another round of division. While the nuclear envelopes (blue) are still intact, the tangled mixture of DNA and proteins have begun to condense into chromosomes (red), which eventually will be divvied up with help from the microtubules (green). Credit: L. Schermelleh/Univ. of Oxford

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TAKING ACTION The tiny diameter and high density of actin filaments are visible in the sheetlike protrusions at this monkey kidney cell’s edge (color-coded by depth, red farthest away). Scientists are still trying to figure out precisely how these sheets form and connect to the cell’s interior so they can understand more about how cells travel. Credit: K. Xu, H.P. Babcock and X. Zhuang/Nature Methods 2012

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SIGNED, SEALED, DELIVERED The processing, packaging and shipping of the numerous proteins and other big molecules made within a cell is the purview of the Golgi body (purple), which along with the chromosomes (green), gets divided up during mitosis. This type of pig kidney cell is often used in drug research and is especially good at making a protein that helps break up blood clots. Credit: T.A. Planchon et al/Nature Methods 2012

SAFETY WRAP Nerve cells that send electrical signals are coated in a protective sheath of myelin (like the insulation surrounding electrical wires), without which the elaborate, efficient nervous system of vertebrates might never have evolved. Precisely how myelin sheaths form is still a mystery, and one that scientists would desperately like to solve, since faulty myelin has been implicated in several neurological diseases including multiple sclerosis. This glial cell will extend to wrap a nerve in myelin, a task likely dependent on the actin skeleton (green) and the associated actin proteins (red). Credit: B. Zuchero and A. Olson, Barres Lab/Stanford

NO TRESPASSING One of the body’s primary bulwarks against outside contaminants are epithelial cells, which make up skin and line cavities such as the intestine. These protective cells (human newborn foreskin shown) can exist as sheets of tissue that are held tightly together by several structures including adherens junctions (green, blue shows cell nuclei). Scientists are investigating how HIV, the virus that causes AIDS, manages to get through the adherens junctions and into the body. Credit: K. Fahrbach and T.J. Hope/Northwestern Univ.

HAVE FEET, WILL TRAVEL Cancer cells become difficult to eradicate once they spread, and the sheetlike cellular feet called lamellipodia make such locomotion possible. By studying how cells like this bone cancer cell (membrane proteins color-coded by depth) sense tension and crawl and stick themselves in tissue, scientists may identify new potential therapeutic targets. Credit: P. Kanchanawong and C. Waterman/NIH; M. Davidson/FSU; G. Shtengel and H. Hess/Janelia Farm Research Campus/HHMI

Then and now

The nitty-gritty of cell biology happens on scales too small to see through a conventional light microscope. But several new imaging techniques at a range of resolutions provide new views — and new understanding — of how cells function.

SIM (~100 nm)

Structured illumination microscopy shines a striped pattern of light onto a sample. That light interacts with light from fluorescent tags on cellular material and generates a pattern of interference called a moiré fringe. Using a series of moiré fringes it’s possible to mathematically extract and reconstruct a super-resolution image. SIM is ideal for looking at entire cells in 3-D, ensembles of cells or multiple cellular structures at once. SIM: Lothar Schermelleh, Univ. of Oxford

STED (~30–70 nm)

When a focused light beam hits a fluorescent-tagged specimen, it generates a blurry halo. With stimulated emission depletion microscopy, a second laser shines a doughnut-shaped beam of light that turns off the excited molecules in the halo. This provides a sharper view that, when scanned across the sample, produces a super-resolution image. STED: R. Medda, D. Wildanger, L. Kastrup and S.W. Hell/Max Planck Institute

PALM (~10–55 nm)

Photoactivated localization microscopy incorporates into a sample special fluorescent proteins that can be toggled between on and off states when hit with a particular wavelength of light. This allows researchers to illuminate a subset of molecules in a sample and eliminate overlapping fluorescence that would blur details if everything in the sample was lit up at once. iPALM (interferometric PALM) provides images in 3-D. PALM: J.A. Galbraith, G. Shtengel, H.F. Hess and C.G. Galbraith/NINDS/NIH, Janelia Farm Research Campus/HHMI and NICHD/NIH

STORM (~20–55 nm)

Stochastic optical reconstruction microscopy, developed around the same time as PALM, also relies on fluorescent tags that can be switched on and off. In STORM’s case, the tags can be dyes or proteins. Using dyes may require an extra step, but they can be switched on and off more quickly and don’t burn out as fast as fluorescent proteins. Dyes can also be attached to genetic material. STORM: M. Bates et al/Science 2007

Light sheet (~100 nm)

Imaging cells can be a violent process. The heat from light can cook cells, and the tinier the object the more light scientists need to see it (or to “interrogate” it). Light sheet microscopy hits a sample with a thin sheet of light that excites only the molecules in a single plane, minimizing damage. Bessel beam imaging uses superthin sheets of light to image live cells — and their innards — in action (see images, “A FLY IS BORN” and “SIGNED, SEALED, DELIVERED,” above). Simultaneous multiview light-sheet microscopy (see image, “PACKING IT IN,” in slideshow above) uses thicker sheets to track ensembles of cells. Light sheet: K. Branson, Y. Wan, W. Lemon and P. Keller/Janelia Farm Research Campus/HHMI


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