Life exists at various scales, some visible and others too small to see—microscopic. Humans are, on average, a meter and a half. Zoom into our bodies and you’ll see organs measurable with a ruler. Keep going and you’ll encounter cells, anywhere from one to 100 micrometers in diameter, about the width of a hair. Zoom in some more and parts of the cell come into view: microtubules, proteins, DNA, and a menagerie of molecules and machines that sustain life. These are just a couple of nanometers in diameter, one thousandth the width of a human hair.
Understanding the complexity of biology cannot be fully achieved without seeing it, so scientists have been trying for decades to increase the precision and resolution of microscopes. In the August edition of Nature Methods, two groups published papers detailing major advances in the field of microscopy. Their techniques improve pre-existing technologies—light sheet microscopy and single molecule localization microscopy—and have the potential to change the way that we look at life.
All the light we now can see
The microscope traces its roots to Antony van Leeuwenhoek, a Dutch haberdasher with a love of lens crafting, but he was far from the first to experiment with the arrangement of small circles of glass. Robert Hooke and others before him had been toying with lenses to observe objects too small to see with the naked eye: pins of needles, specks of dust, and lice clinging to hairs. Their rudimentary microscopes could magnify objects by about thirty times. In 1665, Leeuwenhoek built a microscope with a 270x magnification.
Over the following centuries, physicists and engineers made tweaks to the microscope, playing with the placement of samples, the light sources illuminating them, and the lenses placed above them. Despite these variations, today’s conventional microscopes, called light microscopes, share the same basic architecture as Leeuwenhoek’s first. They gather light emanating from a sample and use a series of lenses to distort the light in a way that magnifies the sample.
But there is one major limitation to this design: light microscopy typically only works for two-dimensional samples flattened by a glass coverslip. If scientists want to capture three-dimensional images, they must look elsewhere.
Slicing cells with light
To overcome this limitation, light-sheet microscopy was developed in the early 20th century. It works by taking stacks of images and converting them into a comprehensive, three-dimensional picture. To do this, it uses a laser to illuminate one section of a sample one-fiftieth the thickness of a sheet of paper. Above the laser is a camera which takes a picture of the illuminated plane. The laser moves up slightly, and another picture is taken. This iterative process continues hundreds of times, and the images are compiled into a three-dimensional model.
Reto Fiolka, a professor at the University of Texas, Southwestern, has been looking for ways to improve light-sheet microscopy for the past decade. A former aerospace engineer, he decided to switch to microscopy after he realized “how important it was to apply optics to the life sciences.” Fiolka’s recent article in Nature Methods outlines ways to upgrade light-sheet microscopy.
According to Fiolka, microscope companies have struggled to commercialize light-sheet microscopy because the machines are too bulky. Light-sheet microscopes illuminate samples with a plane of light that is perpendicular to the objective lenses that detect that light. This is in contrast to conventional light microscopes, which illuminate and gather light from the same angle. As a result, light-sheet microscopes are typically large and expensive. “They are million-dollar machines that just do one task,” Fiolka said.
Fiolka wants to change this. In his paper, he speculated that light-sheet microscopy could be done using a single eye numerical aperture objective—a device that would combine the light source and the lens, like conventional light microscopes. The technology is cutting-edge and involves angling the light beams: “If you use a single objective with an extraordinarily large opening angle of seventy degrees, then you can tilt a beam of light at the edge of that objective that illuminates a sheet within the sample,” Fiolka explained.
Fiolka’s paper is speculative, but its implications are widespread. If companies were to capitalize on his idea, they could tack on this objective lens to existing light microscopes, widening access to basic light-sheet microscopy.
Seeing the building blocks of life
Fiolka’s technique works well for visualizing cells, but what if you want to zoom in further? What if you want to see the molecules inside mouse neurons and the proteins inside fish tails? Professor Ke Xu and his lab at the University of California, Berkeley, have invented a technique called oblique-plane single-molecule localization microscopy, or obSTORM for short, that allows them to do just that.
Single-molecule localization microscopy allows scientists to see, as the name suggests, single molecules. Until a couple of decades ago, it was considered impossible, and it is still very difficult. Light microscopy is limited because light is a wave, and, just like water, it has crests and troughs. The crests and troughs of light waves emitted by molecules that are less than 300 nanometers apart will blend with each other and produce one big blurry mess of a picture. In cells—which are teeming with molecules and proteins in very close proximity—it is impossible to distinguish the location of these particles using conventional techniques.
Xu’s approach is by no means conventional. “It works almost like a blinking Christmas tree,” he said. Cells are injected with fluorescent probes, little lights that attach to molecules and can be selectively turned on. Only a subset of them are turned on at one time. Then a picture is taken. In an iterative process that involves intense mathematical computation, the lights are gradually all turned on, and the once unintelligible mess is resolved into a spectacular picture of molecules.
Xu’s technique, like Fiolka’s, involves shining a laser beam through a sample, which excites the blinking probes. Instead of shining light through flat planes, however, Xu found that the resolution of images could be increased by imaging planes with a slanted angle. Tilting the light source decreased background noise.
Before Xu and his group invented this technique, the molecules deep inside our cells were difficult to visualize. By slanting the sheet of light that hits the cells, they’ve been able to take clear pictures of the molecules inside neurons and fish tails, microscopic particles that were previously elusive.
Fiolka, though interested in a larger scale of life, is also counting on sheets of light to improve pre-existing microscopes. Both he and Xu are pushing the boundaries of microscopy, one light sheet at a time, such that one day we will be able to see the intricacy of life around and inside us.