Groundbreaking Microscope Makes 3D Video of Living Cells in Real Time

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The saying goes, “Lightning never strikes the same place twice.” But what’s in a saying? Dr. Eric Betzig recently showed creating one revolutionary new microscope doesn’t mean he can’t create another.

Betzig, a physicist and engineer at the Janelia Farms Research Campus in Virginia, has invented an incredibly powerful microscope that promises to bring the frenetic activity of cells into full view. And he’s done it after winning the Nobel Prize in Chemistry last month for another advanced microscope he developed less than 10 years ago.

Betzig’s latest breakthrough, called lattice light-sheet microscopy, allows researchers to watch the fast processes taking place in living cells in real-time 3D. Below, we’ve included a selection of the 31 remarkable videos posted by Betzig’s team (go here for the full list). This first video shows an immune system T-cell (red) attacking a target cell (blue).

To understand why Betzig’s technique is so exciting, it’s worth a quick look back at microscopy’s roots.

Microscopes have come a long way since the first prototypes were made in the Netherlands in the 1500s. Conventional microscopes use light and a group of lenses to magnify small items like cells. Yet, these light microscopes are limited by the wavelengths of visible light, making it impossible to study the structure and activity of cells in any great detail.

The most powerful microscopes at our disposal today are electron microscopes. The electron beam in these microscopes has a much smaller wavelength than visible light, allowing incredible resolution and magnification.

However, an electron microscope can only be used on processed and fixed samples, which means that it can’t be used to study live cells. So, most researchers studying biology use an advanced optical microscope that utilizes fluorescence to generate a more detailed image of cells.

How does it work? A fluorescent molecule can be attached to various parts of the cell and excited by a specific wavelength of light, causing it to emit light of a different wavelength. Using sophisticated filters, the two wavelengths are separated so that only the light emitted by the excited fluorescent molecule is used to create the image.

Even fluorescence microscopy, however, is not without its own limitations, mostly resulting from the high intensity of light used to excite the fluorophores. The intense light can result in the fluorophores losing the ability to emit light, a phenomenon called photobleaching. The light can also damage cells in two ways, collectively known as phototoxicity.

First, short wavelength light is high energy and damages biological systems. Think of the UV rays from the sun that sunblock helps protect us from. Second, fluorophores can produce reactive chemicals that damage cells when they are illuminated.

[This second video using Betzig’s new method shows a cancer cell (green) crawling through a collagen matrix (orange).]

Dr. Betzig’s Nobel prize-winning creation addressed some of the limitations from this intense light in a clever way.

Instead of using a powerful beam of light to illuminate the entire cell, it uses a much weaker light focused on a specific part of the cell. So focused in fact that it could “see” individual fluorescent molecules that are only nanometers apart.

Using this approach, anywhere from 40 to 100,000 snapshots of individual molecules are taken and then stitched together to produce a detailed, 3D image. The technology was named PALM, or photo-activated localization microscopy, and quickly made single-molecule microscopy the gold standard for studying live cells with minimal damage.

Not being one to rest on his laurels, however, Dr. Betzig quickly moved to improve the PALM technology, which could produce high-resolution images but was too slow to see biological processes occurring in real time.

His latest creation, called lattice light-sheet microscopy, was published last month in the journal Science.

[This final video shows the first few rounds of cell division of a C. elegans worm embryo. A protein critical for the separation and passage of chromosomes between cells (AIR-2) is localized with fluorescent molecules.]

Lattice light-sheet microscopy uses a non-diffracting light called a Bessel beam that illuminates the sample from the side. But instead of using a single powerful beam, Betzig divided the beam into seven thin beams. Again, this lowered phototoxicity because the samples weren’t being hit with a single powerful beam but rather several, less intense beams.

Further, it permitted faster scanning of the sample—up to 1,000 scans per second—allowing the capture of fast moving cellular processes in multiple thin sections that can be coalesced into stunning high-resolution 3D images and videos.

And the fast scan times combined with the divided Bessel beam has virtually made photobleaching a non-issue.

While the development of super-resolution microscopes coming out of Dr. Betzig’s laboratory are exciting, the real treat will come once the technology makes its way to cellular and molecular biologists around the world. Already in the past year, thirty teams of scientists studying various areas of biology have visited Dr. Betzig’s lab to learn and use the microscope.

The ability to produce high-resolution images of biological samples with minimal damage will only increase our knowledge of what and how things unfold at the cellular and subcellular level. Where that knowledge will eventually lead is unknown, but it’s hard to overstate how valuable it will be in our understanding of life at the molecular level.

Image and Video Credit: Betzig Lab, HHMI/Janelia Research Campus, Bembenek Lab, University of Tennessee; 10/24/14 issue of the journal Science.

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