Three-dimensional imaging of living cells gives researchers a more detailed and accurate spatial visualization of the interplay of cells and their components than was possible with earlier imaging systems, in which the imaging process itself was toxic to the cells and could lead to artifactual observations. Recent improvements have made 3D imaging a valuable tool for many applications, such as cell biology, developmental biology, neuroscience and cancer research. Current techniques are more accurate than ever before, giving data in real time, noninvasively, with little to no cell preparation required. Here are some examples of new and improved 3D-imaging systems for living cells from both commercial and academic sources.
Many options for imaging systems
Live-cell 3D-imaging systems come in many different varieties, such as confocal, photoacoustic, light-sheet and holographic tomography. Recent developments in confocal technologies have reduced phototoxicity to cells and improved the temporal resolution, compared with earlier confocal systems. For example, Revvity’s UltraVIEW VoX 3D Live Cell Imaging System uses a spinning-disk microscope that protects cell health by using less laser light. “Spinning-disk microscopy allows a researcher to look at living cells in 3D over time with time-lapse experiments, whilst submitting cells to minimal photon doses, which can damage them,” says Jacob Tesdorpf, portfolio director for high-content imaging instruments and applications at Revvity. A new Flash4 sCMOS camera gives the UltraVIEW VoX better sensitivity and higher frame rates, enabling “scientists to capture more images per second, gaining sensitivity and speed and resolving intracellular process faster than before,” says Tesdorpf. The UltraVIEW VoX 3D Live Cell Imaging System is used for techniques such as co-localization, ratiometric imaging, fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET).
GE Healthcare’s DeltaVision Elite is a high-resolution fluorescence microscopy system for live-cell 3D imaging. Paul Goodwin, science director at GE Healthcare Life Sciences, says that deconvolution of imaging data allows researchers to improve the images further. “Especially the high-resolution details can be improved by measuring the optical performance of the system,” says Goodwin. “We use deconvolution to get a better estimate of where the fluorescence is, and how much is truly there. We’re allowing the customer to consistently achieve the diffraction limit using complex samples.”
The 3D-SIM super-resolution modality on the DeltaVision OMX wasn’t originally designed for live-cell imaging. But a few years ago, “we realized we could adapt it for live-cell applications,” says Goodwin. “And [we] made it 40 to 50 times faster than it originally was.” The company’s systems are well suited for cell biology and microbiology, with some applications in developmental biology and neurobiology, according to Goodwin; they are less appropriate for deep imaging into the brain or for zebrafish development. “You don’t normally want to use deconvolution microscopy or super resolution in something highly complex, like deep in a brain,” he says. “For these applications, there are other tools more suitable, like confocal or two-photon microscopy.”
Zeiss’s Lightsheet Z.1 imaging system uses light-sheet fluorescence microscopy, which gives 3D images of live cells using less light because of its light-path arrangement. The excitation and detection paths are separated into perpendicular axes. Because the excitation beam illuminates only one thin optical slice at a time—namely, the plane that is in focus—the cells experience less light exposure and thus less photodamage. The software also takes advantage of deconvolution to improve the images.
Endra Life Sciences’ Nexus 128 is a photoacoustic 3D-imaging system for live cells. It uses both optical imaging and ultrasound to provide images without any contrast agents needed. Different types of soft tissues absorb laser light differently, which creates inherent contrasts without dyes or probes. But if contrast agents are required for specialized applications, the Nexus 128 also works with near-infrared dyes or fluorescent probes optimized for photoacoustic imaging.
Nanolive’s 3D Cell Explorer combines holography and rotational scanning to obtain quantitative 3D images of living cells—without staining or sample preparation—in less than a second [1]. “One can measure the refractive index distribution within the cell over time,” says Sebastien Equis, head of product development and cofounder of Nanolive. “Just like computer tomography for human bodies, our product makes a complete tomography of the living cell through a rotational scanning laser head, which illuminates the sample from 360 degrees.” The technology is noninvasive, and it’s useful for measuring cellular processes with real-time kinetics, such as mitosis, cell movements or vesicular trafficking, as well as many other applications.
High-content imaging in 3D
High-content imaging also is benefitting from advances in live-cell 3D imaging. Used to gather high-resolution imaging data—often for rapid screening of large numbers of cells—high-content imaging systems with 3D capabilities are now offered by several vendors. For example, Revvity’s new Opera Phenix™ High Content Screening has a proprietary confocal spinning-disk design called Synchrony™ Optics, which makes it suitable for screening applications and provides high-resolution images of 3D cell-culture models.
GE Healthcare’s IN Cell Analyzer 6000, another high-content 3D-imaging system, has a virtual confocal aperture that can adjust to different lighting conditions to optimize image quality. Although only a couple of years old, IN Cell Analyzer’s line-scanning confocal technology is showing growth, says Goodwin. “Scientists are finding ways of using the ability to image in three dimensions in screening applications.”
Innovations for the future
Recent innovations in academia have produced exciting new live-cell 3D-imaging systems that have yet to be commercialized. White-light tomography, for example, developed in the lab of Gabriel Popescu at the University of Illinois at Urbana-Champaign, images cells with a commonly used phase contrast microscope fitted with a spatial light interference microscopy (SLIM) module added [2]. The technique can generate 3D images over time while the cells remain undisturbed. Another technique, known as SCAPE (swept, confocally aligned, planar excitation) microscopy, has been developed in the lab of Elizabeth Hillman at Columbia University Medical Center [3]. SCAPE is faster than light-sheet, laser scanning confocal and two-photon microscopy and can be performed without disturbing the cells. It is most suitable for imaging superficial layers or transparent organisms, as it doesn’t penetrate as deeply as, say, two-photon microscopy.
Lattice light-sheet microscopy, developed in the lab of Eric Betzig at the Howard Hughes Medical Institute’s Janelia Research Campus, uses a Bessel beam divided seven ways to create a light sheet. The interference between the individual beams creates a 2D lattice of light, which can scan cells at about 1,000 planes per second [4]. The technique is fast and especially gentle on cells; it minimizes phototoxicity and photobleaching.
“Lattice light-sheet technology is phenomenal,” says Goodwin. “The engineering challenge will come as the technology becomes commercialized.” It is no small feat to build a live-cell 3D-imaging system that most biologists—not just the designers and engineers—can use to get useful data. “That will be the real test.”
The available options for researchers performing live-cell 3D imaging have vastly improved with advances in commercial offerings and self-designed systems. Scientists wanting more information from their cells will benefit from these offerings, making new discoveries and generating exciting results.
Caitlin Smith
Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
References
[1] Cotte, Y, et al., “Marker-free phase nanoscopy,” Nature Photonics, 7:113-117, 2013.
[2] Kim, T, et al., “White-light diffraction tomography of unlabelled live cells,” Nature Photonics, 8:256-263, 2014.
[3] Bouchard, MB, et al., “Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms,” Nature Photonics, 9:113-119, 2015. [PubMed ID: 25663846]
[4] Chen, BC, et al., “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science, 346(6208):1257998, 2014. [PubMed ID: 25342881]
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