Two decades ago, biologists analyzed the 3D structure of samples by slicing them like cake—but in very thin slices—and then manually drawing the features on each section as observed under a microscope. Using the drawings of those so-called serial sections, biologists added a depth dimension to the structural information. Beyond its labor-intensive nature, this method left much to be desired in accuracy, and even more in repeatability. Biologists craved an easier and better solution to explore life sciences in 3D.

More recently, scientists—especially ones who are not experts in microscopy and imaging—have had options for performing 3D resolution analysis of cells. For example, the Olympus SD-OSR spinning-disk super-resolution system provides 3D imaging of live cells. A microscope system, however, doesn’t need to be confocal to enable 3D imaging. The EVOS FL Auto Cell Imaging System from Thermo Fisher Scientific, for example, includes an XYZ motorized stage that can automatically collect a stack of images from a sample, and the images can be reconstructed through software to visualize the sample in 3D.

Although only specialists once used such advanced forms of imaging, today’s systems make this technology accessible to almost any researcher, with only minimal training in many cases. In general, any biological question that involves the structure of a sample might benefit from imaging it in 3D. Here we discuss key considerations when transitioning to 3D imaging of cells and how to obtain the greatest resolution and characterization of potential biomarker targets.

Focus on the optics

Like human eyes, which consist of various parts—the cornea, lens and so on—a microscope’s imaging depends on several components working together. In fact, every component from the sample to the imaging sensor impacts the quality of the image.

Imagine a sample on a slide. The light being detected goes from the specific structure being imaged and then through the rest of the sample, the fluid surrounding the sample, the cover slip, the immersion oil between the cover slip and the objective lens, and then the objective itself and the rest of the microscope’s optical components. The parts that the researcher can easily control—the cover slip, immersion oil and objective—all affect the optics of the system, and some combinations will work better than others for a specific sample.

The objective lens in an imaging system is essential to obtaining the best depth information. For one thing, it needs a high numerical aperture. Its numerical aperture represents the amount of light it can collect, and the level of detail it can resolve. So when imaging deeper—through more tissue—a researcher wants as much light and detail as possible, which comes from an objective with a high numerical aperture.

Getting the right overall combination of components usually requires some trial and error with a specific sample. “With live cells,” says Russell Ulbrich, product manager for high-end imaging systems at Olympus Scientific Solutions Americas, “you should try to use an objective with a correction collar, and do your best to use a refractive medium that is well matched to the sample.” He adds, “Many don’t do this, and they suffer the consequences.”

In many imaging applications, researchers select fluorescent tags to identify specific targets, and this can require some adjustments when adding the third dimension. “When going 2D to 3D,” says Brian Almond, senior product manager at Thermo Fisher Scientific, “the reagents still work, but you may need to incubate them longer.” Additionally, when using live cells, make sure the reagents are nontoxic for situations that require longer incubation.

Getting super-resolution

Structured illumination microscopy (SIM) provides better resolution than what is allowed by the diffraction limit of visible light, which is about 200 nanometers. At GE Healthcare’s Life Sciences business, says Trisha Koenke, DV OMX product manager, “We’ve been investing in extending 3D imaging resolution in space and time.” The company’s DeltaVision OMX SR uses less light than most super-resolution techniques, which makes a better environment for cells and enables researchers to use standard fluorescent markers. As Koenke says, “Ultra high-speed image acquisition ensures that previously unseen dynamic physiological processes can be captured and studied.”

To get the best image, especially in super-resolution, scientists often need to try more than one approach. The Blaze SIM module allows the DeltaVision OMX SR to provide 2D- or 3D-SIM images.

In addition to the improvements in resolution, this platform works with standard preparation for fluorescent imaging. Then, scientists can image dynamic cellular events for as long as several hours.

As Koenke explains, “Scientists at the University of Washington were able to use the DeltaVision OMX SR to image a cell going through mitosis in 3D-SIM.” She adds, “The experiment included a 7.5-micron stack—915 images per time point—and the cell still progresses through mitosis, an indication that phototoxicity is minimal.”

Souped-up software

In some cases, researchers can process an image after capturing it, for improved resolution. Deconvolution improves the quality of the depth resolution of both confocal and widefield fluorescent imaging. This image-processing algorithm removes some of the noise from an image. “It’s not required, but deconvolution can dramatically improve the signal-to-noise ratio, improving the image both qualitatively and quantitatively,” says Ulbrich. “And it’s very easy and very fast.” Deconvolution doesn’t come with all imaging software packages, but it usually only costs about half the price of a good objective lens. “Think of deconvolution as a way to make your best objective perform even better,” Ulbrich advises.

Today’s software can also simplify 3D imaging. “The EVOS is supported by an intuitive user interface and software that is accessible by everyday users,” says Magnus Persmark, senior product manager at Thermo Fisher Scientific. Nonetheless, he adds, “It’s never trivial to do automated 3D scans.” That’s especially true with living cells, which can die from excessive exposure. “So there’s a learning curve,” Persmark says, “and it’s more with the cells than the scope.”

Keep in mind that all of microscopy works as a system. “The takeaway is that every aspect of the sample contributes to the quality of a 3D image, and every element—whether glass, water or living cell—becomes part of the optical system,” Ulbrich explains.

Detection and diagnostics

Applications for 3D imaging span much—if not most—of biology, and this technique comes in especially handy when exploring clinical applications. “Biologists go to 3D for a number of reasons,” says Persmark, “and one is that 3D cell models are more physiologically relevant than standard 2D cell-culture models.” Also, the cell-cell and cell-matrix interactions impact many essential processes that reflect biology more faithfully and thus can be observed more clearly in 3D. A more realistic environment and 3D imaging improve the conditions for clinically relevant research, such as identifying biomarkers or developing diagnostics.

Using 3D cultures, though, creates some imaging challenges. “Applying standard microscopy techniques can be challenging on 3D cell-culture systems,” says Lubna Hussain, senior product manager at Lonza. “The more dense, tissue-like structure can block the light.” For such challenges, Lonza developed its RAFT 3D Culture System, which is built from translucent collagen scaffolds. With cells cultured in this system, says Hussain, “fluorescently stained cells can be easily visualized with subcellular resolution under a standard fluorescence microscope.”

This 3D culture system can be used for a variety of research or preclinical applications. Hussain says it has been used for “tissue-engineering and tissue-modeling applications, such as the development of corneal, liver fibrosis or blood-brain barrier models.” All of these could be used to improve identification of biomarkers or even to discover and develop new drug targets.

More than meets the eye in 3D

From basic biological research to medical applications, 3D imaging delivers more information—all in easier-to-gather ways and from samples in more natural and biologically relevant conditions.

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