Scanning through slides of biological tissue, most biologists wonder: Is this how it really looks? Am I getting the entire regulatory picture here? To answer these questions, some platforms provide in vivo imaging, and some even image whole—small, but entire—animals.

At the University of Florida College of Medicine, research coordinator and lab manager Gary Brown and his colleagues have an IVIS Spectrum In Vivo System, which was sold originally by Caliper and is now supported by Revvity. This system captures fluorescent or luminescent signals in living tissue. “This allows for a visual rendition of where genes are targeting activity, immunological agents are active, or tumor location and growth monitoring,” Brown says. “These are but a few of the potential uses for this instrument, which provides the benefit of being able to acquire data from living subjects in a noninvasive manner, which reduces the numbers needed to achieve statistical significance.”

To look more deeply inside living animals, today’s scientists can select from various devices and techniques.

Tracking targets

Using labels that fluoresce or luminesce, biologists can tag and track many molecular targets. 

For example, LI-COR Biosciences developed the Pearl Trilogy Imager about a decade ago to image fluorescent signals inside small animals, and the company added bioluminescent capabilities in 2015. According to Jeff Harford, product marketing manager at LI-COR Biosciences, there is an “increasing interest in moving fluorescence optical imaging into humans.” He adds, “What was speculative 10 years ago has made a lot of progress, and we now see more than 14 clinical trials that are taking place using our dyes labeled to various compounds.” As a result, this technology is expanding into imaging probes for diagnostic, therapeutic and surgical applications.

The reagents also influence how this technology can be used. Harford says, “We have a tremendous amount of expertise in dye development, optical-probe and contrast-agent development and conjugations, histology and current good manufacturing practices [cGMPs].”

Some of the most advanced labels arise from genetic engineering. For instance, Jeffrey Hung, chief commercial officer at Vigene Biosciences, says, “Coupling two-photon imaging with genetically encoded biosensors, such as GCaMP6 and CaMPARI, opens an exciting opportunity to view neuronal activities in an excellent signal-to-noise ratio with a high temporal and spatial fidelity.” These markers can also be easily added to an organism through transduction with Vigene’s adeno-associated viruses that contain these markers and others. “With a ready-to-inject format and high efficiency of gene delivery,” Hung says, “these products empower global life scientists to conduct sophisticated in vivo imaging experiments.”

Listening In

Instead of just looking at live animals, scientists can listen. For example, Brown and his colleagues also use a VisualSonics Vevo 770 Hi-Res Ultrasound Imaging System. “The Vevo acquires ultrasound images noninvasively in small animal species used in research applications,” he explains. “By the use of contrast and micro-bubble reagents, a skilled operator can acquire vasculature diameters, vessel-wall thicknesses, blood flow rate via Doppler, volumetrics data capture and more.”

Other approaches to sound-based imaging, such as photoacoustic imaging, also can be used. One team of scientists used this technique to detect prostate-cancer cells [1]. Also, iThera Medical uses its proprietary multispectral optoacoustic tomography (MSOT), which combines the targeting of dyes used in optical imaging and the spatial and temporal resolution of ultrasound.

Imaging for everyone

One of the key trends driving in vivo imaging is the breadth of applications and users.

Instead of systems that can only be used by imaging experts, today’s systems cater to life scientists in general. 

For example, a growing number of magnetic resonance (MR) users, including basic biologists, are operating the systems, says Todd Sasser, product marketing manager, molecular imaging at Bruker BioSpin. Also, these systems can show more detail than ever. “The MRI Cryo Coil,” Sasser says, “allows in vivo resolutions approaching traditional histology” as well as the use of live organisms.

In addition, many systems offer multimodal imaging, such as positron emission tomography (PET) and MR. “The latest integrated PET-MR technologies combine molecular biology and targeted molecular imaging with excellent software tissue contrast, offering more insight than the sum of the individual modalities,” Sasser explains.

Other experts also see movement toward making systems simpler to operate. “The trend continues toward ease of use and automation for generating images needed for analysis,” says Sean Gallagher, director of research and development at Analytik Jena. “Using one-click applications with our software and iBox systems automatically aligns the filters, lighting, optics and camera settings and then delivers the optimal animal or plant image.”

Optimizing a system and labels adds even more possibilities. As Gallagher says, “With our new introduction of bioluminescence-based imagers with ultracooled, very low-noise, back-thinned CCD technology and f/0.95 optics, we address the wide range of applications using luciferases.”

Upping the interface

As for desirable improvements, Brown says, “The very first thing I would like to see would be well-thought-out software [graphical user interface] for control of the instruments.”

He also wants software that can evolve as needed. “More often than not, the lack of software support is the death knell for many instruments that mechanically and electrically work just fine,” he explains. So he’d like vendors to develop modular software that is “maintained by the manufacturer in general machine or assembly language,” which would allow “license holders to get the code and compile it for whatever system is required, in whatever operating-system version is necessary.”

Researchers have many options when it comes to imaging their samples. With continuous improvements to instrument and reagents as well as the supporting software, scientists are using in vivo imaging technologies in a diverse array of applications. As this field evolves, biologists can look deeper and deeper, all the while watching life in action.

Reference

[1] Dogra, V, et al., “Photoacoustic imaging with an acoustic lens detects prostate cancer cells labeled with PSMA-targeting near-infrared dye-conjugates,” J Biomed Optics, 21:66019, 2016. [PMID: 27367255]

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