In vivo imaging has been an indispensible and powerful tool in biomedical research, enabling a number of significant breakthrough discoveries. These basic research successes have fueled interest among clinicians to apply this technology to advance patient care. In this Q&A, we interviewed five in vivo imaging experts. We asked them about the considerable discoveries enabled by in vivo imaging as well as the new tools and technological updates that researchers and clinicians need to build on advances and enable even more breakthroughs.

We spoke to Alexandra de Lille, Ph.D., Director of Technical Applications for in vivo imaging at Revvity; Sean Gallagher, Ph.D., CTO, Analytik Jena; Joy Kovar, M.S., Principal Research Scientist, LI-COR; and Lori Roberts, Ph.D., Director, Bioscience Biotium.

  • How has in vivo imaging impacted biomedicine and translational research?

Dr. de Lille: In vivo imaging has significantly advanced animal model research as it allows the assessment of disease and therapeutic response noninvasively in live animals. Previously, these observations were only possible in ex vivo tissues, limiting the accuracy of readout. Furthermore, in vivo imaging facilitates longitudinal imaging in more sophisticated animal models, such as genetically engineered mouse models (GEMMs), increasing the data value per subject while saving resources. Researchers now can mimic clinical trials in mice with more relevant endpoints, such as survival, and run these in parallel with patient studies as co-clinical trials to ultimately better treatment efficacy.

Dr. Gallagher: Use fewer animals since the animals-can track over time in the same animal for research into disease treatments and drug challenges. Luminary screening with novel drugs against cancer and cancer metastasis is a good example. In vivo imaging methods have reduced the number of animals needed as well as improved the data because animals serve as their own control over a time course of weeks.

In vivo imaging has also enabled the continually expanding area of fluorescence-guided surgery with fluorescently tagged antibodies and cells. Experimental test procedures indicate that fluorescence-guided surgery is more effective than white-light surgery alone and for removing cancerous tissue. This is a rapidly expanding translational area research.

Drug distribution parameters in research animals are also easily assessed without surgery, enabling a better understanding of timing for distribution—for example, the collection points of a bioharma derived drug such as an antibody. Do these antibodies target the tumors, for example, or do they get bound in the lungs?

Ms. Kovar: In vivo imaging has had a tremendous impact on biomedical research. I work in the area of optical imaging with near-infrared (NIR) dyes, so my responses will primarily be focused on that platform. With the advent of targeted imaging agents, we can employ a dye conjugated to a targeting protein (antibody, peptide, drug, etc.) and then use lasers and light-emitting diodes (LEDs) to visualize various aspects of a disease. Being able to see and evaluate flow, uptake, and expression in cells is exciting. Optical imaging allows us to move from the cellular level to the whole animal and so more closely evaluate molecular biology of a particular disease or cancer. Now that the research community has enhanced and grown the field of optical imaging, the medical community is identifying areas where this technology can be effective and can enhance the level of care they can bring to their patients as well as define better treatments.

  • What are some new (and potentially revolutionary) applications for in vivo imaging?

Dr. de Lille: To date, no single imaging modality is able to address all possible biological questions, such as changes in anatomy, organ function, molecular expression, or metabolism. As a result, multimodality, multiparametric, and ratiometric imaging methods are on the rise. Integrated imaging modalities that combine, for example, optical and micro computed tomography (micro-CT) imaging, offer a molecular readout in an anatomical context. Alternatively, side-by-side modality registration via DICOM® (Digital Imaging and Communications in Medicine), in combination with animal imaging shuttles (positron emission tomography, or PET, and optical), is gaining practice. Specific probe development, especially targeted PET probes beyond [18]F-flourodeoxyglucose (18F-FDG) imaging, is gaining attention with goals to improve diagnostics and therapeutic efficacy assessment in the clinic.

Dr. Gallagher: Some of the most interesting applications for in vivo imaging include personalized medicine. This includes using patient-derived cancer xenografts in mice to create customized cancer treatments based on the patient’s tumor.

In regard to detection and analysis, artificial intelligence and, in particular, machine learning for detecting features in areas of importance found in an image will continue to be an extremely important part of software development activities.

Ms. Kovar: One great example is the potential role optical imaging can play in tumor margin detection. This area is currently receiving a lot of attention by surgeons and others for its potential use and benefits in the clinic.

  • Are there new products/tools that are helping enable in vivo studies?

Dr. de Lille: Novel techniques, like CRISPR (for clustered regularly interspaced short palindromic repeats) technology, help generate advanced animal models with built-in reporters. New labeling luciferases, luciferin substrates, fluorescent proteins, probes, targeted PET probes, and so forth continuously expand the toolbox of the in vivo scientist. High-throughput and ease-of-use imaging combined with automated data analysis are increasingly important in today’s competitive drug discovery landscape.

Dr. Gallagher: The recent development of an improved brighter bacterial lux operon (ilux) has the potential to eliminate most of the drawbacks to the use of current luciferases as bioluminescent reporter genes. The ilux-transformed Escherichia coli cells can be imaged at single-cell resolution without exogenous substrates, such as luciferin. This methodology is potentially adaptable to eukaryotic systems.

The development of genetically modified luciferases combined with novel substrates has created bioluminescence in the NIR range, dramatically extending the depth and sensitivity of detection of an expressing cell in an animal.

Ms. Kovar: I would have to say NIR dyes are a key tool in the advance of optical imaging and eventual translation to the clinic. NIR dyes allow us to visualize regions over the inherent autofluorescence that exists in the visible spectral region.

Dr. Roberts: Our CellBriteTM NIR stains are new NIR lipophilic membrane dyes. They are similar to the popular membrane dye DiR, but with better water solubility for staining the plasma membrane for long-term cell tracing and post-transplantation tracking, and a wider selection of NIR emission wavelengths.

  • Describe a specific in vivo imaging challenge and how your company is tackling/solving it.

Dr. de Lille: Revvity specifically emphasizes high-throughput, high-sensitivity, and high-resolution imaging in combination with advanced data analysis. For specific optical imaging, we recently launched IVIS® LuminaTM S5 and Lumina X5, which feature a high-throughput imaging solution and a set of smart animal handling accessories designed with imaging throughput and safety in mind. Smart loading trays allow users to pose animals on the benchtop before placing the tray into the IVIS instrument. This augments the number of animals imaged per hour. No nose cones are required, thus minimizing cleanup.

All tray parts have been tested and are resistant to repeated use with common laboratory disinfectants. Additionally, when used with the next-generation anesthesia unit (RAS-4), strong vacuum capabilities minimize excess gas from escaping, thus preventing exposure of users to anesthetic gas. Using fiducials built into the tray, the software can automatically recognize and draw regions of interest (ROIs), providing automated animal identification and facilitating faster image data analysis.

As such, Living Image® software brings IVIS technology to life by facilitating an intuitive workflow for in vivo optical analysis and data organization. The software supports input of unique animal IDs when using chip technologies and readers, thus streamlining labeling, setup, and subsequent export of data for analysis.

For micro-CT imaging on the other hand, the Quantum GX2 micro CT scanner uniquely features the AccuCT™ bone software plugin that facilitates a fully automated framework for workflows in bone segmentation and bone analysis like orthopedic studies.

Measurement and analysis of bone morphometry in 3D micro-CT volumes using automated image processing and analysis improve the accuracy, consistency, reproducibility, and speed of preclinical osteological research studies. This is no small feat, as automating segmentation and separation of individual bones in 3D micro-CT volumes of murine models presents significant challenges considering partial volume effects and joints with thin spacing, i.e., 50 to 100 μm. With this software, the widely used morphometric American Society for Bone and Mineral Research (ASBMR) analysis of cortical and trabecular components can be carried out without user interaction or guidance to readily quantify bone turnover and bone mineral density. This saves vast amounts of time in user training and data analysis.

Dr. Gallagher: Rapid detection and documentation of a reporter gene or antibody-tagged tissues and organs of interest can be quite complicated. We have created new imaging methodology combining internal ultrabright filtered LEDs and lasers with internal on-board software for simple and rapid one-touch capture of fluorescent images, and at the same time creating the pseudo-colored and composited image for visualization. We also focus on generating raw unaltered images so the researcher can then apply noise reduction and other applications in visualization as needed.

In vivo imagers can be quite expensive. We are just now introducing a new much lower-cost, high-performance fluorescence imaging system designed for 400- to 900-nm detection with one touch. The new system—the iBox® studio—handles mice and rats and is designed for injectable as well as gaseous anesthesia. It is a perfect complement for the more expensive bioluminescence systems, such as our iBox® ScientiaTM.

Dr. Roberts: NIR dyes are large and hydrophobic compared to other fluorophores. First-generation NIR dyes were designed with many negatively charged sulfonate groups to make them water soluble for biological applications. The negative charge on the dyes can cause nonspecific binding of probes as well as short in vivo half-life and increased immunogenicity.

Biotium's expertise is in fluroescent dye design. Our chemists engineered our near-IR CF® dyes to be water-soluble using a combination of sulfonate and poly(ethylene glycol) (PEG) modifications to limit the number of negative charges. As a consequence, the dyes have better solubility and brightness with less effect on probe specificity and in vivo stability.

Ms. Kovar: How do we improve on our ability to see deeper? This already is a question for the optical imaging community. Many are thinking how can we move deeper into the NIR and how can we improve on our imaging. I think there has been some very exciting research in the area of NIR-II or wavelengths above 900 nm that show promise.

  • What are some trends on the horizon in in vivo imaging?

Dr. de Lille: The imaging instrument of the future will be label free, without depth penetration limitation, without radiation exposure, with high sensitivity and resolution, and also inexpensive, fast, and user friendly with automated image acquisition, reconstruction, and data analysis. Revvity’s R&D team strives to attain this goal by offering the most advanced hardware, such as the CMOS (or complementary metal-oxide-semiconductor) detector panel in the QuantumGX2 microCT scanner.

Revvity implements machine learning algorithms for image recognition and automated data analysis, and also features a molecular and chemistry lab combined with a vivarium to create the best biological solutions. Last, the integrated offering of Revvity’s “omics” solutions, high-content 3D cell and organoid screening, in vivo imaging, and quantitative pathology creates a circle of intel in drug discovery, while bridging the gap from bench to bedside.

Dr. Gallagher: Probably the most interesting development for in vivo imaging is the increasing availability of shortwave infrared (SWIR) cameras, optics, and the associated nascent development of shortwave infrared tags. Existing fluorescent tags in the NIR frequently have fluorescent emission tails that go out to the SWIR region and can also be used. The SWIR allows very deep and high-resolution images relative to visible wavelengths and NIR. With the lowering cost of the SWIR cameras and the increasing availability of SWIR optics and tags, this will be an ongoing and exciting area.

For the current generation of in vivo imaging, the development of bioluminescent and fluorescent NIR capabilities continues, as noted earlier. Scientific CMOS cameras will continue to improve and are already quite useful in the fluorescence arena.

On the entry-level, more features and ease of use at lower cost will continue to be a demand from scientists.

Ms. Kovar: I would suggest there is a clear trend to move NIR dyes into the clinic. Currently, they are used for tumor margin detection, but I would have to assume someone is out there thinking of new ways to take advantage of optical imaging to help enhance care over and above just visualization, for example, for theranostics, therapy, etc.

Dr. Roberts: Researchers are looking to SWIR dyes with 1000- to 2000-nm emission to increase the number of NIR detection channels. Longer emission wavelengths also offer higher contrast, sensitivity, and penetration depths compared to NIR dyes with 700- to 900-nm emission.