Dynamic near-infrared fluorescence (DNIF) whole-body imaging of small animals has become a popular tool in experimental biomedical research. In humans, however, the field of view has been limited to body parts, such as rheumatoid hands, diabetic feet or sentinel lymph nodes. Here we present a new whole-body DNIF-system suitable for adult subjects. We explored whether this system (i) allows dynamic whole-body fluorescence imaging and (ii) can detect modulations in skin perfusion. The non-specific fluorescent probe indocyanine green (ICG) was injected intravenously into two subjects, and fluorescence images were obtained at 5 Hz. The in- and out-flow kinetics of ICG have been shown to correlate with tissue perfusion. To validate the system, skin perfusion was modulated by warming and cooling distinct areas on the chest and the abdomen. Movies of fluorescence images show a bolus passage first in the face, then in the chest, abdomen and finally in the periphery (~10, 15, 20 and 30 seconds, respectively). When skin perfusion is augmented by warming, bolus arrives about 5 seconds earlier than when the skin is cooled and perfusion decreased. Calculating bolus arrival times and spatial fitting of basis time courses extracted from different regions of interest allowed a mapping of local differences in subcutaneous skin perfusion. This experiment is the first to demonstrate the feasibility of whole-body dynamic fluorescence imaging in humans. Since the whole-body approach demonstrates sensitivity to circumscribed alterations in skinperfusion, it may be used to target autonomous changes in polyneuropathy and to screen for peripheral vascular diseases.

Segmentation of anatomical structures and particularly abdominal organs is a fundamental problem for quantitative image analysis in preclinical research. This paper presents a novel approach for whole body segmentation of small animals in a multimodal setting of MR, CT and optical imaging. The algorithm integrates multiple imaging sequences into a machine learning framework, which generates supervoxels by an efficient hierarchical agglomerative strategy and utilizes multiple SVM-kNN classifiers each constrained by a heatmap prior region to compose the segmentation. We demonstrate results showing segmentation of mice images into several structures including the heart, lungs, liver, kidneys, stomach, vena cava, bladder, tumor, and skeleton structures. Experimental validation on a large set of mice and organs, indicated that our system outperforms alternative state of the art approaches. The system proposed can be generalized to various tissues and imaging modalities to produce automatic atlas-free segmentation, thereby enabling a wide range of applications in preclinical studies of small animal imaging.

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca(2+), cyclic adenosine monophosphate and adenosine 5'-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.

Microbubble (MB) contrast agents have revolutionalised the way ultrasound (US) imaging can be used clinically and pre-clinically. Contrast-enhanced US offers improvements in soft-tissue contrast, as well as the ability to visualise disease processes at the molecular level. However, its inability to provide in vivo whole-body imaging can hamper the development of new MB formulations. Herein, we describe a fast and efficient method for achieving 68Ga-labelling of MBs after a direct comparison of two different strategies. The optimised approach produces 68Ga-labelled MBs in good yields through the bioorthogonal inverse-electron-demand Diel-Alder reaction between a trans-cyclooctene-modified phospholipid and a new tetrazine-bearing HBED-CC chelator. The ability to noninvasively study the whole-body distribution of 68Ga-labelled MBs was demonstrated in vivo using positron emission tomography (PET). This method could be broadly applicable to other phospholipid-based formulations, providing accessible solutions for in vivo tracking of MBs.

Tendency is to moderate the injected activity and/or reduce acquisition time in PET examinations to minimize potential radiation hazards and increase patient comfort. This work aims to assess the performance of regular full-dose (FD) synthesis from fast/low-dose (LD) whole-body (WB) PET images using deep learning techniques.

Fast Field-Cycling (FFC) is a well-established Nuclear Magnetic Resonance (NMR) technique that exploits varying magnetic fields to quantify molecular motion over a wide range of time scales, providing rich structural information from nanometres to micrometres, non-invasively. Previous work demonstrated great potential for FFC-NMR biomarkers in medical applications; our research group has now ported this technology to medical imaging by designing a whole-body FFC Magnetic Resonance Imaging (FFC-MRI) scanner capable of performing accurate measurements non-invasively over the entire body, using signals from water and fat protons. This is a unique tool to explore new biomarkers related to disease-induced tissue remodelling. Our approach required making radical changes in the design, construction and control of MRI hardware so that the magnetic field is switched within 12.5 ms to reach any field strength from 50 μT to 0.2 T, providing clinically useful images within minutes. Pilot studies demonstrated endogenous field-dependant contrast in biological tissues in good agreement with reference data from other imaging modalities, confirming that our system can perform multiscale structural imaging of biological tissues, from nanometres to micrometres. It is now possible to confirm ex vivo results obtained from previous clinical studies, offering applications in diagnosis, staging and monitoring treatment for cancer, stroke, osteoarthritis and oedema.

Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.

The use of computed tomography (CT) to evaluate obesity in canines is limited. Traditional CT image analysis is cumbersome and uses prediction equations that require manual calculations. In order to overcome this, our study investigated the use of advanced image analysis software programs to determine body composition in dogs with an application to canine obesity research. Beagles and greyhounds were chosen for their differences in morphology and propensity to obesity. Whole body CT scans with regular intervals were performed on six beagles and six greyhounds that were subjected to a 28-day weight-gain protocol. The CT images obtained at days 0 and 28 were analyzed using software programs OsiriX, ImageJ, and AutoCAT. The CT scanning technique was able to differentiate bone, lean, and fat tissue in dogs and proved sensitive enough to detect increases in both lean and fat during weight gain over a short period. A significant difference in lean : fat ratio was observed between the two breeds on both days 0 and 28 (P < 0.01). Therefore, CT and advanced image analysis proved useful in the current study for the estimation of body composition in dogs and has the potential to be used in canine obesity research.

Whole-body magnetic resonance imaging (MRI) is increasingly being used for a number of indications. Our aim was to review and describe indications and scan protocols for diagnostic value of whole-body MRI for multifocal disease in children and adolescents, we conducted a systematic search in Medline, Embase and Cochrane for all published papers until November 2018. Relevant subject headings and free text words were used for the following concepts: 1) whole-body, 2) magnetic resonance imaging and 3) child and/or adolescent. Included were papers in English with a relevant study design that reported on the use and/or findings from whole-body MRI examinations in children and adolescents. This review includes 54 of 1,609 papers identified from literature searches. Chronic nonbacterial osteomyelitis, lymphoma and metastasis were the most frequent indications for performing a whole-body MRI. The typical protocol included a coronal STIR (short tau inversion recovery) sequence with or without a coronal T1-weighted sequence. Numerous studies lacked sufficient data for calculating images resolution and only a few studies reported the acquired voxel volume, making it impossible for others to reproduce the protocol/images. Only a minority of the included papers assessed reliability tests and none of the studies documented whether the use of whole-body MRI affected mortality and/or morbidity. Our systematic review confirms significant variability of technique and the lack of proven validity of MRI findings. The information could potentially be used to boost attempts towards standardization of technique, reporting and guidelines development.

When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins) or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r²  =  0.996 and 0.969, respectively). Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20-50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation into tissue by IgG1. These studies demonstrate the ability of quantitative FMT imaging of FR/NIR agents to non-invasively visualize and quantify the biodistribution of different agents over time.

A single contrast agent that offers whole-body non-invasive imaging along with the superior sensitivity and spatial resolution of surface-enhanced resonance Raman scattering (SERRS) imaging would allow both pre-operative mapping and intraoperative imaging and thus be highly desirable. We hypothesized that labeling our recently reported ultrabright SERRS nanoparticles with a suitable radiotracer would enable pre-operative identification of regions of interest with whole body imaging that can be rapidly corroborated with a Raman imaging device or handheld Raman scanner in order to provide high precision guidance during surgical procedures. Here we present a straightforward new method that produces radiolabeled SERRS nanoparticles for combined positron emission tomography (PET)-SERRS tumor imaging without requiring the attachment of molecular chelators. We demonstrate the utility of these PET-SERRS nanoparticles in several proof-of-concept studies including lymph node (LN) tracking, intraoperative guidance for LN resection, and cancer imaging after intravenous injection. We anticipate that the radiolabeling method presented herein can be applied generally to nanoparticle substrates of various materials by first coating them with a silica shell and then applying the chelator-free protocol.

Distinct physiological states arise from complex interactions among the various organs present in the human body. PET is a non-invasive modality with numerous successful applications in oncology, neurology, and cardiology. However, while PET imaging has been applied extensively in detecting focal lesions or diseases, its potential in detecting systemic abnormalities is seldom explored, mostly because total-body imaging was not possible until recently.

Fluorochrome-labelled iron oxide magnetic nanoparticles (MNP) have been of great help in elucidating biological processes. Here, we used dually-fluorochrome-labelled MNP and studied to what extent fluorescence detection could reflect their fate in living animals. One day after application in mice (200 µmol Fe/kg body weight), the fluorescence of the dye attached to the core (DY-730) was very prominent and in agreement with the increase of iron in the liver and spleen of mice, but inconspicuous at time points thereafter. We attribute this fluorescence behavior to early degradation processes of the MNP´s core in the cellular lysosomal compartment. In contrast, the fluorescence of the dye DY-555 stuck to the PEG coating was not detectable in vivo. In summary, labelling of MNP with dyes at their metallic core could be of help when detecting first incidences of MNP biodegradation in vivo, as opposed to dyes attached to the MNP coating.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

Organism-level systems biology aims to identify, analyze, control and design cellular circuits in organisms. Many experimental and computational approaches have been developed over the years to allow us to conduct these studies. Some of the most powerful methods are based on using optical imaging in combination with fluorescent labeling, and for those one of the long-standing stumbling blocks has been tissue opacity. Recently, the solutions to this problem have started to emerge based on whole-body and whole-organ clearing techniques that employ innovative tissue-clearing chemistry. Here, we review these advancements and discuss how combining new clearing techniques with high-performing fluorescent proteins or small molecule tags, rapid volume imaging and efficient image informatics is resulting in comprehensive and quantitative organ-wide, single-cell resolution experimental data. These technologies are starting to yield information on connectivity and dynamics in cellular circuits at unprecedented resolution, and bring us closer to system-level understanding of physiology and diseases of complex mammalian systems.

Advances in fluorescence imaging coupled with the generation of near infrared probes have significantly improved the capabilities of non-invasive, real-time imaging in whole animals. In this study we were able to overcome a limitation of in vivo fluorescence imaging and have established a dual cell tracking method where two different cell types can be monitored according to the spectral signature of the cell labelling fluorophore. Using a mouse model of acute liver injury, we have characterised the in vivo migration patterns of wild type and transgenic neutrophils with impaired chemotaxis. Here, we were able to demonstrate that IVIS provides a sensitive multiplexing technology to differentiate two different cell populations based on the spectral signature of the cell labelling fluorophores. This spectral unmixing methodology has the potential to uncover multidimensional cellular interactions involved in many diseases such as fibrosis and cancer. In vivo spectral un-mixing provides a useful tool for monitoring multiple biological process in real-time in the same animal.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

To prospectively compare artefacts and image quality in testicular stage I cancer patients using different combinations of breathing schemes and Multi-band (MB) in whole-body DWIBS at 1.5 T.Diffusion-Weighted whole-body Imaging with Background body signal Suppression (DWIBS) using inversion recovery (IR) fat saturation is a cornerstone in oncologic whole-body MRI, but implementation is restrained by long acquisition times. The new Multi-Band (MB) technique reduces scan time which can be reinvested in respiratory compensation.

Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.