Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative. This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease

A novel method to allow noninvasive, longitudinal imaging of the murine immune system in vivo

In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior

Techniques for RNA in vivo imaging in plants

Since the discovery of small RNAs and RNA silencing, RNA biology has taken a centre stage in cell and developmental biology. Small RNAs, but also mRNAs and other types of cellular and viral RNAs are processed at specific subcellular localizations. To fully understand cellular RNA metabolism and the various processes influenced byit, techniques are required that permit the sequence-specific tracking of RNAs in living cells. A variety of methods for RNA visualization have been developed since the 1990s, but plant cells pose particular challenges and not all approaches are applicable to them. On the other hand, plant RNA metabolism is particularly diverse and RNAs are even transported between cells, so RNA imaging can potentially provide many valuable insights into plant function at the cellular and tissue level. This Short Review briefly introduces the currently available techniques for plant RNA in vivo imaging and discusses their suitability for different biological questions.PostprintPeer reviewe

Second harmonic generating (SHG) nanoprobes for in vivo imaging

Fluorescence microscopy has profoundly changed cell and molecular biology studies by permitting tagged gene products to be followed as they function and interact. The ability of a fluorescent dye to absorb and emit light of different wavelengths allows it to generate startling contrast that, in the best cases, can permit single molecule detection and tracking. However, in many experimental settings, fluorescent probes fall short of their potential due to dye bleaching, dye signal saturation, and tissue autofluorescence. Here, we demonstrate that second harmonic generating (SHG) nanoprobes can be used for in vivo imaging, circumventing many of the limitations of classical fluorescence probes. Under intense illumination, such as at the focus of a laser-scanning microscope, these SHG nanocrystals convert two photons into one photon of half the wavelength; thus, when imaged by conventional two-photon microscopy, SHG nanoprobes appear to generate a signal with an inverse Stokes shift like a fluorescent dye, but with a narrower emission. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach nor blink, and the signal they generate does not saturate with increasing illumination intensity. The resulting contrast and detectability of SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues

In vivo imaging of protease activity by Probody therapeutic activation.

Probody™ therapeutics are recombinant, proteolytically-activated antibody prodrugs, engineered to remain inert until activated locally by tumor-associated proteases. Probody therapeutics exploit the fundamental dysregulation of extracellular protease activity that exists in tumors relative to healthy tissue. Leveraging the ability of a Probody therapeutic to bind its target at the site of disease after proteolytic cleavage, we developed a novel method for profiling protease activity in living animals. Using NIR optical imaging, we demonstrated that a non-labeled anti-EGFR Probody therapeutic can become activated and compete for binding to tumor cells in vivo with a labeled anti-EGFR monoclonal antibody. Furthermore, by inhibiting matriptase activity in vivo with a blocking-matriptase antibody, we show that the ability of the Probody therapeutic to bind EGFR in vivo was dependent on protease activity. These results demonstrate that in vivo imaging of Probody therapeutic activation can be used for screening and characterization of protease activity in living animals, and provide a method that avoids some of the limitations of prior methods. This approach can improve our understanding of the activity of proteases in disease models and help to develop efficient strategies for cancer diagnosis and treatment

In vivo imaging of pyrrole-imidazole polyamides with positron emission tomography

The biodistribution profiles in mice of two pyrrole-imidazole polyamides were determined by PET. Pyrrole-imidazole polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor-DNA interfaces, and modulate gene expression pathways in cell culture experiments. The 18F-radiolabeled polyamides were prepared by oxime ligation between 4-[18F]-fluorobenzaldehyde and a hydroxylamine moiety at the polyamide C terminus. Small animal PET imaging of radiolabeled polyamides administered to mice revealed distinct differences in the biodistribution of a 5-ring β-linked polyamide versus an 8-ring hairpin, which exhibited better overall bioavailability. In vivo imaging of pyrrole-imidazole polyamides by PET is a minimum first step toward the translation of polyamide-based gene regulation from cell culture to small animal studies

Handheld 3D scanning system for in-vivo imaging of skin cancer

Postprint (published version

Human platelets repurposed as vehicles for in vivo imaging of myeloma xenotransplants.

Human platelets were identified in tumors by Trousseau in 1865, although their roles in tumor microenvironments have only recently attracted the attention of cancer researchers. In this study we exploit and enhance platelet interactions in tumor microenvironments by introducing tumor-targeting and imaging functions. The first step in repurposing human platelets as vehicles for tumor-targeting was to inhibit platelet-aggregation by cytoplasmic-loading of kabiramide (KabC), a potent inhibitor of actin polymerization and membrane protrusion. KabC-Platelets can accumulate high levels of other membrane-permeable cytoxins and probes, including epidoxorubicin, carboxyfluorescein di-ester and chlorin-e6. Finally, mild reaction conditions were developed to couple tumor-targeting proteins and antibodies to KabC-platelets. Fluorescence microscopy studies showed KabC-platelets, surface-coupled with transferrin and Cy5, bind specifically to RPMI8226 and K562 cells, both of which over-express the transferrin receptor. Repurposed platelets circulate for upto 9-days a feature that increases their chance of interacting with target cells. KabC-platelets, surface-coupled with transferrin and Cy7, or chlorin-e6, and injected in immuno-compromised mice were shown to accumulate specifically in sub-cutaneous and intra-cranial myeloma xenotransplants. The high-contrast, in vivo fluorescence images recorded from repurposed platelets within early-stage myeloma is a consequence in part of their large size (φ~2µm), which allows them to transport 100 to 1000-times more targeting-protein and probe molecules respectively. Human platelets can be configured with a plurality of therapeutic and targeting antibodies to help stage tumor environments for an immunotherapy, or with combinations of therapeutic antibodies and therapeutic agents to target and treat cardiovascular and neurologic diseases

In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots

Background: Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known. Results: We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures. Conclusion: We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs