Aster glehni Extract, Including Caffeoylquinic Acids as the Main Constituents, Induces PPAR ß/δ-Dependent Muscle-Type Change and Myogenesis in Apolipoprotein E Knockout Mice.

To probe the functions of Aster glehni (AG) extract containing various caffeoylquinic acids on dyslipidemia, obesity, and skeletal muscle-related diseases focused on the roles of skeletal muscle, we measured the levels of biomarkers involved in oxidative phosphorylation and type change of skeletal muscle in C2C12 cells and skeletal muscle tissues from apolipoprotein E knockout (ApoE KO) mice. After AG extract treatment in cell and animal experiments, western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used to estimate the levels of proteins that participated in skeletal muscle type change and oxidative phosphorylation. AG extract elevated protein expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), phosphorylated 5'-AMP-activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor beta/delta (PPARß/δ), myoblast determination protein 1 (MyoD), and myoglobin in skeletal muscle tissues. Furthermore, it elevated the ATP concentration. However, protein expression of myostatin was decreased by AG treatment. In C2C12 cells, increments of MyoD, myoglobin, myosin, ATP-producing pathway, and differentiation degree by AG were dependent on PPARß/δ and caffeoylquinic acids. AG extract can contribute to the amelioration of skeletal muscle inactivity and sarcopenia through myogenesis in skeletal muscle tissues from ApoE KO mice, and function of AG extract may be dependent on PPARß/δ, and the main functional constituents of AG are trans-5-O-caffeoylquinic acid and 3,5-O-dicaffeoylquinic acid. In addition, in skeletal muscle, AG has potent efficacies against dyslipidemia and obesity through the increase of the type 1 muscle fiber content to produce more ATP by oxidative phosphorylation in skeletal muscle tissues from ApoE KO mice.

Single-Cell PET Imaging and Tracking.

Positron emission tomography (PET) is one of the most sensitive whole-body molecular imaging techniques available in the clinic, able to detect picomolar levels of probe. As such, it was recently demonstrated that PET could also be used to track single radiolabeled cells in small animals. In this protocol, we present detailed procedures for radiolabeling cells using mesoporous silica nanoparticles (MSNs) and for tracking these cells in real time using in vivo PET. This includes static imaging of single cells as well as dynamic tracking of moving cells directly from the list-mode data. The protocol provides detailed instructions and examples for each step.

Pentafluorobenzene: Promising Applications in Diagnostics and Therapeutics.

Pentafluorobenzene (PFB) represents a class of aromatic fluorine compounds employed exclusively across a spectrum of chemical and biological applications. PFBs are credited with developing various chemical synthesis techniques, networks and biopolymers, bioactive materials, and targeted drug delivery systems. The first part of this review delves into recent developments in PFB-derived molecules for diagnostic purposes. In the latter segment, PFB's role in the domain of theragnostic applications is discussed. The review elucidates different mechanisms and interaction strategies applied in leveraging PFBs to formulate diagnostic and theragnostic tools, substantiated by proper examples. The utilization of PFBs emerges as an enabler, facilitating manifold reactions, improving materials' properties, and even opening avenues for explorative research.

In Vivo Stem Cell Imaging Principles and Applications.

Stem cells are the foundational cells for every organ and tissue in our body. Cell-based therapeutics using stem cells in regenerative medicine have received attracting attention as a possible treatment for various diseases caused by congenital defects. Stem cells such as induced pluripotent stem cells (iPSCs) as well as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and neuroprogenitors stem cells (NSCs) have recently been studied in various ways as a cell-based therapeutic agent. When various stem cells are transplanted into a living body, they can differentiate and perform complex functions. For stem cell transplantation, it is essential to determine the suitability of the stem cell-based treatment by evaluating the origin of stem, the route of administration, in vivo bio-distribution, transplanted cell survival, function, and mobility. Currently, these various stem cells are being imaged in vivo through various molecular imaging methods. Various imaging modalities such as optical imaging, magnetic resonance imaging (MRI), ultrasound (US), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) have been introduced for the application of various stem cell imaging. In this review, we discuss the principles and recent advances of in vivo molecular imaging for application of stem cell research.

Buspirone Induces Weight Loss and Normalization of Blood Pressure via the Stimulation of PPARδ Dependent Energy Producing Pathway in Spontaneously Hypertensive Rats.

Introduction: Buspirone, as a partial agonist for a 5-hydroxytryptamine (serotonin) receptor 1A (5-HT1A), has been prescribed as an anxiolytic drug for patients. In addition, the lowering effect of serotonin on blood pressure was reported in hypertensive animal model. We investigated the therapeutic mechanism of buspirone against lipid metabolism disturbed by hypertension of early stage via hypertensive and obese animal model. Methods: The levels of various biomarkers related to lipid metabolism and hypertension were estimated through the measurement of body weight and fat weight, blood analysis, western blotting, immunohistochemistry, and staining methods. Results: The lipid accumulation was lowered in differentiated 3T3-L1 cells by buspirone treatments of 50 and 100 µM compared with untreated differentiated control. Body weight and abdominal fat weight were lowered in spontaneously hypertensive rats (SHRs) administered with buspirone of 10 mg/kg/day for 4 weeks than 8-week untreated group. Triglyceride (TG) level was decreased in SHRs administered with buspirone of 5 and 10 mg/kg/day compared to 8-week untreated group. High-density lipoprotein (HDL)-cholesterol concentration was elevated by buspirone 10 mg/kg/day treatment compared to 8-week untreated group. Blood pressures in SHRs were lowered by buspirone treatments of 5 and 10 mg/kg/day compared with 8-week untreated group. Protein levels for peroxisome proliferator-activated receptor δ (PPARδ), 5' adenosine monophosphate-activated protein kinase (AMPK), and PPARγ coactivator-1 alpha (PGC-1α) were increased both in C2C12 cells treated by buspirone of 100 µM and in SHRs administered by buspirone of 1, 5, and 10 mg/kg/day compared to untreated control cells and 8-week untreated group. Fat cell numbers decreased in 8-week untreated group were increased in SHRs administered by buspirone treats of 1, 5, and 10 mg/kg/day. Protein expression levels for angiotensin II type 1 receptor (AT1R) and vascular cell adhesion molecule 1 (VCAM1) were increased in 8-week untreated group compared to 4-week group, however, they were decreased by buspirone treatments of 1, 5, and 10 mg/kg/day. Conclusion: Buspirone may induce the losses of body weight and abdominal fat weight through the activation of PPARδ dependent catabolic metabolism producing energy, and eventually, the ameliorated lipid metabolism could normalize high blood pressure.

Immune-Enhancing Effect of Sargassum horneri on Cyclophosphamide-Induced Immunosuppression in BALB/c Mice and Primary Cultured Splenocytes.

Sargassum horneri (SH) is a seaweed that has several features that benefit health. In this study, we investigated the immune-enhancing effect of SH, focusing on the role of spleen-mediated immune functions. Chromatographic analysis of SH identified six types of monosaccharide contents, including mannose, rhamnose glucose, galactose xylose and fucose. SH increased cell proliferation of primary cultured naïve splenocytes treated with or without cyclophosphamide (CPA), an immunosuppression agent. SH also reversed the CPA-induced decrease in Th1 cytokines. In vivo investigation revealed that SH administration can increase the tissue weight of major immune organs, such as the spleen and thymus. A similar effect was observed in CPA-injected immunosuppressed BALB/c mice. SH treatment increased the weight of the spleen and thymus, blood immune cell count and Th1 cytokine expression. Additionally, the YAC-1-targeting activities of natural killer cells, which are important in innate immunity, were upregulated upon SH treatment. Overall, our study demonstrates the immune-enhancing effect of SH, suggesting its potential as a medicinal or therapeutic agent for pathologic conditions involving immunosuppression.

In vivo molecular and single cell imaging.

Molecular imaging is used to improve the disease diagnosis, prognosis, monitoring of treatment in living subjects. Numerous molecular targets have been developed for various cellular and molecular processes in genetic, metabolic, proteomic, and cellular biologic level. Molecular imaging modalities such as Optical Imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Computed Tomography (CT) can be used to visualize anatomic, genetic, biochemical, and physiologic changes in vivo. For in vivo cell imaging, certain cells such as cancer cells, immune cells, stem cells could be labeled by direct and indirect labeling methods to monitor cell migration, cell activity, and cell effects in cell-based therapy. In case of cancer, it could be used to investigate biological processes such as cancer metastasis and to analyze the drug treatment process. In addition, transplanted stem cells and immune cells in cell-based therapy could be visualized and tracked to confirm the fate, activity, and function of cells. In conventional molecular imaging, cells can be monitored in vivo in bulk non-invasively with optical imaging, MRI, PET, and SPECT imaging. However, single cell imaging in vivo has been a great challenge due to an extremely high sensitive detection of single cell. Recently, there has been great attention for in vivo single cell imaging due to the development of single cell study. In vivo single imaging could analyze the survival or death, movement direction, and characteristics of a single cell in live subjects. In this article, we reviewed basic principle of in vivo molecular imaging and introduced recent studies for in vivo single cell imaging based on the concept of in vivo molecular imaging. [BMB Reports 2022; 55(6): 267-274].

Mechanoporation enables rapid and efficient radiolabeling of stem cells for PET imaging.

Regenerative medicine uses the patient own stem cells to regenerate damaged tissues. Molecular imaging techniques are commonly used to image the transplanted cells, either right after surgery or at a later time. However, few techniques are fast or straightforward enough to label cells intraoperatively. Adipose tissue-derived stem cells (ADSCs) were harvested from knee joints of minipigs. The cells were labeled with PET contrast agent by flowing mechanoporation using a microfluidic device. While flowing through a series of microchannels, cells are compressed repeatedly by micro-ridges, which open transient pores in their membranes and induce convective transport, intended to facilitate the transport of 68Ga-labeled and lipid-coated mesoporous nanoparticles (MSNs) into the cells. This process enables cells to be labeled in a matter of seconds. Cells labeled with this approach were then implanted into cartilage defects, and the implant was imaged using positron emission tomography (PET) post-surgery. The microfluidic device can efficiently label millions of cells with 68Ga-labeled MSNs in as little as 15 min. The method achieved labeling efficiency greater than 5 Bq/cell on average, comparable to 30 min-long passive co-incubation with 68Ga-MSNs, but with improved biocompatibility due to the reduced exposure to ionizing radiation. Labeling time could also be accelerated by increasing throughput through more parallel channels. Finally, as a proof of concept, ADSCs were labeled with 68Ga-MSNs and quantitatively assessed using clinical PET/MR in a mock transplant operation in pig knee joints. MSN-assisted mechanoporation is a rapid, effective and straightforward approach to label cells with 68Ga. Given its high efficiency, this labeling method can be used to track small cells populations without significant effects on viability. The system is applicable to a variety of cell tracking studies for cancer therapy, regenerative therapy, and immunotherapy.

Noninvasive and Highly Multiplexed Five-Color Tumor Imaging of Multicore Near-Infrared Resonant Surface-Enhanced Raman Nanoparticles In Vivo.

In vivo multiplexed imaging aims for noninvasive monitoring of tumors with multiple channels without excision of the tissue. While most of the preclinical imaging has provided a number of multiplexing channels up to three, Raman imaging with surface-enhanced Raman scattering (SERS) nanoparticles was suggested to offer higher multiplexing capability originating from their narrow spectral width. However, in vivo multiplexed SERS imaging is still in its infancy for multichannel visualization of tumors, which require both sufficient multiplicity and high sensitivity concurrently. Here we create multispectral palettes of gold multicore-near-infrared (NIR) resonant Raman dyes-silica shell SERS (NIR-SERRS) nanoparticle oligomers and demonstrate noninvasive and five-plex SERS imaging of the nanoparticle accumulation in tumors of living mice. We perform the five-plex ratiometric imaging of tumors by varying the administered ratio of the nanoparticles, which simulates the detection of multiple biomarkers with different expression levels in the tumor environment. Furthermore, since this method does not require the excision of tumor tissues at the imaging condition, we perform noninvasive and longitudinal imaging of the five-color nanoparticles in the tumors, which is not feasible with current ex vivo multiplexed tissue analysis platforms. Our work surpasses the multiplicity limit of previous preclinical tumor imaging methods while keeping enough sensitivity for tumor-targeted in vivo imaging and could enable the noninvasive assessment of multiple biological targets within the tumor microenvironment in living subjects.

Identification of Lymphatic and Hematogenous Routes of Rapidly Labeled Radioactive and Fluorescent Exosomes through Highly Sensitive Multimodal Imaging.

There has been considerable interest in the clinical use of exosomes as delivery vehicles for treatments as well as for promising diagnostic biomarkers, but the physiological distribution of exosomes must be further elucidated to validate their efficacy and safety. Here, we aimed to develop novel methods to monitor exosome biodistribution in vivo using positron emission tomography (PET) and optical imaging. Exosomes were isolated from cultured mouse breast cancer cells and labeled for PET and optical imaging. In mice, radiolabeled and fluorescently labeled exosomes were injected both via lymphatic and hematogenous metastatic routes. PET and fluorescence images were obtained and quantified. Radioactivity and fluorescence intensity of ex vivo organs were measured. PET signals from exosomes in the lymphatic metastatic route were observed in the draining sentinel lymph nodes. Immunohistochemistry revealed greater exosome uptake in brachial and axillary versus inguinal lymph nodes. Following administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in the lungs, liver, and spleen. Exosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and fluorescence and were visualized via PET and optical imaging, suggesting that this simultaneous and rapid labeling method could provide valuable information for further exosome translational research and clinical applications.

Instant labeling of therapeutic cells for multimodality imaging.

Autologous therapeutic cells are typically harvested and transplanted in one single surgery. This makes it impossible to label them with imaging biomarkers through classical transfection techniques in a laboratory. To solve this problem, we developed a novel microfluidic device, which provides highly efficient labeling of therapeutic cells with imaging biomarkers through mechanoporation. Methods: Studies were performed with a new, custom-designed microfluidic device, which contains ridges, which compress adipose tissue-derived stem cells (ADSCs) during their device passage. Cell relaxation after compression leads to cell volume exchange for convective transfer of nanoparticles and nanoparticle uptake into the cell. ADSCs were passed through the microfluidic device doped with iron oxide nanoparticles and 18F-fluorodeoxyglucose (FDG). The cellular nanoparticle and radiotracer uptake was evaluated with DAB-Prussian blue, fluorescent microscopy, and inductively coupled plasma spectrometry (ICP). Labeled and unlabeled ADSCs were imaged in vitro as well as ex vivo in pig knee specimen with magnetic resonance imaging (MRI) and positron emission tomography (PET). T2 relaxation times and radiotracer signal were compared between labeled and unlabeled cell transplants using Student T-test with p<0.05. Results: We report significant labeling of ADSCs with iron oxide nanoparticles and 18F-FDG within 12+/-3 minutes. Mechanoporation of ADSCs with our microfluidic device led to significant nanoparticle (> 1 pg iron per cell) and 18F-FDG uptake (61 mBq/cell), with a labeling efficiency of 95%. The labeled ADSCs could be detected with MRI and PET imaging technologies: Nanoparticle labeled ADSC demonstrated significantly shorter T2 relaxation times (24.2±2.1 ms) compared to unlabeled cells (79.6±0.8 ms) on MRI (p<0.05) and 18F-FDG labeled ADSC showed significantly higher radiotracer uptake (614.3 ± 9.5 Bq / 1×104 cells) compared to controls (0.0 ± 0.0 Bq/ 1×104 cells) on gamma counting (p<0.05). After implantation of dual-labeled ADSCs into pig knee specimen, the labeled ADSCs revealed significantly shorter T2 relaxation times (41±0.6 ms) compared to unlabeled controls (90±1.8 ms) (p<0.05). Conclusion: The labeling of therapeutic cells with our new microfluidic device does not require any chemical intervention, therefore it is broadly and immediately clinically applicable. Cellular labeling using mechanoporation can improve our understanding of in vivo biodistributions of therapeutic cells and ultimately improve long-term outcomes of therapeutic cell transplants.

Whole-body tracking of single cells via positron emission tomography.

In vivo molecular imaging can measure the average kinetics and movement routes of injected cells through the body. However, owing to non-specific accumulation of the contrast agent and its efflux from the cells, most of these imaging methods inaccurately estimate the distribution of the cells. Here, we show that single human breast cancer cells loaded with mesoporous silica nanoparticles concentrating the 68Ga radioisotope and injected into immunodeficient mice can be tracked in real time from the pattern of annihilation photons detected using positron emission tomography, with respect to anatomical landmarks derived from X-ray computed tomography. The cells travelled at an average velocity of 50 mm s-1 and arrested in the lungs 2-3 s after tail-vein injection into the mice, which is consistent with the blood-flow rate. Single-cell tracking could be used to determine the kinetics of cell trafficking and arrest during the earliest phase of the metastatic cascade, the trafficking of immune cells during cancer immunotherapy and the distribution of cells after transplantation.

Hard X-ray excited optical luminescence from protein-directed Au∼20 clusters.

Hard X-ray excited optical luminescence is a unique property of materials, which makes them promising for biological imaging applications. However, the preparation of biocompatible contrast agents for hard X-ray excited optical luminescence remains a considerable challenge that has, to date, not been overcome. In this study, we investigated the luminescence properties of protein-directed Au∼20 clusters upon hard X-ray irradiation, both in solution and when embedded in films.

A gold nanoparticle system for the enhancement of radiotherapy and simultaneous monitoring of reactive-oxygen-species formation.

Gold nanoparticles (AuNPs) are known to sensitize cancer cells to radiation therapy (RT) by increasing the deposition of ionizing energy in their immediate vicinity. However, this process of dose enhancement is challenging to monitor because it is heterogeneous at the sub-cellular scale. Furthermore, radiation damage is primarily mediated by reactive oxygen species (ROS) that are produced following water radiolysis. Here, radiation-responsive PEGylated gold nanoparticles (RPAuNPs) were synthesized for the enhanced generation and concurrent detection of ROS in cancer cells and tumors. PEGylated gold particles (20 nm diameter) were functionalized with dihydrorhodamine 123 (DHR-123), a known ROS sensor, to monitor ROS generation in their immediate vicinity. These NPs were able to effectively radiosensitize cells, as measured by increased cell apoptosis following RT. Furthermore, the fluorescence of these RPAuNPs was 7-fold higher after 6 Gy RT due to the local production of ROS near the surface of the NP. Finally, multispectral fluorescence imaging was used to monitor NP-induced ROS in vivo, following conformal RT, in a xenograft model of breast cancer. This theranostic NP system provides a novel approach for monitoring the nanoscale enhancement of RT by high-Z metal NPs.

Flexible optically stimulated luminescence band for 1D in vivo radiation dosimetry.

In vivo dosimetry helps ensure the accuracy of radiation treatments. However, standard techniques are only capable of point sampling, making it difficult to accurately measure dose variation along curved surfaces in a continuous manner. The purpose of this work is to introduce a flexible dosimeter band and validate its performance using pre-clinical and clinical x-ray sources. Dosimeter bands were fabricated by uniformly mixing BaFBr:Eu storage phosphor powders into a silicone based elastomer. An optical readout device with dual-wavelength excitation was designed and built to correct for non-uniform phosphor density and extract accurate dose information. Results demonstrated significant correction of the non-uniform readout signal and excellent dose linearity up to 8 Gy irradiation using a pre-clinical 320 kV x-ray system. Beam profile measurements were demonstrated over a long distance of ~30 cm by placing multiple dosimeters in a single line and stitching the results. The performance of the dosimeters was also tested using a clinical linear accelerator (6 MV) and compared to radiochromic film. Once bias corrected, the bands displayed a linear dose response over the 1.02-9.36 Gy range (R 2 > 0.99). The proposed system can be further improved by reducing the size of the readout beam and by more uniformly mixing the phosphor powder with the elastomer. We expect this technique to find application for large-field treatments such as total-skin irradiation and total-body irradiation.

Development and MPI tracking of novel hypoxia-targeted theranostic exosomes.

Treating the hypoxic region of the tumor remains a significant challenge. The goals of this study are to develop an exosome platform that can target regions of tumor hypoxia and that can be monitored in vivo using magnetic particle imaging (MPI). Four types of exosomes (generated under hypoxic or normoxic conditions, and with or without exposure to X-ray radiation) were isolated from MDA-MB-231 human breast cancer cells. Exosomes were labeled by DiO, a fluorescent lipophilic tracer, to quantify their uptake by hypoxic cancer cells. Subsequently, the exosomes were modified to carry SPIO (superparamagnetic iron oxide) nanoparticles and Olaparib (PARP inhibitor). FACS and fluorescence microscopy showed that hypoxic cells preferentially take up exosomes released by hypoxic cells, compared with other exosome formulations. In addition, the distribution of SPIO-labeled exosomes was successively imaged in vivo using MPI. Finally, the therapeutic efficacy of Olaparib-loaded exosomes was demonstrated by increased apoptosis and slower tumor growth in vivo. Our novel theranostic platform could be used as an effective strategy to monitor exosomes in vivo and deliver therapeutics to hypoxic tumors.

Toward redesigning the PEG surface of nanocarriers for tumor targeting: impact of inner functionalities on size, charge, multivalent binding, and biodistribution.

Achieving accurate and efficacious tumor targeting with minimal off-target effects is of paramount importance in designing diagnostic and therapeutic agents for cancer. In this respect, nanocarriers have gained enormous popularity because of their attainable multifunctional features, as well as tumor-targeting potential by extravasation. However, once administered into the bloodstream, nanocarriers face various in vivo obstacles that may significantly impair their performance needed for clinical translation. Herein, we demonstrate a strategy to enhance tumor-targeting efficiency by embedding functionalities in the interior region of partially PEGylated nanocarriers (ca. 10 nm in diameter), intended for active or passive targeting. The cooperative impact of these topologically inner functional groups (IFGs) was marked: enhancements of >100-fold in IC50in vitro (e.g., a high-avidity ligand with cationic IFGs) and >2-fold in tumor accumulation at 2 h post-injection in vivo (e.g., a high-avidity ligand with anionic IFGs), both against the fully PEGylated counterpart. Analogous to allosteric modulators, properly employed IFGs may substantially improve the process of effectively directing nanocarriers to tumors, which is otherwise solely dependent on avidity or extravasation.

Simultaneous Detection of EGFR and VEGF in Colorectal Cancer using Fluorescence-Raman Endoscopy.

Fluorescence endomicroscopy provides quick access to molecular targets, while Raman spectroscopy allows the detection of multiple molecular targets. Using a simultaneous fluorescence-Raman endoscopic system (FRES), we herein demonstrate its potential in cancer diagnosis in an orthotopically induced colorectal cancer (CRC) xenograft model. In the model, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were targeted with antibody-conjugated fluorescence and surface-enhanced Raman scattering (F-SERS) dots. FRES demonstrated fast signal detection and multiplex targeting ability using fluorescence and Raman signals to detect the F-SERS dots. In addition, FRES showed a multiplex targeting ability even on a subcentimeter-sized CRC after spraying with a dose of 50 µg F-SERS dots. In conclusion, molecular characteristics of tumor cells (EGFR in cancer cell membranes) and tumor microenvironments (VEGF in the extracellular matrix) could be simultaneously investigated when performing a colonoscopy.

Visualization of exosome-mediated miR-210 transfer from hypoxic tumor cells.

Cancer cells actively release exosomes carrying specific cellular components, such as proteins, mRNA, and miRNA, to communicate with various cells in the tumor microenvironment. We visualized exosome-mediated transfer of miR-210 from hypoxic breast cancer cells to neighboring cells using a miR-210 specific reporter system. By in vitro and in vivo visualization, we found that exosomes with miR-210 were transferred to cells in the tumor microenvironment and that miR-210 was involved in expression of vascular remodeling related genes, such as Ephrin A3 and PTP1B, to promote angiogenesis. These results indicate that cellular components, such as miRNAs from hypoxic cancer cells, spread to adjacent cancer cells in the tumor microenvironment via exosomes and influence tumor progression.

A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM.

Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and (64)Cu. HT29 (hTERT+) and U2OS (hTERT-) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo.