Methods for dynamic and whole volume imaging of the zebrafish heart.

Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration.

Rehydration of Freeze Substituted Brain Tissue for Pre-embedding Immunoelectron Microscopy.

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.

Re-Evaluations of Zr-DFO Complex Coordination Chemistry for the Estimation of Radiochemical Yields and Chelator-to-Antibody Ratios of 89Zr Immune-PET Tracers.

(1) Background: Deferoxamine B (DFO) is the most widely used chelator for labeling of zirconium-89 (89Zr) to monoclonal antibody (mAb). Despite the remarkable developments of the clinical 89Zr-immuno-PET, chemical species and stability constants of the Zr-DFO complexes remain controversial. The aim of this study was to re-evaluate their stability constants by identifying species of Zr-DFO complexes and demonstrate that the stability constants can estimate radiochemical yield (RCY) and chelator-to-antibody ratio (CAR). (2) Methods: Zr-DFO species were determined by UV and ESI-MS spectroscopy. Stability constants and speciation of the Zr-DFO complex were redetermined by potentiometric titration. Complexation inhibition of Zr-DFO by residual impurities was investigated by competition titration. (3) Results: Unknown species, ZrHqDFO2, were successfully detected by nano-ESI-Q-MS analysis. We revealed that a dominant specie under radiolabeling condition (pH 7) was ZrHDFO, and its stability constant (logß111) was 49.1 ± 0.3. Competition titration revealed that residual oxalate inhibits Zr-DFO complex formation. RCYs in different oxalate concentration (0.1 and 0.04 mol/L) were estimated to be 86% and >99%, which was in good agreement with reported results (87%, 97%). (4) Conclusion: This study succeeded in obtaining accurate stability constants of Zr-DFO complexes and estimating RCY and CAR from accurate stability constants established in this study.

Novel Time-Resolved Fluorescence Immunochromatography Paper-Based Sensor with Signal Amplification Strategy for Detection of Deoxynivalenol.

Immunoassay has the advantages of high sensitivity, high specificity, and simple operation, and has been widely used in the detection of mycotoxins. For several years, time-resolved fluorescence immunochromatography (TRFIA) paper-based sensors have attracted much attention as a simple and low-cost field detection technology. However, a traditional TRFIA paper-based sensor is based on antibody labeling, which cannot easily meet the current detection requirements. A second antibody labeling method was used to amplify the fluorescence signal and improve the detection sensitivity. Polystyrene fluorescent microspheres were combined with sheep anti-mouse IgG to prepare fluorescent probes (Eu-IgGs). After the probe fully reacted with the antibody (Eu-IgGs-Abs) in the sample cell, it was deployed on the paper-based sensor using chromatography. Eu-IgGs-Abs that were not bound to the target were captured on the T-line, while those that were bound were captured on the C-line. The paper-based sensor reflected the corresponding fluorescence intensity change. Because a single molecule of the deoxynivalenol antibody could bind to multiple Eu-IgGs, this method could amplify the fluorescence signal intensity on the unit antibody and improve the detection sensitivity. The working standard curve of the sensor was established under the optimum working conditions. It showed the lower limit of detection and higher recovery rate when it was applied to actual samples and compared with other methods. This sensor has the advantages of high sensitivity, good accuracy, and good specificity, saving the amount of antibody consumed and being suitable for rapid field detection of deoxynivalenol.

Molecular endoscopic imaging in cancer.

Cancer is one of the major causes of death in both the USA and Europe. Molecular imaging is a novel field that is revolutionizing cancer management. It is based on the molecular signature of cells in order to study the human body both in normal and diseased conditions. The emergence of molecular imaging has been driven by the difficulties associated with cancer detection, particularly early-stage premalignant lesions which are often unnoticed as a result of minimal or no structural changes. Endoscopic surveillance is the standard method for early-stage cancer detection. In addition to recent major advancements in endoscopic instruments, significant progress has been achieved in the exploration of highly specific molecular probes and the combination of both will permit significant improvement of patient care. In this review, we provide an outline of the current status of endoscopic imaging and focus on recent applications of molecular imaging in gastrointestinal, hepatic and other cancers in the context of detection, targeted therapy and personalized medicine. As new imaging agents have the potential to broadly expand our cancer diagnostic capability, we will also present an overview of the main types of optical molecular probes with their pros and cons. We conclude by discussing the challenges and future prospects of the field.

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii.

Cronobacter sakazakii is a foodborne pathogen that can cause a rare, septicemia, life-threatening meningitis, and necrotizing enterocolitis in infants. In general, standard methods for pathogen detection rely on culture, plating, colony counting and polymerase chain reaction DNA-sequencing for identification, which are time, equipment and skill demanding. Recently, nanoparticle- and surface-based immunoassays have increasingly been explored for pathogen detection. We investigate the functionalization of gold nanoparticles optimized for irreversible and specific binding to C. sakazakii and their use for spectroscopic detection of the pathogen. We demonstrate how 40-nm gold nanoparticles grafted with a poly(ethylene glycol) brush and functionalized with polyclonal antibodies raised against C. sakazakii can be used to specifically target C. sakazakii. The strong extinction peak of the Au nanoparticle plasmon polariton resonance in the optical range is used as a label for detection of the pathogens. Individual binding of the nanoparticles to the C. sakazakii surface is also verified by transmission electron microscopy. We show that a high degree of surface functionalization with anti-C. sakazakii optimizes the detection and leads to a detection limit as low as 10 CFU/mL within 2 h using a simple cuvette-based UV-Vis spectrometric readout that has great potential for further optimization.

Development of a rapid and ultrasensitive magnetic chemiluminescence immunoassay for the detection of adiponectin and its clinical application.

Adiponectin (ADPN), which serum/plasma adiponectin levels are closely associated with insulin resistance and type 2 diabetes, and lower adiponectin levels predict an increased risk of diabetes, is a strong indicator of diabetes risk in people at high risk of diabetes in different races. Using the unique principle and performance advantages of chemiluminescence immunoassay (CLIA), an ADPN-CLIA method with high sensitivity, high specificity and wide detection range was established based on the principle of two-steps method of sandwich-type, with the magnetic particles (MPs) as the solid phase carrier and acridinium ester (AE) as the chemiluminescence reaction system. The selection of the main raw materials required, the preparation conditions of MPs-coated antibodies, the methods of AE-labeled antibodies, sample requirements and reaction modes were optimized and evaluated. AE labeling experiment was successfully performed with the labeling efficiency of 8.366 and the antibody utilization rate of 96.8%. The chemiluminescent immunoassay for ADPN had a good linear relationship from 0 ng/mL to 250 ng/mL (R2 =0.9993), with the detection limit of 0.05 ng/mL. The coefficient of variation (CV) of intra-assay and inter-assay precision were both less than 5% respectively. The recovery rates for accuracy were from 91.26% to 107.46%. The comparison experiment of 80 clinical serum samples between the developed ADPN-CLIA with the immunoturbidimetry showed that the correlation coefficient was 0.956, and the Bland-Altman analysis showed that the limits of agreement were - 0.364 and 0.433.

Conjugation of Fluorochromes to Monoclonal Antibodies.

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is the most widely used application of flow cytometry. Here, we present protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, we provide a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Labeling an antibody with fluorescein isothiocyanate (FITC) Basic Protocol 2: Labeling an antibody with long-armed biotin Basic Protocol 3: Labeling an antibody with Texas Red-X Basic Protocol 4: Labeling an antibody with a synthetic organic fluor kit Basic Protocol 5: Labeling an antibody with phycobiliproteins Basic Protocol 6: Conjugation of Texas Red to R-phycoerythrin to produce an energy transfer fluorochrome.

Ten-fold Robust Expansion Microscopy.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Identification of Immune Cells in Murine Olfactory Mucosa.

Olfactory immunology is an emerging field in the context of infectious disease and neuroimmunology, yet characterization of immune cells within the murine olfactory mucosa remains sparse. This is partially due to the difficulty in distinguishing olfactory-resident immune cells from immune cells that reside within nasal turbinate bone marrow. Using techniques like intranasal antibody labeling, we have developed methods to definitively identify olfactory immune cells via flow cytometry and immunofluorescent confocal microscopy. This protocol will describe the best practices for these methods, as well as detail how intravenous antibody labeling can be used to study the blood-olfactory barrier, an important determinant of olfactory immunity. We also include validated markers for the identification of major olfactory immune populations.

Single-Molecule Localization Microscopy Using Time-Lapse Imaging of Single-Antibody Labeling.

In single-molecule localization microscopy (SMLM), immunofluorescence (IF) staining affects the quality of the reconstructed superresolution images. However, optimizing IF staining remains challenging because IF staining is a one-step, irreversible process. Sample labeling through reversible binding presents an alternative strategy, but such techniques require significant technological advancements to enhance the dissociation of labels without sacrificing their binding specificity. In this article, we introduce time-lapse imaging of single-antibody labeling. Our versatile technique utilizes commercially available dye-conjugated antibodies. The method controls the antibody concentrations to capture single-antibody labeling of subcellular targets, thereby achieving SMLM through the labeling process. We further demonstrate dual-color single-antibody labeling to enhance the sample labeling density. The new approach allows the evaluation of antibody binding at the single-antibody level and within the cellular environment. This comprehensive guide offers step-by-step instructions for time-lapse imaging of single-antibody labeling experiments and enables the application of the single-antibody labeling technique to a wide range of targets. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for single-antibody labeling Basic Protocol 2: Data acquisition for single-molecule localization microscopy Alternate Protocol: Dual-color single-antibody labeling using OptoSplit II equation Basic Protocol 3: Image analysis.

Differential in vivo labeling with barcoded antibodies allows for simultaneous transcriptomic profiling of airway, lung tissue and intravascular immune cells.

Single-cell RNA sequencing (scRNA-seq) is the state-of-the-art approach to study transcriptomic signatures in individual cells in respiratory health and disease. However, classical scRNA-seq approaches provide no spatial information and are performed using either bronchoalveolar lavage fluid (BAL) or lung single cell suspensions to assess transcript levels in airway and tissue immune cells, respectively. Herein we describe a simple method to simultaneously characterize transcriptomic features of airway, lung parenchymal and intravascular immune cells based on differential in vivo labeling with barcoded antibodies. In addition to gaining basic spatial information, this approach allows for direct comparison of cells within different anatomical compartments. Furthermore, this method provides a time- and cost-effective alternative to classical scRNA-seq where lung and BAL samples are processed individually, reducing animal and reagent use. We demonstrate the feasibility of this approach in a preclinical mouse model of bacterial lung infection comparing airway, parenchymal and vasculature neutrophils early after infection.

Protocol for Measuring Concentrations of Extracellular Vesicles in Human Blood Plasma with Flow Cytometry.

Extracellular vesicles (EVs) are lipid membrane enclosed particles that are released from cells into body fluids, such as blood. EVs offer potential new biomarkers of diseases, because the cellular origin, composition, concentration, and function of EVs change in health and disease. The concentration of EVs from specific cell types in blood can be determined with flow cytometry. A flow cytometer measures fluorescence and light scattering signals from single EVs, but only if these signals are sufficiently bright to be detected. Measured concentrations of EVs are therefore only reproducible and comparable if the detection ranges are known and reported in standard units, such as molecules of equivalent soluble fluorophore (MESF) for fluorescence signals and the diameter in nm for scatter signals. The goal of this protocol is to discuss all steps needed to derive the concentration of cell-type specific EVs within a known diameter range and fluorescence range. More specifically, this protocol describes how to determine the concentration of CD61+ (Integrin beta-3, platelet marker), CD235a+ (Glycophorin A, erythrocyte marker), and CD45+ (leukocyte common antigen) EVs in human blood plasma with an Apogee A60-Micro flow cytometer using scatter-based triggering. The principles behind this protocol could lay a firm basis for the design of a protocol suitable for other flow cytometers and body fluids.

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion.

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

Development of a novel chemiluminescence immunoassay for the detection of procalcitonin.

OBJECTIVE: To evaluate the analytical performance of our previously developed chemiluminescence immunoassay (CLIA) kit for the detection of procalcitonin (PCT) and compare with the results obtained using the Vidas B.R.A.H.M.S. PCT™ test (PCT-V). DESIGN AND METHODS: Our laboratory previously designed a novel CLIA kit and supporting instrument (AE-180) for the detection of PCT. We analyzed the clinical performance of this system, including the imprecision, limit of detection, and linearity of analyses of 305 serum specimens. The results were compared with measurements of the same serum samples obtained with PCT-V. RESULTS: The limit of detection and blank of our kit were 0.0075 and 0.0039 ng/mL, respectively. The intra- and inter-assay coefficient of variation of the kit were both between 0.8% and 3.9%. The equation of linearity was found to be y = 1.03× + 0.06 (r = 0.99) for concentrations in the range of 0.01-110 ng/mL. The correlation coefficient with the results of PCT-V was 0.995, and the equation obtained for Passing and Bablok regression analysis was 1.061 for our CLIA PCT kit and - 0.003 for PCT-V. Our kit slightly overestimated the concentration according to comparison with PCT-V results. CONCLUSION: The kit that was previously developed in our laboratory for the measurement of serum PCT concentration using CLIA technology shows excellent performance, just that the functional sensitivity is not as good as the PCT-V; therefore, we suggest that this kit is suitable for clinical use.

A Nut-and-Bolt Microfluidic Mixing System for the Rapid Labeling of Immune Cells with Antibodies.

A nut-and-bolt microfluidic system was previously developed for a point-of-care (POC) human immunodeficiency virus (HIV) test and was able to acquire images of CD4 (cluster of differentiation 4) + T-lymphocytes in a sample drop of blood followed by image analysis. However, as the system was not fully integrated with a sample reaction module, the mixing of the sample with the antibody reagent was carried out manually. To achieve a rapid reaction with a reduced amount of costly reagent in a POC diagnostic system, an efficient sample mixing function must be implemented. Here, we propose a novel method to drastically accelerate the process of sample mixing and increase the reaction rate in the nut-and-bolt microfluidic system, where the sample is mixed with the reagent in a reaction chamber formed by connecting a nut with a bolt-like sample cartridge. The mixing is facilitated by rotating the sample cartridge bidirectionally using a DC motor, which agitates the sample in a chaotic manner. A microbead complex formed by the avidin-biotin interaction was used as a model reaction system to examine the feasibility of our mixing module. We found that the reaction time for the avidin-biotin binding by mixing was 7.5 times shorter than in the incubation method, achieving a reaction efficiency of over 95%. The performance of our mixing system was further demonstrated by measuring the concentration of CD4 cells labeled with a fluorescent antibody in the blood sample. The antigen-antibody reaction mixing was faster by a factor of 20, reaching a reaction efficiency comparable to the conventional incubation method.

Fast Detection of 2,4,6-Trinitrotoluene (TNT) at ppt Level by a Laser-Induced Immunofluorometric Biosensor.

The illegal use of explosives by terrorists and other criminals is an increasing issue in public spaces, such as airports, railway stations, highways, sports venues, theaters, and other large buildings. Security in these environments can be achieved by different means, including the installation of scanners and other analytical devices to detect ultra-small traces of explosives in a very short time-frame to be able to take action as early as possible to prevent the detonation of such devices. Unfortunately, an ideal explosive detection system still does not exist, which means that a compromise is needed in practice. Most detection devices lack the extreme analytical sensitivity, which is nevertheless necessary due to the low vapor pressure of nearly all explosives. In addition, the rate of false positives needs to be virtually zero, which is also very difficult to achieve. Here we present an immunosensor system based on kinetic competition, which is known to be very fast and may even overcome affinity limitation, which impairs the performance of many traditional competitive assays. This immunosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. In the case of the explosive 2,4,6-trinitrotoluene (TNT), some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and chip-based mixing-devices and flow-cells. The system achieved limits of detection of 1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 min. A cross-reactivity test with 5000 pM solutions showed no signal by pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). This immunosensor belongs to the most sensitive and fastest detectors for TNT with no significant cross-reactivity by non-related compounds. The consumption of the labeled antibody is surprisingly low: 1 mg of the reagent would be sufficient for more than one year of continuous biosensor operation.

Site-Specific Photocrosslinking to Immunoglobulin G Using Photoreactive Antibody-Binding Domains.

The high specificity and strong binding affinity of antibodies, most commonly immunoglobulin G (IgG), have led to their use in a wide range of research, diagnostic and therapeutic applications. Many of these applications require the antibody to be labeled with additional chemical or biological moieties. Here, we describe a method for the rapid and site-specific labeling of nearly any "off-the-shelf" IgG. Our method utilizes small photoreactive antibody-binding domains (pAbBDs) that are produced by modifying the IgG-binding domains of Protein A and Protein G with the unnatural amino acid benzoylphenylalanine (BPA). The pAbBDs are covalently linked to IgG heavy chains upon exposure to ultraviolet light. Fusion of pAbBDs to a given protein of interest or conjugation of pAbBDs with drugs, fluorophores, and/or other chemical moieties, enables the facile production of a diverse range of antibody conjugates.

Fully Automated 89Zr Labeling and Purification of Antibodies.

Dozens of monoclonal antibodies (mAbs) have been approved for clinical use, and hundreds more are under development. To support these developments and facilitate a personalized medicine approach, PET imaging and quantification of mAbs, after chelation with desferrioxamine B (DFO) and radiolabeling with 89Zr, has become attractive. Also, the use of 89Zr-mAbs in preclinical and clinical studies is expanding rapidly. Despite these rapid developments, 89Zr radiolabeling is still performed manually. Therefore, we aimed to develop a simple, fully automated, good-manufacturing-practice (GMP)-compliant production procedure for the 89Zr labeling of mAbs. Such procedures should increase the robustness and capacity of 89Zr-mAb production while minimizing the radiation dose to the operator. Here, the procedures for fully automated 89Zr-mAb production are described and applied to produce batches of 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab suitable for clinical use. Both products had to meet the GMP-compliant quality standards with respect to yield, radiochemical purity, protein integrity, antigen binding, sterility, and endotoxin levels. Methods: Automated 89Zr labeling of mAbs was developed on a Scintomics GRP 2V module and comprised the following steps: reagent transfer to the 89Zr-containing reaction vial, mixing of the reagents followed by a 60-min reaction at room temperature to obtain optimal radiolabeling yields, and product purification using a PD-10 desalting column. Results: Radiochemical yields of 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab were all more than 90% according to instant thin-layer chromatography. Isolated yields were 74.6% ± 2.0% and 62.6% ± 3.0% for 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab, respectively, which are similar to isolated yields obtained using GMP protocols for manual 89Zr labeling of mAbs. To meet the GMP-compliant quality standards, only the radiochemically pure fractions were collected from PD-10, resulting in a lower isolated yield than the radiochemical yield according to instant thin-layer chromatography. The radiochemical purity and protein integrity were more than 95% for both products, and the antigen binding was 95.6% ± 0.6% and 87.1% ± 2.2% for 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab, respectively. The products were sterile, and the endotoxin levels were within acceptable limits, allowing future clinical production using this procedure. Conclusion: Procedures for fully automated GMP-compliant production of 89Zr-mAbs were developed on a commercially available synthesis module, which also allows the GMP production of other radiolabeled mAbs.

Molecular endoscopic imaging: the future is bright.

The prediction and final survival rate of gastrointestinal cancers are dependent on the stage of disease. The ideal would be to detect those gastrointestinal lesions at early stage or even premalignant forms which are difficult to detect by conventional endoscopy with white light optical imaging as they show minimum or no changes in morphological characteristics and are thus left untreated. The introduction of molecular imaging has greatly changed the pattern for detecting gastrointestinal lesions from purely macroscopic structural imaging to the molecular level. It allows microscopic examination of the gastrointestinal mucosa with endoscopy after the topical or systemic application of molecular probes. In recent years, major advancements in endoscopic instruments and specific molecular probes have been achieved. This review focuses on the current status of endoscopic imaging and highlights the application of molecular imaging in gastrointestinal and hepatic disease in the context of diagnosis and therapy based on recently published literature in this field. We also discuss the challenges of molecular endoscopic imaging, its future directions and potential that could have a tremendous impact on endoscopic research and clinical practice in future.