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Background This prospective cohort study aimed to assess sentinel lymph node (SLN) mapping using isosulfan blue (ISB) compared with ISB plus indocyanine green (ICG) and near-infrared imaging (NIR) for patients with endometrial cancer. Methods In this study, 200 patients with endometrial cancer underwent SLN assessments and were randomized to ISB + ICG ( n  = 180) or ISB alone ( n  = 20). Blue dye determinations were recorded for all 200 cases followed by NIR imaging of ICG for 180 randomized subjects. All the patients underwent robotically assisted hysterectomy with pelvic ± aortic lymphadenectomy. Results The mean age of the patients was 64.5 ± 8.4 years, and the mean body mass index (BMI) was 33 ± 7.6 kg/m 2 . The histologies were endometrioid G1 (43%), G2 (30%), G3 (7%), and type 2 (20%). The mean time from dye injection to initiation of mapping was 13.4 ± 6.2 min, and the time to removal of SLN was 17.4 ± 11.2 min. Detection of SLN for the 20 ISB control cases did not differ from that for the 180 ISB + ICG cases ( p  > 0.05). The rates of SLN detection for ISB + ICG/NIR ( n  = 180) versus ISB ( n  = 200) were as follows: bilateral (83.9 vs. 40%), unilateral (12.2 vs. 36%), and none (3.9 vs. 24%) ( p  < 0.001). The median SLN per case was 2 (range 0–4). Positive SLNs were found in 21.1% ( n  = 38) of the ISB + ICG cases compared with 13.5% ( n  = 27) of the ISB cases ( p  = 0.056). The false-negative rate for SLN biopsy was 2.5% (95% confidence interval, 0.1–14.7%). In 61% (25/41) of the node-positive cases, SLN was the only positive lymph node (LN). Isolated tumor cells were found in 39.5% (15/38) of the SLN metastasis cases compared with 26.7% (4/15) of the non-SLN metastasis cases ( p  = 0.528). Conclusions In this prospective study, ISB + ICG and NIR detected more SLNs and more LN metastases than ISB alone. Assessment of SLN with ICG + ISB/NIR imaging had excellent sensitivity for detection of metastasis and no safety issues.

Theranostic principles utilize a molecular biomarker specific for a tumor target, initially for imaging to assess target expression and, if deemed suitable, for targeted therapy. This presents an exciting opportunity for a highly personalized treatment strategy in the era of precision medicine. Prostate-specific membrane antigen (PSMA) theranostics has attracted increasing attention as a promising targeted treatment in metastatic prostate cancer (PC). 177Lu-DOTA-PSMA-617 (177Lu-PSMA-617) is a PSMA-targeted small molecule with favorable properties and is the most extensively investigated PSMA radioligand for radionuclide therapy (RNT) in PC. Since 2014 multiple retrospective studies and more recently a phase II prospective study demonstrated safety and impressive efficacy of 177Lu-PSMA RNT. The evidence generated by these trials led to two currently underway randomized trials in metastatic castrate-resistant PC: TheraP (NCT03392428) and VISION (NCT03511664). While we wait for these pivotal trials to read out, nuclear medicine physicians, medical oncologists, radiation oncologists, and urologists are facing a steep learning curve to master the intricacies and nuances of this novel therapeutic strategy. This review article aims to share and discuss the evolving experience in practical aspects of PSMA theranostics.

We report development of the first genetically encoded bioluminescent indicator for membrane voltage called LOTUS-V. Since it is bioluminescent, imaging LOTUS-V does not require external light illumination. This allows bidirectional optogenetic control of cellular activity triggered by Channelrhodopsin2 and Halorhodopsin during voltage imaging. The other advantage of LOTUS-V is the robustness of a signal-to-background ratio (SBR) wherever it expressed, even in the specimens where autofluorescence from environment severely interferes fluorescence imaging. Through imaging of moving cardiomyocyte aggregates, we demonstrated the advantages of LOTUS-V in long-term imaging are attributable to the absence of phototoxicity, and photobleaching in bioluminescent imaging, combined with the ratiometric aspect of LOTUS-V design. Collectively LOTUS-V extends the scope of excitable cell control and simultaneous voltage phenotyping, which should enable applications in bioscience, medicine and pharmacology previously not possible.

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF = 0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.

In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k -means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.

Localization of subcentimeter ground glass opacities during minimally invasive thoracoscopic lung cancer resections is a significant challenge in thoracic oncology. Intraoperative molecular imaging has emerged as a potential solution, but the availability of suitable fluorescence agents is a limiting factor. To evaluate the suitability of SGM-101, a carcinoembryonic antigen-related cell adhesion molecule type 5 (CEACAM5) receptor-targeted near-infrared fluorochrome, for molecular imaging-guided lung cancer resections, because glycoprotein is expressed in more than 80% of adenocarcinomas. For this nonrandomized, proof-of-principal, phase 1 controlled trial, patients were divided into 2 groups between August 1, 2020, and January 31, 2022. Patients with known CEACAM5-positive gastrointestinal tumors suggestive of lung metastasis were selected as proof-of-principle positive controls. The investigative group included patients with lung nodules suggestive of primary lung malignant neoplasms. Patients 18 years or older without significant comorbidities that precluded surgical exploration with suspicious pulmonary nodules requiring surgical biopsy were included in the study. SGM-101 (10 mg) was infused up to 5 days before index operation, and pulmonary nodules were imaged using a near-infrared camera system with a dedicated thoracoscope. SGM-101 localization to pulmonary nodules and its correlation with CEACAM5 glycoprotein expression by the tumor as quantified by tumor and normal pulmonary parenchymal fluorescence. Ten patients (5 per group; 5 male and 5 female; median [IQR] age, 66 [58-69] years) with 14 total lesions (median [range] lesion size, 0.91 [0.90-2.00] cm) were enrolled in the study. In the control group of 4 patients (1 patient did not undergo surgical resection because of abnormal preoperative cardiac clearance findings that were not deemed related to SGM-101 infusion), the mean (SD) lesion size was 1.33 (0.48) cm, 2 patients had elevated serum CEA markers, and 2 patients had normal serum CEA levels. Of the 4 patients who underwent surgical intervention, those with 2+ and 3+ tissue CEACAM5 expression had excellent tumor fluorescence, with a mean (SD) tumor to background ratio of 3.11 (0.45). In the patient cohort, the mean (SD) lesion size was 0.68 (0.22) cm, and no elevations in serum CEA levels were found. Lack of SGM-101 fluorescence was associated with benign lesions and with lack of CEACAM5 staining. This in-human proof-of-principle nonrandomized controlled trial demonstrated SGM-101 localization to CEACAM5-positive tumors with the detection of real-time near-infrared fluorescence in situ, ex vivo, and by immunofluorescence microscopy. These findings suggest that SGM-101 is a safe, receptor-specific, and feasible intraoperative molecular imaging fluorochrome that should be further evaluated in randomized clinical trials. ClinicalTrials.gov identifier: NCT04315467.

Phosphodiesterase 10A (PDE10A) is selectively expressed in the striatal regions in the brain and may play a role in modulating dopaminergic and glutamatergic second messenger pathways. PDE10A inhibitors are expected to be useful in treating neuropsychiatric disorders such as schizophrenia and Huntington’s disease. In this study, the brain kinetics of [11C]T-773 in the human brain and test-retest reproducibility of the outcome measures were evaluated. Subsequently, the occupancy of a novel PDE10A inhibitor, TAK-063, was measured using [11C]T-773. Dynamic PET measurements were conducted three times for 12 healthy male subjects after intravenous bolus injection of [11C]T-773: two baseline PETs and one postdose PET (3hours) after oral administration of TAK-063 for four subjects, and one baseline PET and two postdose PET (3hours and 23hours) for eight subjects. Kinetic model analysis was performed with arterial input functions. PDE10A occupancy was calculated as the percent change of the binding specific to PDE10A (Vs) total distribution volume (VT), which was calculated as the VT of the putamen minus the VT of the cerebellum. Regional brain uptake was highest in the putamen. Time-activity curves of the brain regions were described with two tissue-compartment (2TC) models. The mean VT was 5.5±0.7 in the putamen and 2.3±0.5 in the cerebellum in the baseline PET. Absolute VT variability between the two baseline scans was less than 7%. Reproducibility of VT was excellent. PDE10A occupancy in the putamen ranged from 2.8% to 72.1% at 3hours after a single administration of 3 to 1000mg of TAK-063, and increased in a dose- and plasma concentration-dependent manner. At 23hours postdose, PDE10A occupancy in the putamen was 0 to 42.8% following administration of 3 to 100mg of TAK-063. In conclusion, [11C]T-773 showed good characteristics as a PET radioligand for PDE10A in the human brain.

Key Points Fluorescence nanoscopy (also known as super-resolution microscopy) methods have expanded optical imaging to reach the nanometre resolution range, typically 20–50 nm and even down to the 1 nm level. Diffraction-unlimited nanoscopy methods, which neutralize the resolution-limiting role of diffraction, separate fluorophores by transiently transferring them between (at least) two discernible states, typically an 'on' and an 'off' state of fluorescence. The counting of molecules in nanoscale settings such as within organelles is a crucially important development, along with labelling strategies to reliably pinpoint the locations and spatial proximities of all the molecules investigated in an imaging experiment. Dynamic nanoscopy and extensions of nanoscopy imaging to tissue and in vivo contexts are further frontiers. Examples taken from mitochondrial biology and neurobiology illustrate the capabilities and discovery potential of nanoscale molecule-specific imaging with focused light. Fluorescence nanoscopy enables the optical imaging of cellular components with resolutions at the nanometre scale. With the growing availability of super-resolution microscopes, nanoscopy methods are being increasingly applied. Quantitative, multicolour, live-cell nanoscopy and the corresponding labelling strategies are under continuous development. Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.

Human epidermal growth factor receptor2/Neu, which is overexpressed in about 30% of human breast cancers, transduces growth signals in large part via the Ras–Raf–MEK–ERK pathway. Nevertheless, it is a matter of controversy whether high ERK activity in breast cancer tissues correlates with better or worse prognosis, leaving the role of ERK activity in the progression of breast cancers unresolved. To address this issue, we live-imaged ERK activity in mammary tumors developed in mouse mammary tumor virus-Neu transgenic mice, which had been crossed with transgenic mice expressing a Förster resonance energy transfer biosensor for ERK. Observation of the tumor by two-photon microscopy revealed significant heterogeneity in ERK activity among the mammary tumor cells. The level of ERK activity in each cell was stable up to several hours, implying a robust mechanism that maintained the ERK activity within a limited range. By sorting the mammary tumor cells on the basis of their ERK activity, we found that ERK high cells less efficiently generated tumorspheres in vitro and tumors in vivo than did ERK low cells. In agreement with this finding, the expressions of the cancer stem cell markers CD49f, CD24 and CD61 were decreased in ERK high cells. These observations suggest that high ERK activity may suppress the self-renewal of mammary cancer stem cells.

Super-resolution microscopy has overcome a long-held resolution barrier—the diffraction limit—in light microscopy and enabled visualization of previously invisible molecular details in biological systems. Since their conception, super-resolution imaging methods have continually evolved and can now be used to image cellular structures in three dimensions, multiple colors, and living systems with nanometer-scale resolution. These methods have been applied to answer questions involving the organization, interaction, stoichiometry, and dynamics of individual molecular building blocks and their integration into functional machineries in cells and tissues. In this Review, we provide an overview of super-resolution methods, their state-of-the-art capabilities, and their constantly expanding applications to biology, with a focus on the latter. We will also describe the current technical challenges and future advances anticipated in super-resolution imaging.