Atomically precise Fluorescent Gold Nanocluster as a barrier-permeable and brain-specific imaging probe.

Photonic nanomaterials play a crucial role in facilitating the necessary signal for optical brain imaging, presenting a promising avenue for early diagnosis of brain-related disorders. However, the blood-brain barrier (BBB) presents a significant challenge, blocking the entry of most molecules or materials from the bloodstream into the brain. To overcome this, photonic nanocrystals in the form of gold clusters (LAuC) with size less than 3nm, have been developed, with Levodopa conjugated to LAuC (Dop@LAuC) for targeted brain imaging. Dop@LAuC crosses the BBB and emits in the near-infrared (NIR) wavelength, enabling real-time optical brain imaging. An in vitro BBB model using brain endothelial cells showed that 50% of Dop@LAuC crossed the barrier within 3 hours, compared to only 10% of LAuC, highlighting the enhanced ability of L-dopa-conjugated gold clusters to penetrate the BBB. In vivo optical imaging in healthy mice further confirmed the material's efficacy to cross BBB without compromising the barrier integrity.

Rapid and specific detection of nanoparticles and viruses one at a time using microfluidic laminar flow and confocal fluorescence microscopy.

Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we introduce an approach for rapid and specific detection of single nanoparticles using a confocal-based flow virometer. The combination of laminar flow in a microfluidic channel and correlated fluorescence signals emerging from both free dyes and fluorescently labeled primary antibodies provide insights into nanoparticle volumes and specificities. We evaluate and validate the assay using fluorescent beads and viruses, including SARS-CoV-2 with fluorescently labeled primary antibodies. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting viruses at relevant loads. Based on our results, we envision the future use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup.

Optimized intravital three-photon imaging of intact mouse tibia links plasma cell motility to functional states.

Intravital deep bone marrow imaging is crucial to studying cellular dynamics and functions but remains challenging, and minimally invasive methods are needed. We employed a high pulse-energy 1650 nm laser to perform three-photon microscopy in vivo, reaching ≈400 µm depth in intact mouse tibia. Repetition rates of 3 and 4 MHz allowed us to analyze motility patterns of fast and rare cells within unperturbed marrow and to identify a bi-modal migratory behavior for plasma cells. Third harmonic generation (THG) was identified as a label-free marker for cellular organelles, particularly endoplasmic reticulum, indicating protein synthesis capacity. We found a strong THG signal, suggesting high antibody secretion, in one-third of plasma cells while the rest showed low signals. We discovered an inverse relationship between migratory behavior and THG signal, linking motility to functional plasma cell states. This method may enhance our understanding of marrow microenvironment effects on cellular functions.

What's behind Margin Status in Oral Cancer?

In the 2nd century AD, Galen argued that the failure to remove any single 'root' of a malignant tumor could result in a local relapse. After nearly 2 millennia, this problem appears to be even more challenging due to our increased understanding of the complexity of tumor formation and spread. Pathological analysis of tumor margins under a microscope remains the primary and only accepted method for confirming the complete tumor removal. However, this method is not an all-or-nothing test, and it can be compromised by various intrinsic and extrinsic limitations. Among the intrinsic limitations of pathological analysis we recall the pathologist handling, tissue shrinkage, the detection of minimal residual disease and the persistence of a precancerous field. Extrinsic limitations relate to surgical tools and their thermal damage, the different kinds of surgical resections and frozen sections collection. Surgeons, as well as oncologists and radiotherapists, should be well aware of and deeply understand these limitations to avoid misinterpretation of margin status, which can have serious consequences. Meanwhile, new technologies such as Narrow band imaging have shown promising results in assisting with the achievement of clear superficial resection margins. More recently, emerging techniques like Raman spectroscopy and near-infrared fluorescence have shown potential as real-time guides for surgical resection. The aim of this narrative review is to provide valuable insights into the complex process of margin analysis and underscore the importance of interdisciplinary collaboration between pathologists, surgeons, oncologists, and radiotherapists to optimize patient outcomes in oral cancer surgery.

A novel HER2-specific sensor based on DARPin_9-29 and albumin binding domain for real-time fluorescence-guided tumor detection in animal model of cancer.

In animal models of cancer, targeted fluorescence bioimaging, performed non-invasively and in real time, is indispensable tool for assessing tumor location, spread of metastasis, and the therapeutic potential of anticancer drugs under development. To overcome the limitation of antibodies in bioimaging applications, small artificial scaffold proteins based on ankyrin repeats (DARPins, designed ankyrin repeat proteins) are used as tumor-associated antigen binders. In this study for the first time, we assessed the potential of DARPin_9-29, the human epidermal growth factor receptor 2 (HER2) subdomain I-specific protein, genetically fused with albumin binding domain (ABD) and conjugated with Cyanine5.5 as a NIR sensor for fluorescence bioimaging of HER2-positive cancer in animal model. In vivo biodistribution studies have revealed sufficient tumor-to-background ratios at 48 h (3.17 ± 0.55) and 72 h (3.49 ± 0.64) postinjection, providing excellent contrast between the primary tumor and tissue background and allowing clear breast tumor detection. Ex vivo biodistribution has shown that ABD module in DARP-ABD sensor prevents renal reabsorption and increases tumor accumulation in more than 10-folds compared to excreting organs. To verify if DARP-ABD-Cy5.5 can demarcate HER2-positive tumor in vivo, HER2-positive syngeneic breast cancer cell line with constitutive gene expression of luciferase eFFLuc, was created. The powerful combination of bioluminescence and fluorescence imaging let to track the fluorescent anti-HER2 DARP-ABD sensor in bioluminescent HER2-positive breast tumors. Our results validate DARP-ABD as a promising sensor for fluorescence-guided imaging of HER2-positive solid cancer, which can be used in the development of improved anticancer treatment strategies.

State of the art medical devices for fluorescence-guided surgery (FGS): technical review and future developments.

BACKGROUND: Medical devices for fluorescence-guided surgery (FGS) are becoming available at a fast pace. The main challenge for surgeons lies in the lack of in-depth knowledge of optical imaging, different technical specifications and poor standardisation, and the selection of the best device based on clinical application. METHODS: This manuscript aims to provide an up-to-date description of the commercially available fluorescence imaging platforms by comparing their mode of use, required settings, image types, compatible fluorophores, regulatory approval, and cost. We obtained this information by performing a broad literature search on PubMed and by contacting medical companies directly. The data for this review were collected up to November 2023. RESULTS: Thirty-two devices made by 19 medical companies were identified. Ten systems are surgical microscopes, 5 can be used for both open and minimally invasive surgery (MIS), 6 can only be used for open surgery, and 10 only for MIS. One is a fluorescence system available for the Da Vinci robot. Nineteen devices can provide an overlay between fluorescence and white light image. All devices are compatible with Indocyanine Green, the most common fluorescence dye used intraoperatively. There is significant variability in the hardware and software of each device, which resulted in different sensitivity, fluorescence intensity, and image quality. All devices are CE-mark regulated, and 30 were FDA-approved. CONCLUSION: There is a prolific market of devices for FGS and healthcare professionals should have basic knowledge of their technical specifications to use it at best for each clinical indication. Standardisation across devices must be a priority in the field of FGS, and it will enhance external validity for future clinical trials in the field.

Mesoscale Brain Mapping: Bridging Scales and Modalities in Neuroimaging - A Symposium Review.

Advances in the spatiotemporal resolution and field-of-view of neuroimaging tools are driving mesoscale studies for translational neuroscience. On October 10, 2023, the Center for Mesoscale Mapping (CMM) at the Massachusetts General Hospital (MGH) Athinoula A. Martinos Center for Biomedical Imaging and the Massachusetts Institute of Technology (MIT) Health Sciences Technology based Neuroimaging Training Program (NTP) hosted a symposium exploring the state-of-the-art in this rapidly growing area of research. "Mesoscale Brain Mapping: Bridging Scales and Modalities in Neuroimaging" brought together researchers who use a broad range of imaging techniques to study brain structure and function at the convergence of the microscopic and macroscopic scales. The day-long event centered on areas in which the CMM has established expertise, including the development of emerging technologies and their application to clinical translational needs and basic neuroscience questions. The in-person symposium welcomed more than 150 attendees, including 57 faculty members, 61 postdoctoral fellows, 35 students, and four industry professionals, who represented institutions at the local, regional, and international levels. The symposium also served the training goals of both the CMM and the NTP. The event content, organization, and format were planned collaboratively by the faculty and trainees. Many CMM faculty presented or participated in a panel discussion, thus contributing to the dissemination of both the technologies they have developed under the auspices of the CMM and the findings they have obtained using those technologies. NTP trainees who benefited from the symposium included those who helped to organize the symposium and/or presented posters and gave "flash" oral presentations. In addition to gaining experience from presenting their work, they had opportunities throughout the day to engage in one-on-one discussions with visiting scientists and other faculty, potentially opening the door to future collaborations. The symposium presentations provided a deep exploration of the many technological advances enabling progress in structural and functional mesoscale brain imaging. Finally, students worked closely with the presenting faculty to develop this report summarizing the content of the symposium and putting it in the broader context of the current state of the field to share with the scientific community. We note that the references cited here include conference abstracts corresponding to the symposium poster presentations.

Optimized in vivo multispectral bioluminescent imaging of tumor biology using engineered BRET reporters.

The ability to visualize and track multiple biological processes in vivo in real time is highly desirable. Bioluminescence imaging (BLI) has emerged as an attractive modality for non-invasive cell tracking, with various luciferase reporters enabling parallel monitoring of several processes. However, simultaneous multiplexed imaging in vivo is challenging due to suboptimal reporter intensities and the need to image one luciferase at a time. We report a multiplexed BLI approach using a single substrate that leverages bioluminescence resonance energy transfer (BRET)-based reporters with distinct spectral profiles for triple-color BLI. These luciferase-fluorophore fusion reporters address light transmission challenges and use optimized coelenterazine substrates. Comparing BRET reporters across two substrate analogs identified a green-yellow-orange combination that allows simultaneous imaging of three distinct cell populations in vitro and in vivo. These tools provide a template for imaging other biological processes in vivo during a single BLI session using a single reporter substrate.

Trends and Future Directions in Margin Analysis for Head and Neck Cancers.

Margin status in head and neck cancer has important prognostic implications. Currently, resection is based on manual palpation and gross visualization followed by intraoperative specimen or tumor bed-based margin analysis using frozen sections. While generally effective, this protocol has several limitations including margin sampling and close and positive margin re-localization. There is a lack of evidence on the association of use of frozen section analysis with improved survival in head and neck cancer. This article reviews novel technologies in head and neck margin analysis such as 3-dimensional scanning, augmented reality, molecular margins, optical imaging, spectroscopy, and artificial intelligence.

Neurotoxin-Derived Optical Probes for Elucidating Molecular and Developmental Biology of Neurons and Synaptic Connections : Toxin-Derived Optical Probes for Neuroimaging.

Botulinum neurotoxins (BoNTs) and tetanus toxin (TeTX) are the deadliest biological substances that cause botulism and tetanus, respectively. Their astonishing potency and capacity to enter neurons and interfere with neurotransmitter release at presynaptic terminals have attracted much interest in experimental neurobiology and clinical research. Fused with reporter proteins or labelled with fluorophores, BoNTs and TeTX and their non-toxic fragments also offer remarkable opportunities to visualize cellular processes and functions in neurons and synaptic connections. This study presents the state-of-the-art optical probes derived from BoNTs and TeTX and discusses their applications in molecular and synaptic biology and neurodevelopmental research. It reviews the principles of the design and production of probes, revisits their applications with advantages and limitations and considers prospects for future improvements. The versatile characteristics of discussed probes and reporters make them an integral part of the expanding toolkit for molecular neuroimaging, promoting the discovery process in neurobiology and translational neurosciences.

Spatiotemporal resonance in mouse primary visual cortex.

Human primary visual cortex (V1) responds more strongly, or resonates, when exposed to ∼10, ∼15-20, and ∼40-50 Hz rhythmic flickering light. Full-field flicker also evokes the perception of hallucinatory geometric patterns, which mathematical models explain as standing-wave formations emerging from periodic forcing at resonant frequencies of the simulated neural network. However, empirical evidence for such flicker-induced standing waves in the visual cortex was missing. We recorded cortical responses to flicker in awake mice using high-spatial-resolution widefield imaging in combination with high-temporal-resolution glutamate-sensing fluorescent reporter (iGluSnFR). The temporal frequency tuning curves in the mouse V1 were similar to those observed in humans, showing a banded structure with multiple resonance peaks (8, 15, and 33 Hz). Spatially, all flicker frequencies evoked responses in V1 corresponding to retinotopic stimulus location, but some evoked additional peaks. These flicker-induced cortical patterns displayed standing-wave characteristics and matched linear wave equation solutions in an area restricted to the visual cortex. Taken together, the interaction of periodic traveling waves with cortical area boundaries leads to spatiotemporal activity patterns that may affect perception.

Ultrafast Response and Broad Detection Range of a Ternary Cation Perovskite Single-Crystal Thin Film Photodetector for Imaging.

FA-MA-Cs ternary cation perovskite exhibits excellent optoelectronic properties and high stabilities against humidity and light soaking and thus has aroused extensive attention in polycrystalline thin film solar cells. Compared with polycrystalline counterparts, FA-MA-Cs ternary cation perovskite single-crystal thin films (SCTFs) have lower defects, better carrier transport capacity, and stability because of lacking grain boundary defects. However, the immature growth technology of SCTFs restricts digging out its optoelectronic potential. Here, we proposed an improved space-confined method to grow large area FA0.9 MA0.05Cs0.05PbI2.7Br0.3 SCTFs using a tunable heating area to control the nucleation and growth process. Its area reaches 64 mm2 with a thickness of 26 µm. The SCTF exhibits high crystallinity, low defect density, long carrier lifetime, and high moisture resistance stability. Besides, a photosensitive chip based on a planar metal-semiconductor-metal photodetector demonstrates linear response to the three primary colors, with a photosensitive range that is 1.5 times that of protocol 3 wide color gamut. Under high-frequency light sources, the on/off ratio reaches 3.9 × 103, and the response time can be as low as 400 ns. Such ultrafast response speed and broad photosensitive range are successfully achieved for imaging applications.

Optical Transmission in Single-Layer Brain Tissues under Different Optical Source Types: Modelling and Simulation.

The human brain is a complex organ controlling daily activity. Present technique models have mostly focused on multi-layer brain tissues, which lack understanding of the propagation characteristics of various single brain tissues. To better understand the influence of different optical source types on individual brain tissues, we constructed single-layer brain models and simulated optical propagation using the Monte Carlo method. Based on the optical simulation results, sixteen optical source types had different optical energy distributions, and the distribution in cerebrospinal fluid had obvious characteristics. Five brain tissues (scalp, skull, cerebrospinal fluid, gray matter, and blood vessel) had the same set of the first three optical source types with maximum depth, while white matter had a different set of the first three optical source types with maximum depth. Each brain tissue had different optical source types with the maximum and minimum full width at half maximum. The study on single-layer brain tissues under different optical source types lays the foundation for constructing complex brain models with multiple tissue layers. It provides a theoretical reference for optimizing the selection of optical source devices for brain imaging.

Dual-mode optical projection mapping system: integration of laser speckle contrast and subcutaneous vein imaging.

Dual-mode optical imaging can simultaneously provide morphological and functional information. Furthermore, it can be integrated with projection mapping method to directly observe the images in the region of interest. This study was aimed to develop a dual-mode optical projection mapping system (DOPMS) that obtains laser speckle contrast image (LSCI) and subcutaneous vein image (SVI) and projects onto the region of interest, minimizing the spatial misalignment between the regions captured by the camera and projected by a projector. In in vitro and in vivo studies, LSCI and SVI were obtained and projected under single-mode illumination, where either the laser or light-emitting diode (LED) was activated, and under dual-mode illumination, where the laser and LED were activated simultaneously. In addition, fusion image (FI) of LSCI and SVI was implemented to selectively observe blood perfusion in the vein. DOPMS successfully obtained LSCI, SVI, and FI and projected them onto the identical region of interest, minimizing spatial misalignment. Single-mode illumination resulted in relatively clearer and noise-free images. Dual-mode illumination introduced speckle noise to SVI and FI but enabled real-time imaging by simultaneously employing LSCI, SVI, and FI. FI may be more effective for quasi-static evaluations before and after treatment under single-mode illumination and for real-time evaluation during treatment under dual-mode illumination owing to its faster image processing, albeit with a potential tradeoff in image quality.

Gold Nanoparticles for Retinal Molecular Optical Imaging.

The incorporation of gold nanoparticles (GNPs) into retinal imaging signifies a notable advancement in ophthalmology, offering improved accuracy in diagnosis and patient outcomes. This review explores the synthesis and unique properties of GNPs, highlighting their adjustable surface plasmon resonance, biocompatibility, and excellent optical absorption and scattering abilities. These features make GNPs advantageous contrast agents, enhancing the precision and quality of various imaging modalities, including photoacoustic imaging, optical coherence tomography, and fluorescence imaging. This paper analyzes the unique properties and corresponding mechanisms based on the morphological features of GNPs, highlighting the potential of GNPs in retinal disease diagnosis and management. Given the limitations currently encountered in clinical applications of GNPs, the approaches and strategies to overcome these limitations are also discussed. These findings suggest that the properties and efficacy of GNPs have innovative applications in retinal disease imaging.

Long-working-distance high-collection-efficiency three-photon microscopy for in vivo long-term imaging of zebrafish and organoids.

Zebrafish and organoids, crucial for complex biological studies, necessitate an imaging system with deep tissue penetration, sample protection from environmental interference, and ample operational space. Traditional three-photon microscopy is constrained by short-working-distance objectives and falls short. Our long-working-distance high-collection-efficiency three-photon microscopy (LH-3PM) addresses these challenges, achieving a 58% fluorescence collection efficiency at a 20 mm working distance. LH-3PM significantly outperforms existing three-photon systems equipped with the same long working distance objective, enhancing fluorescence collection and dramatically reducing phototoxicity and photobleaching. These improvements facilitate accurate capture of neuronal activity and an enhanced detection of activity spikes, which are vital for comprehensive, long-term imaging. LH-3PM's imaging of epileptic zebrafish not only showed sustained neuron activity over an hour but also highlighted increased neural synchronization and spike numbers, marking a notable shift in neural coding mechanisms. This breakthrough paves the way for new explorations of biological phenomena in small model organisms.

Volumetric Analyses of Dysmorphic Maxillofacial Structures Using 3D Surface-Based Approaches: A Scoping Review.

Background/Objectives: Three-dimensional (3D) analysis of maxillofacial structures in dysmorphic patients offers clinical advantages over 2D analysis due to its high accuracy and precision in measuring many morphological parameters. Currently, no reliable gold standard exists for calculating 3D volumetric measurements of maxillofacial structures when captured by 3D surface imaging techniques. The aim of this scoping review is to provide an overview of the scientific literature related to 3D surface imaging methods used for volumetric analysis of the dysmorphic maxillofacial structures of patients affected by CL/P or other syndromes and to provide an update on the existing protocols, methods, and, when available, reference data. Methods: A total of 17 papers selected according to strict inclusion and exclusion criteria were reviewed for the qualitative analysis out of more than 4500 articles published between 2002 and 2024 that were retrieved from the main electronic scientific databases according to the PRISMA-ScR guidelines. A qualitative synthesis of the protocols used for the selection of the anatomical areas of interest and details on the methods used for the calculation of their volume was completed. Results: The results suggest a great degree of heterogeneity between the reviewed studies in all the aspects analysed (patient population, anatomical structure, area selection, and volume calculation), which prevents any chance of direct comparison between the reported volumetric data. Conclusions: Our qualitative analysis revealed dissimilarities in the procedures specified in the studies, highlighting the need to develop uniform methods and protocols and the need for comparative studies to verify the validity of methods in order to achieve high levels of scientific evidence, homogeneity of volumetric data, and clinical consensus on the methods to use for 3D volumetric surface-based analysis.

Naturalistic movies and encoding analysis define areal borders in marmoset third-tier visual cortex.

Accurate definition of the borders of cortical visual areas is essential for the study of neuronal processes leading to perception. However, data used for definition of areal boundaries have suffered from issues related to resolution, uniform coverage, or suitability for objective analysis, leading to ambiguity. Here, we present a novel approach that combines widefield optical imaging, presentation of naturalistic movies, and encoding model analysis, to objectively define borders in the primate extrastriate cortex. We applied this method to test conflicting hypotheses about the third-tier visual cortex, where areal boundaries have remained controversial. We demonstrate pronounced tuning preferences in the third-tier areas, and an organizational structure in which the dorsomedial area (DM) contains representations of both the upper and lower contralateral quadrants, and is located immediate anterior to V2. High-density electrophysiological recordings with a Neuropixels probe confirm these findings. Our encoding-model approach offers a powerful, objective way to disambiguate areal boundaries.

Beyond primary visual cortex: The leading role of lateral occipital complex in early conscious visual processing.

The study of the neural substrates that serve conscious vision is one of the unsolved questions of cognitive neuroscience. So far, consciousness literature has endeavoured to disentangle which brain areas and in what order are involved in giving rise to visual awareness, but the problem of consciousness still remains unsolved. Availing of two different but complementary sources of data (i.e., Fast Optical Imaging and EEG), we sought to unravel the neural dynamics responsible for the emergence of a conscious visual experience. Our results revealed that conscious vision is characterized by a significant increase of activation in extra-striate visual areas, specifically in the Lateral Occipital Complex (LOC), and that, more interestingly, such activity occurred in the temporal window of the ERP component commonly thought to represent the electrophysiological signature of visual awareness, i.e., the Visual Awareness Negativity (VAN). Furthermore, Granger causality analysis, performed to further investigate the flow of activity occurring in the investigated areas, unveiled that neural processes relating to conscious perception mainly originated in LOC and subsequently spread towards visual and motor areas. In general, the results of the present study seem to advocate for an early contribution of LOC in conscious vision, thus suggesting that it could represent a reliable neural correlate of visual awareness. Conversely, striate visual areas, showing awareness-related activity only in later stages of stimulus processing, could be part of the cascade of neural events following awareness emergence.

Simulation of predicting atrial fibrosis in patients with paroxysmal atrial fibrillation during sinus node recovery time in optical imaging.

Paroxysmal atrial fibrillation is a common arrhythmia, and its development process and prediction of the degree of atrial fibrosis are of great significance for treatment and management. Optical imaging technology provides a new means for non-invasive observation of atrial electrical activity. The aim of this study is to investigate the predictive effect of sinus node recovery time on the degree of atrial fibrosis in patients with paroxysmal atrial fibrillation, and to provide a basis for the application of optical imaging technology in the study of atrial fibrosis. The study collected clinical and optical imaging data from a group of patients with paroxysmal atrial fibrillation, and used statistical analysis methods to investigate the relationship between sinus node recovery time and the degree of atrial fibrosis. The research results indicate that there is a significant correlation between the recovery time of the sinus node and the degree of atrial fibrosis, that is, there is a positive correlation between the prolonged recovery time of the sinus node and the aggravation of atrial fibrosis. SNRT can serve as an effective indicator for evaluating atrial matrix and can be applied to predict recurrence after catheter ablation of paroxysmal atrial fibrillation. Shortening SNRT through catheter ablation can become an important predictor of effective catheter ablation.