Impact of signal-to-noise ratio and contrast definition on the sensitivity assessment and benchmarking of fluorescence molecular imaging systems.

Significance: Standardization of fluorescence molecular imaging (FMI) is critical for ensuring quality control in guiding surgical procedures. To accurately evaluate system performance, two metrics, the signal-to-noise ratio (SNR) and contrast, are widely employed. However, there is currently no consensus on how these metrics can be computed. Aim: We aim to examine the impact of SNR and contrast definitions on the performance assessment of FMI systems. Approach: We quantified the SNR and contrast of six near-infrared FMI systems by imaging a multi-parametric phantom. Based on approaches commonly used in the literature, we quantified seven SNRs and four contrast values considering different background regions and/or formulas. Then, we calculated benchmarking (BM) scores and respective rank values for each system. Results: We show that the performance assessment of an FMI system changes depending on the background locations and the applied quantification method. For a single system, the different metrics can vary up to ∼ 35 dB (SNR), ∼ 8.65 a . u . (contrast), and ∼ 0.67 a . u . (BM score). Conclusions: The definition of precise guidelines for FMI performance assessment is imperative to ensure successful clinical translation of the technology. Such guidelines can also enable quality control for the already clinically approved indocyanine green-based fluorescence image-guided surgery.

Implementation of PSMA PET/CT and alignment of ordering to SNMMI appropriate use criteria in a large network system.

INTRODUCTION: The Society of Nuclear Medicine and Molecular Imaging (SNMMI) provides appropriate use criteria (AUC) for prostate-specific membrane antigen positron emission tomography/computed tomography (PSMA PET/CT) which include guidance on imaging in newly diagnosed prostate cancer and in patients with biochemically recurrent (BCR) disease. This study aims to examine trends in PSMA implementation and the prevalence and outcomes of scans ordered in scenarios deemed rarely appropriate or not meeting SNMMI AUC. METHODS: We retrospectively identified patients who were diagnosed with presumptive National Comprehensive Cancer Network unfavorable intermediate, high, or very high risk prostate cancer, patients who underwent staging for BCR, and all patients staged with PSMA between July 2021 and March 2023. Positivity was validated by adherence to a predetermined reference standard. RESULTS: The frequency of PSMA use increased in initial staging from 24% to 80% and work-up of BCR from 91% to 99% over our study period. In addition, 5% (17/340) of PSMA scans ordered for initial staging did not meet AUC and 3% (15/557) of posttreatment scans were deemed rarely appropriate. Initial staging orders not meeting SNMMI AUC resulted in no positivity (0/17), while rarely appropriate posttreatment scans were falsely positive in 75% (3/4) of cases. Urologists (53%, 17/32) comprised the largest ordering specialty in rarely appropriate use. CONCLUSION: The frequency of PSMA use rose across the study period. A significant minority of patients received PSMA PET/CT in rarely appropriate scenarios yielding no positivity in initial staging and significant false positivity post-therapy. Further education of providers and electronic medical record-based interventions could help limit the rarely appropriate use of PET imaging.

The sensitivity of magnetic particle imaging and fluorine-19 magnetic resonance imaging for cell tracking.

Magnetic particle imaging (MPI) and fluorine-19 (19F) MRI produce images which allow for quantification of labeled cells. MPI is an emerging instrument for cell tracking, which is expected to have superior sensitivity compared to 19F MRI. Our objective is to assess the cellular sensitivity of MPI and 19F MRI for detection of mesenchymal stem cells (MSC) and breast cancer cells. Cells were labeled with ferucarbotran or perfluoropolyether, for imaging on a preclinical MPI system or 3 Tesla clinical MRI, respectively. Using the same imaging time, as few as 4000 MSC (76 ng iron) and 8000 breast cancer cells (74 ng iron) were reliably detected with MPI, and 256,000 MSC (9.01 × 1016 19F atoms) were detected with 19F MRI, with SNR > 5. MPI has the potential to be more sensitive than 19F MRI for cell tracking. In vivo sensitivity with MPI and 19F MRI was evaluated by imaging MSC that were administered by different routes. In vivo imaging revealed reduced sensitivity compared to ex vivo cell pellets of the same cell number. We attribute reduced MPI and 19F MRI cell detection in vivo to the effect of cell dispersion among other factors, which are described.

Adjacent Cell Marker Lateral Spillover Compensation and Reinforcement for Multiplexed Images.

Multiplex imaging technologies are now routinely capable of measuring more than 40 antibody-labeled parameters in single cells. However, lateral spillage of signals in densely packed tissues presents an obstacle to the assignment of high-dimensional spatial features to individual cells for accurate cell-type annotation. We devised a method to correct for lateral spillage of cell surface markers between adjacent cells termed REinforcement Dynamic Spillover EliminAtion (REDSEA). The use of REDSEA decreased contaminating signals from neighboring cells. It improved the recovery of marker signals across both isotopic (i.e., Multiplexed Ion Beam Imaging) and immunofluorescent (i.e., Cyclic Immunofluorescence) multiplexed images resulting in a marked improvement in cell-type classification.

Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research.

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.

Introduction to Infrared and Raman-Based Biomedical Molecular Imaging and Comparison with Other Modalities.

Molecular imaging has rapidly developed to answer the need of image contrast in medical diagnostic imaging to go beyond morphological information to include functional differences in imaged tissues at the cellular and molecular levels. Vibrational (infrared (IR) and Raman) imaging has rapidly emerged among the molecular imaging modalities available, due to its label-free combination of high spatial resolution with chemical specificity. This article presents the physical basis of vibrational spectroscopy and imaging, followed by illustration of their preclinical in vitro applications in body fluids and cells, ex vivo tissues and in vivo small animals and ending with a brief discussion of their clinical translation. After comparing the advantages and disadvantages of IR/Raman imaging with the other main modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography/single-photon emission-computed tomography (PET/SPECT), ultrasound (US) and photoacoustic imaging (PAI), the design of multimodal probes combining vibrational imaging with other modalities is discussed, illustrated by some preclinical proof-of-concept examples.

High content imaging for monitoring signalling dynamics in single cells.

All living cells are sensors of their environment: they sense signals, hormones, cytokines, and growth factors, among others. Binding of these signals to cell surface receptors initiates the transmission of messages along intracellular signalling pathways through protein-protein interactions, enzymatic modifications and conformational changes. Typically, the activation of signalling pathways are monitored in whole populations of cells, giving population average measures, often using experimental methods that destroy and homogenise the cell population. High content imaging is an automated, high-throughput fluorescence microscopy method that enables measurements of signal transduction pathways to be taken from live cells. It can be used to measure signalling dynamics, how the abundance of particular proteins of interest change over time, or to record how particular proteins move and change their localisation in response to a signal from their environment. Using this, and other single cell methods, it is becoming increasingly clear that cells are in fact very variable in their response to a given stimulus and in the quantities of cellular components they express, even in clonal (isogenic) cell lines. This review will discuss how high content imaging has contributed to our growing understanding of cellular heterogeneity. It will discuss how data generated has been combined with information theoretic approaches to quantify the amount of information transferred through noisy signalling pathways. Lastly, the relevance of heterogeneity to our understanding and treatment of disease will be considered, highlighting the importance of single cell measurements.

LIVE-PAINT allows super-resolution microscopy inside living cells using reversible peptide-protein interactions.

We present LIVE-PAINT, a new approach to super-resolution fluorescent imaging inside live cells. In LIVE-PAINT only a short peptide sequence is fused to the protein being studied, unlike conventional super-resolution methods, which rely on directly fusing the biomolecule of interest to a large fluorescent protein, organic fluorophore, or oligonucleotide. LIVE-PAINT works by observing the blinking of localized fluorescence as this peptide is reversibly bound by a protein that is fused to a fluorescent protein. We have demonstrated the effectiveness of LIVE-PAINT by imaging a number of different proteins inside live S. cerevisiae. Not only is LIVE-PAINT widely applicable, easily implemented, and the modifications minimally perturbing, but we also anticipate it will extend data acquisition times compared to those previously possible with methods that involve direct fusion to a fluorescent protein.

Post-COVID-19 New Normal for Molecular Imaging Departments: A United Kingdom Perspective.

Coronavirus disease 2019 has changed the way the world is navigated and has had a massive impact on health care. Depending on where you are in the world, the guidance on dealing with potential infected patients is varied. With the high risk of a second wave, it is important to learn from initial responses to plan for the future. With proper preparation, it is possible to minimize exposure and risk of contamination to individuals visiting molecular imaging departments. Such precautions will help departments operate at full capacity. From the widespread nature of this pandemic, a global perspective can be useful; what follows is the United Kingdom's perspective.

Effectiveness of Photodynamic Screening Using 5-Aminolevulinic Acid for the Diagnosis of Pancreatic Cancer.

BACKGROUND/AIM: We evaluated urinary levels of porphyrin metabolites, such as uroporphyrin (UP) and coproporphyrin (CP), after 5-Aminolevulinic acid (ALA) administration in patients with or without pancreatic cancer (PaC). PATIENTS AND METHODS: Sixty-seven subjects with PaC, 11 with pancreatitis, and 9 with normal pancreas (NP) were enrolled. Urine samples from all subjects were collected prior to ALA administration and at more than 4 hours after ALA administration. We measured the urinary levels of UP and CP by high-performance liquid chromatography analysis. RESULTS: The PaC group showed significantly higher UP levels compared to NP groups (104.9 nmol/g Cre vs. 53.4 nmol/g Cre, p=0.014). Moreover, PaC patients with long-term survival had significantly lower urinary levels of UP at diagnosis (98.8 nmol/gCre) than the short-term survival group (125.2 nmol/gCre) (p=0.042). CONCLUSION: The urinary levels of UP after ALA administration might serve as a promising biomarker for diagnosis and prognosis prediction of PaC.

Advances in tissue-based imaging: impact on oncology research and clinical practice.

INTRODUCTION: Tissue-based imaging has emerged as a critical tool in translational cancer research and is rapidly gaining traction within a clinical context. Significant progress has been made in the digital pathology arena, particularly in respect of brightfield and fluorescent imaging. Critically, the cellular context of molecular alterations occurring at DNA, RNA, or protein level within tumor tissue is now being more fully appreciated. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumor microenvironment, including the potential interplay between various cell types. AREAS COVERED: This review summarizes the recent developments within the field of tissue-based imaging, centering on the application of these approaches in oncology research and clinical practice. EXPERT OPINION: Significant advances have been made in digital pathology during the last 10 years. These include the use of quantitative image analysis algorithms, predictive artificial intelligence (AI) on large datasets of H&E images, and quantification of fluorescence multiplexed tissue imaging data. We believe that new methodologies that can integrate AI-derived histologic data with omic data, together with other forms of imaging data (such as radiologic image data), will enhance our ability to deliver better diagnostics and treatment decisions to the cancer patient.

Simultaneous Monitoring of Multi-Enzyme Activity and Concentration in Tumor Using a Triply Labeled Fluorescent In Vivo Imaging Probe.

The use of fluorescent imaging probes that monitor the activity of proteases that experience an increase in expression and activity in tumors is well established. These probes can be conjugated to nanoparticles of iron oxide, creating a multimodal probe serving as both a magnetic resonance imaging (MRI) agent and an indicator of local protease activity. Previous works describe probes for cathepsin D (CatD) and metalloproteinase-2 (MMP2) protease activity grafted to cross-linked iron oxide nanoparticles (CLIO). Herein, we have synthesized a triply labeled fluorescent iron oxide nanoparticle molecular imaging (MI) probe, including an AF750 substrate concentration reporter along with probes for cathepsin B (CatB) sand MMP2 protease activity. The reporter provides a baseline signal from which to compare the activity of the two proteases. The activity of the MI probe was verified through incubation with the proteases and tested in vitro using the human HT29 tumor cell line and in vivo using female nude mice injected with HT29 cells. We found the MI probe had the appropriate specificity to the activity of their respective proteases, and the reporter dye did not activate when incubated in the presence of only MMP2 and CatB. Probe fluorescent activity was confirmed in vitro, and reporter signal activation was also noted. The fluorescent activity was also visible in vivo, with injected HT29 cells exhibiting fluorescence, distinguishing them from the rest of the animal. The reporter signal was also observable in vivo, which allowed the signal intensities of the protease probes to be corrected; this is a unique feature of this MI probe design.

A Practical Guide to Live-Cell Imaging of Meiosis in Arabidopsis.

Plants are powerful model systems to study meiosis. Our knowledge about the cytology of plant meiosis is mainly based on the analysis of fixed material. Although highly informative, this approach is limited in understanding the dynamics of meiosis. Here, we present a step-by-step instruction for a newly developed method to follow meiosis in male meiocytes of Arabidopsis in real time by confocal laser scanning microscopy. We envision that this method can be easily translated to other plant species and especially crops (e.g., Brassica, maize, and potato).

Multi-Parametric Standardization of Fluorescence Imaging Systems Based on a Composite Phantom.

OBJECTIVE: Fluorescence molecular imaging (FMI) has emerged as a promising tool for surgical guidance in oncology, with one of the few remaining challenges being the ability to offer quality control and data referencing. This paper investigates the use of a novel composite phantom to correct and benchmark FMI systems. METHODS: This paper extends on previous work by describing a phantom design that can provide a more complete assessment of FMI systems through quantification of dynamic range and determination of spatial illumination patterns for both reflectance and fluorescence imaging. Various performance metrics are combined into a robust and descriptive "system benchmarking score," enabling not only the comprehensive comparison of different systems, but also for the first time, correction of the acquired data. RESULTS: We show that systems developed for targeted fluorescence imaging can achieve benchmarking scores of up to 70%, while clinically available systems optimized for indocyanine green are limited to 50%, mostly due to greater leakage of ambient and excitation illumination and lower resolution. The image uniformity can also be approximated and employed for image flat-fielding, an important milestone toward data referencing. In addition, we demonstrate composite phantom use in assessing the performance of a surgical microscope and of a raster-scan imaging system. CONCLUSION: Our results suggest that the new phantom has the potential to support high-fidelity FMI through benchmarking and image correction. SIGNIFICANCE: Standardization of the FMI is a necessary process for establishing good imaging practices in clinical environments and for enabling high-fidelity imaging across patients and multi-center imaging studies.

ASNC/AHA/ASE/EANM/HFSA/ISA/SCMR/SNMMI Expert Consensus Recommendations for Multimodality Imaging in Cardiac Amyloidosis: Part 2 of 2-Diagnostic Criteria and Appropriate Utilization.

Cardiac amyloidosis is emerging as an underdiagnosed cause of heart failure and mortality. Growing literature suggests that a noninvasive diagnosis of cardiac amyloidosis is now feasible. However, the diagnostic criteria and utilization of imaging in cardiac amyloidosis are not standardized. In this paper, Part 2 of a series, a panel of international experts from multiple societies define the diagnostic criteria for cardiac amyloidosis and appropriate utilization of echocardiography, cardiovascular magnetic resonance imaging, and radionuclide imaging in the evaluation of patients with known or suspected cardiac amyloidosis.

An Easily Available Ratiometric Reaction-Based AIE Probe for Carbon Monoxide Light-up Imaging.

Carbon monoxide (CO) is a significant gasotransmitter that naturally modulates inflammatory responses. Visualization of CO in situ would help to reveal its physiological/pathological functions. Unfortunately, most existing CO fluorescent probes show aggregation-caused quenching (ACQ) properties. Herein, we report the reaction-based fluorescent probe (BTCV-CO) with aggregation-induced emission (AIE) characteristics for CO detection and imaging. This ratiometric AIE probe showed excellent stability, high sensitivity (detection limit of 30.8 nM), and superior selectivity. More importantly, this CO-responsive AIE probe could be facilely designed and easily obtained by two-step synthesis with high yield, providing an easy-to-handle AIE toolbox for real-time visualization of CO in a living system.