Long-term Imaging Research Articles

CT surveillance for type 1 autoimmune pancreatitis: cumulative radiation dose and diagnostic performance for disease relapse.

Long-term follow-up is essential for type 1 autoimmune pancreatitis (AIP) patients due to high relapse rates. The cumulative radiation dose from repeated CT scans during follow-up should not be ignored. We aim to investigate the cumulative radiation dose in AIP patients undergoing CT surveillance and the diagnostic performance of CT in detecting disease relapse. The diagnostic performance of MRI from a secondary cohort during the same period was also investigated. This retrospective single-institutional study included 247 type 1 AIP patients with one or more follow-up CT scans, and 120 patients with MR follow-ups. Four metrics were utilized to report the radiation dose, including the volume computed tomography dose index, the dose length product, size-specific dose estimate and effective dose. The diagnostic performance for AIP relapse was assessed, taking the final clinical diagnosis in retrospect as the reference standard. With a median 2.3-year follow-up period, AIP patients followed up with CT exhibited a median cumulative radiation dose of 37.5 mSv. 11.3% of patients have accumulated doses exceeding 100 mSv. For the 169 patients followed over a year, 30.8% sustained an average annual radiation dose surpassing 20 mSv. The sensitivity/specificity/accuracy of CT for detecting abdominal organ relapse was 64.1%/99.6%/97.0%. For AIP patients followed up with MRI, the sensitivity for detecting disease relapse was 90.5%. Considering the accumulation of radiation dose in AIP patients and the insufficient sensitivity in detecting disease relapse with CT, safer and more sensitive imaging follow-up strategies should be explored. Question CT, as the primary imaging modality for autoimmune pancreatitis (AIP) follow-up, raises concerns regarding radiation exposure and lacks reported diagnostic performance in detecting AIP relapse. Findings CT in AIP follow-up causes significant cumulative radiation exposure and exhibits insufficient sensitivity in relapse detection. Clinical relevance Type 1 AIP necessitates long-term imaging follow-up, yet current guidelines lack consensus regarding the prioritization of CT or MRI for such follow-up. CT is widely used but has radiation concerns and limited sensitivity, calling for safer, efficient strategies.

Clog P-Guided Development of Multi-Colored Buffering Fluorescent Probes for Super-Resolution Imaging of Lipid Droplet Dynamics.

Super-resolution fluorescence imaging of live cells increasingly demands fluorescent probes capable of multi-color and long-term dynamic imaging. Understanding the mechanisms of probe-target recognition is essential for the engineered development of such probes. In this study, it is discovered that the molecular lipid solubility parameter, Clog P, determines the staining performance of fluorescent dyes on lipid droplets (LDs). Fluorescent dyes with Clog P values between 2.5 and 4 can form buffering pools outside LDs, replacing photobleached dyes within LDs to maintain constant fluorescence intensity in LDs, thereby enabling dynamic super-resolution imaging of LDs. Guided by Clog P, four different colored buffering LD probes spanning the visible light spectrum have been developed. Using Structured Illumination Microscopy (SIM), the role of LD dynamics have been tracked during cellular ferroptosis with the secretion, storage, and degradation of overexpressed ACSL3 proteins. It is found that LDs serve as storage sites for these proteins through membrane fusion, and further degrade overexpressed proteins via interactions with organelles like lysosomes or through lipophagy, thereby maintaining cellular homeostasis.

Long-term clinical and imaging outcomes of patients undergoing percutaneous femoral intervention following TAVI-related vascular access complications

Abstract Introdcution: Vascular access complications (VAC) are the most common in-hospital adverse event following transcatheter aortic valve implantation (TAVI). Often, ileo-femoral VAC requires bail-out percutaneous interventions with either prolonged balloon inflation and/or stent implantation. The long-term outcomes of these procedures have not been characterized. We aim to assess the clinical and imaging outcomes of patients who underwent bailout percutaneous vascular interventions due to VAC following TAVI. Methods Single-centre prospective study including all consecutive patients with a VAC following trans-femoral TAVI, between 2015-July 2023. VAC was defined as uncontrolled bleeding, acute occlusion or flow limiting stenosis of the femoral vessel leading to percutaneous treatment. Clinical and imaging follow-up was performed with bilateral arterial ultrasound (US) Doppler to assess patency and peak systolic velocity of common femoral arteries. Results A total of 1518 patients underwent TF-TAVI, of which 107 (7%) underwent concomitant ileo-femoral percutaneous angioplasty (PTA) intervention in the same day as TAVI. In patients submitted to PTA, 7 were treated to gain access for TAVI, and 100 (7%) were performed to treat vascular access complications. At a mean follow-up time of 23±3 months, 37 patients died (37%), 6 (16%) from in-hospital complications and 31 (84%) from non-vascular causes after discharge. From the surviving 63 patients (study group) – mean age 83±6 years-old, 29% male, 46% with previous peripheral arterial disease –, 24 underwent bilateral US. Unable to perform imaging on 39 patients due to geographical or mobility issues. At a mean follow-up time of 30±23 months, only 6 patients (10%) reported intermittent claudication, of which 4 (67%) had pre-TAVI symptoms. No patient had at rest symptoms or amputation history. All but one had patent vessel on US. The mean peak systolic velocity of the treated vessel and the contralateral vessel were 71.1±22.9cm/s and 73.1±23.8cm/s, respectively, with no statistically significant difference between them in each patient (mean difference 1.9±25.4, p=0.709). We found no statistically significant differences between peak systolic velocities between both vessels in each patient, accordingly with the treated vessel (Table 2). Conclusion This prospective study shows that patients submitted to percutaneous bailout iliofemoral interventions have a very low rate of lower limb adverse events with very good patency rates at mean follow up of 2.5 years.

Miniaturized photoacoustic microscope for multi-segmental spinal cord imaging in freely moving mice.

Long-term and non-narcotic hemodynamic imaging is indispensable for observing factual physiological information of the spinal cord. Unfortunately, achieving label-free, high-resolution, and widefield spinal cord imaging for mice under freely moving conditions is challenging. In this study, we developed a miniaturized photoacoustic microscope along with a corresponding photoacoustic spinal window to realize high-resolution, multi-segmental hemodynamic imaging of the spinal cord for freely moving mice. The microscope has an outer size of 32 mm × 23 mm × 10 mm, a weight of 5.8 g, and a 4.4 µm lateral resolution within an effective field of view (FOV) of 2.6 mm × 1.8 mm. To eliminate the off-focus phenomena during spinal imaging, the microscope is equipped with a miniature motor to adapt the focal plane. Besides, the microscope is slidable along a customized rail on the window to expand the FOV. We evaluated the stability of the microscope and analyzed vascular images of the spinal cord under various physiological states. The results suggest that the microscope is capable of performing stable, multi-segmental spinal cord imaging in freely moving mice, offering new insights into spinal cord hemodynamics and neurovascular coupling research.

7242 Diffuse Idiopathic Pulmonary Neuroendocrine Cell Hyperplasia (DIPNECH) In A Patient With Recurrent Carcinoid

Abstract Disclosure: F. Woron: None. T.L. Anderson: None. P. Madhavan: None. Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is a rare generalized proliferation of pulmonary neuroendocrine cells. It is considered to be a pre-invasive lesion for pulmonary carcinoid, a rare neuroendocrine tumor (NET). DIPNECH is thought to be underdiagnosed, as patients can be asymptomatic or present with nonspecific cough and dyspnea, though it is also associated with fibrotic lung changes. Long-term annual surveillance imaging is recommended for due to the potential for development and metastasis of carcinoid tumors. We present a patient with recurrent pulmonary carcinoid later diagnosed with probable DIPNECH based on CT findings. The patient is a 72-year-old female with a prior 15 pack-year smoking history, who initially had a left lower lobe typical carcinoid tumor diagnosed and resected in 2018, with no evidence of lymph node metastasis. The patient’s medical history also includes pulmonary sarcoidosis, adrenal adenoma, thyroid nodule, hiatal hernia, and prediabetes. She has no family history of neuroendocrine tumors. Physical exam showed stable vitals with blood pressure of 112/70 mmHg in the right upper arm, heart rate of 84 bpm, and a body mass index of 39. Physical exam was unremarkable except for thyroid nodularity and obesity. Extensive biochemical evaluation for markers including chromogranin A and 24-hour urine 5-HIAA levels were within normal limits. In 2022, surveillance CT imaging showed a new 9 mm nodule in the right lobe within the bronchus, which was shown to be carcinoid on FNA. She complained of dyspnea and wheezing at this time, which has been managed with an albuterol inhaler. In 2023, PET/CT copper 64 dotatate scan showed a focal area of activity related to the nodule in the superior segment, also identified on recent CT scan, with the nodule measuring approximately 2.05 cm x 1.8 cm with a maximal SUV of 4.2. Interestingly, somatostatin receptor scintigraphy did not show any pick-up in the lung in 2018 or 2022. In 2023, the patient underwent right lower lobe superior segmentectomy after the nodule was found to have increased in size. Pathology showed G1, well-differentiated 1.1 cm typical carcinoid tumor with margins negative for invasive carcinoma. All 18 regional lymph nodes were negative for tumor. Immunohistochemical tests show that the tumor cells were positive for INSM1 and chromogranin with a low Ki-67 proliferation index (<2%), supporting the diagnosis. The patient is currently being monitored by yearly CT imaging and followed by both endocrinology and pulmonology. Given this case, we propose that DIPNECH should be considered as a differential in cases of pulmonary carcinoid, especially recurrent ones. Given that DIPNECH is thought to be underdiagnosed and a precursor for carcinoid associated with fibrotic lung changes, it is essential for this disease to be identified in order for patients to receive appropriate long-term monitoring. Presentation: 6/1/2024

8263 Long-term Imaging Follow-up of Non-functioning Pituitary Adenomas

Abstract Disclosure: N. Mehrotra: None. M.M. Ross: None. A.S. Chilukuri: None. S. Eljamri: None. C. Ewing: None. S. Patel: None. V. Patel: None. L.B. Siegel: None. D. Sistla: None. R.K. Shariff: None. P.K. Fazeli: None. Pituitary adenomas are common, affecting ∼15% of the population. Once a pituitary adenoma has been identified on imaging, an evaluation is undertaken for pituitary hormone excess and for pituitary hormone deficiency in the setting of larger adenomas (typically 1cm in size or greater). Hormone-producing or functioning pituitary adenomas are typically treated with medical or surgical therapy, but non-functioning pituitary adenomas can be followed conservatively if they are not contributing to morbidity. The natural history of non-functioning pituitary adenomas has not been well characterized and therefore although guidelines recommend follow-up pituitary imaging, there are no specific guidelines addressing how frequently or for how long these lesions should be followed. Our aim was to describe the clinical history of non-functioning pituitary adenomas using long-term clinical data from a large academic health system. We identified 791 patients who met our inclusion criteria of having more than one brain/pituitary MRI performed at least 3 months apart with a suspected non-functioning adenoma identified on the initial MRI. Each medical record was individually reviewed to confirm whether the adenoma was non-functioning based on clinical/biochemical evaluation. Patients were a median [interquartile range] of 48.3 [34.4, 63.6] years of age at the time of first MRI and 65% (n=517) were women. At presentation, 66% (n=523) of the pituitary adenomas were microadenomas (< 1 cm in size) and 34% were macroadenomas (> 1 cm). All patients had an MRI repeated at least 3 months after the initial MRI with median time of total imaging follow-up of 39.8 [17.0, 75.6] months. During follow-up, 658 patients were noted to have a stable lesion or a decrease in size of the adenoma. Patients whose lesion was noted to grow on MRI were slightly older than those who did not have growth (growth: 57.1 [41.9, 70.3] years versus no growth: 46.8 [33.7, 62.3] years, p< 0.0001). There were no differences with respect to BMI, hypopituitarism, smoking history or alcohol use between those with growth versus those without growth. Seventy-eight patients (9.9%) had surgical resection of their adenoma during follow-up. The most common indication for surgery was growth of the lesion (n=27), followed by visual changes (n=22). In the patients with a microadenoma at presentation, 90.8% remained stable in size or decreased in size during a median follow-up of 43.2 [20.5, 78.6] months. Only 2.3% of patients with a microadenoma at presentation underwent surgical resection during follow-up. In conclusion, the majority of non-functioning pituitary adenomas remain stable during prolonged imaging follow-up. Further study is needed to determine predictors of growth of non-functioning pituitary adenomas. Presentation: 6/2/2024

β-galactosidase instructed in situ self-assembly fluorogenic probe for prolonged and accurate imaging of ovarian tumor

Fluorescent probes are essential for optical imaging and have been extensively employed for precise cancer diagnosis studies. β-galactosidase (β-gal) serves as a primary biomarker for ovarian cancer and has been utilized to develop imaging probes for accurate tumor diagnosis. However, traditional small molecular probes have limitations in terms of rapid diffusion and metabolic clearance from the target lesion, resulting in a short imaging window and compromised tumor-to-background ratios (TBR). Herein, we integrated an enzyme-instructed in situ self-assembly strategy to construct Gal-IRFF, a small molecule-based activatable near-infrared (NIR) fluorogenic probe. Upon cleavage by endogenous β-gal overexpressed in ovarian cancer cells, IRFF exhibited enhanced NIR fluorescence signals and self-assembled into nanoparticles through intermolecular interactions of the Phe-Phe (FF) dipeptide moiety, which facilitated probe accumulation and retention within the tumor lesion. Compared with the small molecule probe Gal-IR, our proposed self-assembly probe Gal-IRFF demonstrated a lower limit of detection (LOD) towards β-gal and showed remarkable improvements in distribution and retention time within SKOV3 cells in vitro and tumors in vivo, thereby providing a long-term imaging window for real-time monitoring β-gal levels in ovarian tumors. Therefore, this study highlights the potential of an enzyme-instructed self-assembly fluorogenic probe design approach for achieving precise tumor diagnosis in vivo.

Long-term three-dimensional high-resolution imaging of live unlabeled small intestinal organoids via low-coherence holotomography.

Organoids, which are miniature in vitro versions of organs, possess significant potential for studying human diseases and elucidating their underlying mechanisms. Live imaging techniques play a crucial role in organoid research and contribute to elucidating the complex structure and dynamic biological phenomena of organoids. However, live, unlabeled high-resolution imaging of native organoids is challenging, primarily owing to the complexities of sample handling and optical scattering inherent in three-dimensional (3D) structures. Additionally, conventional imaging methods fail to capture the real-time dynamic processes of growing organoids. In this study, we introduce low-coherence holotomography as an advanced, label-free, quantitative imaging modality designed to overcome several technical obstacles for long-term live imaging of 3D organoids. We demonstrate the efficacy of low-coherence holotomography by capturing high-resolution morphological details and dynamic activities within mouse small intestinal organoids at subcellular resolution. Moreover, our approach facilitates the distinction between viable and nonviable organoids, significantly enhancing its utility in organoid-based research. This advancement underscores the critical role of live imaging in organoid studies, offering a more comprehensive understanding of these complex systems.

Amino-Acid-Encoded Supramolecular Nanostructures for Persistent Bioluminescence Imaging of Tumor.

Bioluminescence imaging (BLI) is a powerful technique for noninvasive monitoring of biological processes and cell transplantation. Nonetheless, the application of D-luciferin, which is widely employed as a bioluminescent probe, is restricted in long-term in vivo tracking due to its short half-life. This study presents a novel approach using amino acid-encoded building blocks to accumulate and preserve luciferin within tumor cells, through a supramolecular self-assembly strategy. The building block platform called Cys(SEt)-X-CBT (CXCBT, with X representing any amino acid) utilizes a covalent-noncovalent hybrid self-assembly mechanism to generate diverse luciferin-containing nanostructures in tumor cells after glutathione reduction. These nanostructures exhibit efficient tumor-targeted delivery as well as sequence-dependent well-designed morphologies and prolonged bioluminescence performance. Among the selected amino acids (X = Glu, Lys, Leu, Phe), Cys(SEt)-Lys-CBT (CKCBT) exhibits the superior long-lasting bioluminescence signal (up to 72h) and good biocompatibility. This study demonstrates the potential of amino-acid-encoded supramolecular self-assembly as a convenient and effective method for developing BLI probes for long-term biological tracking and disease imaging.

Development of brain metastases in patients managed with non-curative thoracic radiotherapy for stage II/III non-small cell lung cancer

BackgroundThis retrospective study analyzed the incidence of subsequent brain metastases after palliative radiotherapy or chemoradiation in patients with stage II/III non-small cell lung cancer (NSCLC). Risk factors for brain metastases development and survival after diagnosis were evaluated.MethodsDifferent baseline parameters including but not limited to age, stage and target volume size were assessed. Outcomes were abstracted from electronic health records. Uni- and multivariate tests were performed.ResultsThe study included 102 patients and found an actuarial risk of brain metastases of 15% (standard error ± 4) at one year and 20% (± 5) at two years. The maximum time interval was 15 months from start of radiation treatment. A non-significant survival difference was observed (median 12 months without versus 8.3 months with brain metastases, p = 0.21). Incidence was higher in patients with N2/3 stage, larger planning target volume size, and younger age (univariately significant factors). Trends were seen for stage III and adenocarcinoma histology. The multivariate analysis confirmed age as the most important risk factor.ConclusionThe risk of brain metastases development was comparable to that reported in studies of curative chemoradiation. All events occurred within 15 months of follow-up, suggesting that long-term surveillance imaging may not be warranted. Patients younger than 60 years had a very high risk of brain metastases development.

Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging.

Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune cell migration. Microfluidic devices are used for entrapment to support long-term imaging of multicellular organisms, including zebrafish. However, the fabrication of these devices using soft lithography requires specialized facilities and competency in 3D printing, which may not be accessible to every lab. Our adaptation of a previously developed low-cost polyethylene terephthalate lamination method for constructing microfluidic devices increases accessibility by enabling design fabrication and iteration for a fraction of the technical investment of conventional techniques. We use a device made with this method, the Rotational Assistant for Danio Imaging of Subsequent Healing (RADISH), to accommodate drug treatment, manual wounding, and long-term imaging of up to four embryos in the same field of view. With this new design, we successfully capture gross morphological characteristics of the calciumsignal around laser ablation and manual transection wounds for multiple embryos in the 2 h immediately following injury, as well as neutrophil recruitment to the wound edge for 24 h.

Craters on the melanoma surface facilitate tumor-immune interactions and demonstrate pathologic response to checkpoint blockade in humans.

Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8+ T cells in the tumor. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8+ T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response.

Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet.

Multi-target single-molecule super-resolution fluorescence microscopy offers a powerful means of understanding the distributions and interplay between multiple subcellular structures at the nanoscale. However, single-molecule super-resolution imaging of whole mammalian cells is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D. In this work, we have mitigated these issues by developing a steerable, dithered, single-objective tilted light sheet for optical sectioning to reduce fluorescence background and a pipeline for 3D nanoprinting microfluidic systems for reflection of the light sheet into the sample. This easily adaptable novel microfluidic fabrication pipeline allows for the incorporation of reflective optics into microfluidic channels without disrupting efficient and automated solution exchange. By combining these innovations with point spread function engineering for nanoscale localization of individual molecules in 3D, deep learning for analysis of overlapping emitters, active 3D stabilization for drift correction and long-term imaging, and Exchange-PAINT for sequential multi-target imaging without chromatic offsets, we demonstrate whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed.

Long-term mesoscale imaging of 3D intercellular dynamics across a mammalian organ

A comprehensive understanding of physio-pathological processes necessitates non-invasive intravital three-dimensional (3D) imaging over varying spatial and temporal scales. However, huge data throughput, optical heterogeneity, surface irregularity, and phototoxicity pose great challenges, leading to an inevitable trade-off between volume size, resolution, speed, sample health, and system complexity. Here, we introduce a compact real-time, ultra-large-scale, high-resolution 3D mesoscope (RUSH3D), achieving uniform resolutions of 2.6×2.6×6μm3 across a volume of 8,000×6,000×400μm3 at 20Hz with low phototoxicity. Through the integration of multiple computational imaging techniques, RUSH3D facilitates a 13-fold improvement in data throughput and an orders-of-magnitude reduction in system size and cost. With these advantages, we observed premovement neural activity and cross-day visual representational drift across the mouse cortex, the formation and progression of multiple germinal centers in mouse inguinal lymph nodes, and heterogeneous immune responses following traumatic brain injury-all at single-cell resolution, opening up a horizon for intravital mesoscale study of large-scale intercellular interactions at the organ level.

A low-voltage-driven MEMS ultrasonic phased-array transducer for fast 3D volumetric imaging

Wearable ultrasound imaging technology has become an emerging modality for the continuous monitoring of deep-tissue physiology, providing crucial health and disease information. Fast volumetric imaging that can provide a full spatiotemporal view of intrinsic 3D targets is desirable for interpreting internal organ dynamics. However, existing 1D ultrasound transducer arrays provide 2D images, making it challenging to overcome the trade-off between the temporal resolution and volumetric coverage. In addition, the high driving voltage limits their implementation in wearable settings. With the use of microelectromechanical system (MEMS) technology, we report an ultrasonic phased-array transducer, i.e., a 2D piezoelectric micromachined ultrasound transducer (pMUT) array, which is driven by a low voltage and is chip-compatible for fast 3D volumetric imaging. By grouping multiple pMUT cells into one single drive channel/element, we propose an innovative cell–element–array design and operation of a pMUT array that can be used to quantitatively characterize the key coupling effects between each pMUT cell, allowing 3D imaging with 5-V actuation. The pMUT array demonstrates fast volumetric imaging covering a range of 40 mm × 40 mm × 70 mm in wire phantom and vascular phantom experiments, achieving a high temporal frame rate of 11 kHz. The proposed solution offers a full volumetric view of deep-tissue disorders in a fast manner, paving the way for long-term wearable imaging technology for various organs in deep tissues.

Liver injury and recovery following radiation therapy for hepatocellular carcinoma: insights from functional liver imaging

Aim: A critical need for hepatocellular carcinoma (HCC) management is understanding how the liver recovers following radiotherapy (RT). We hypothesized that functional liver imaging with 99mTc-sulfur colloid (SC) SPECT/CT provides additional information on liver injury and recovery after RT compared to conventional imaging. Methods: The liver function of patients with HCC was assessed using 99mTc-SC SPECT/CT imaging before and after definitive RT. The anatomical liver volume (ALV) was segmented on CT imaging. Liver function was measured as the total liver function (TLF) encompassing 30% of maximum SC uptake. Changes in ALV and TLF were compared to clinical characteristics. Results: Of 31 patients with evaluable post-RT SC SPECT/CT scans (total of 32), 23 had pre-treatment Child-Pugh (CP)-A and 9 had CP-B/C scores. The median follow-up post-RT was 57 days. The median change in ALV was -1.7% with no significant difference between CP-A and CP-B/C patients (P = 0.26) or between short- (32-99 days) and long-term (271-1120 days) follow-up imaging groups (P = 0.28). The median change in TLF post-RT was -24% and was significantly different between short- and long-term groups (-39% vs. 2%, P = 0.001) and between CP-A and CP-B/C patients (-19% vs. -57%, P = 0.002). TLF significantly decreased following treatment at all radiation dose levels, with the decline correlating with the dose (P < 0.001). Conclusion: Functional imaging provides additional information regarding liver injury and recovery following RT that conventional imaging cannot reveal. Patients with CP-A liver status showed less decline following RT and most had liver function near or above pre-treatment levels.

Calibration of FRET-based biosensors using multiplexed biosensor barcoding.

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is widely used in the design of genetically encoded fluorescent biosensors, which are powerful tools for monitoring the dynamics of biochemical activities in live cells. FRET ratio, defined as the ratio between acceptor and donor signals, is often used as a proxy for the actual FRET efficiency, which must be corrected for signal crosstalk using donor-only and acceptor-only samples. However, the FRET ratio is highly sensitive to imaging conditions, making direct comparisons across different experiments and over time challenging. Inspired by a method for multiplexed biosensor imaging using barcoded cells, we reasoned that calibration standards with fixed FRET efficiency can be introduced into a subset of cells for normalization of biosensor signals. Our theoretical analysis indicated that the FRET ratio of high-FRET species relative to non-FRET species slightly decreases at high excitation intensity, suggesting the need for calibration using both high and low FRET standards. To test these predictions, we created FRET donor-acceptor pairs locked in "FRET-ON" and "FRET-OFF" conformations and introduced them into a subset of barcoded cells. Our results confirmed the theoretical predictions and showed that the calibrated FRET ratio is independent of imaging settings. We also provided a strategy for calculating the FRET efficiency. Together, our study presents a simple strategy for calibrated and highly multiplexed imaging of FRET biosensors, facilitating reliable comparisons across experiments and supporting long-term imaging applications.

Photobleaching analysis of fluorescent proteins in two-photon microscopy at high repetition rates

Severe photobleaching in two-photon excitation fluorescence microscopy (TPM) has limited its potential for long-term and quantitative imaging of live cells and tissues. One solution is to excite fluorophores with a high-repetition rate which reduces the peak intensity of irradiance. However, there is a lack of knowledge about the general utility of this strategy for fluorescent proteins. Here, using simple photobleaching assay, we studied the photobleaching of EGFP and tdTomato at 80 MHz and 640 MHz repetition rates. Our analysis shows that the impact of high repetition rate excitation on reducing photobleaching in TPM is highly dependent on the excitation wavelength and fluorophores. Our results are useful for selecting optimal parameters to minimize photobleaching and photodamage in TPM.

ANew Perspective On Arterioectatic Spinal Angiopathy with aReversible Pattern: Cause or Consequence?

In 2022, arterioectatic spinal angiopathy (AESA) of childhood was reported as afatal, progressive, multi-segment myelopathy associated with aunique form of non-inflammatory spinal angiopathy involving diffuse dilatation of the anterior spinal artery and cord congestion in children. In this study, we present four more cases of AESA, using early and long-term conventional imaging and flat detector computed tomography angiography (FDCTA) imaging to assess the probability of disease regression and prevent unnecessary interventions. We retrospectively reviewed the clinical and radiological findings of four patients with AESA seen in two neuroradiology departments between 2014 and 2023. The study included three boys and one girl. Two of the boys were siblings. Although the clinical and radiological presentation in the early stages of the clinical course overlapped the definition of AESA, the clinical course was more benign in three of the cases. The clinical courses of the two siblings with monosegmental cord involvement and largely reversible radiological findings suggest that some of the features in the initial definition of the disease cannot be standardized for all patients. The siblings had amutation of the NDUFS gene, which is involved in mitochondrial function and clinical-radiological reversibility in these patients. Many mitochondrial diseases, such as this NDUFS mutation, present with myelopathy, and mitochondrial diseases can sometimes show spontaneous recovery. It is crucial to identify other genetic mutations or environmental factors that trigger the accompanying vascular ectatic findings in AESA in larger multicenter studies to prevent its potential lethal course and possible unnecessary surgical-endovascular interventions.

The Use of a Suture Bridge Technique for Arthroscopic Rotator Cuff Repair in Patients Under 40 Years of Age Resulted in Successful Tendon Healing, Pain Relief, Improved Shoulder Function, and High Patient Satisfaction at a Minimum of 5-Year Follow-Up

PurposeThe purpose of this study was to evaluate the long-term clinical and imaging outcomes after arthroscopic repair of rotator cuff tear (RCT) using a suture bridge technique in patients under 40 years of age. MethodA consecutive series of patients who were treated arthroscopically for RCT by a single surgeon between 2016 and 2018 were retrospectively identified. Both full thickness tears and partial tears were included. Arthroscopic cuff repair employed a knotless double-row suture bridge technique, utilizing braided suture tapes. To assess clinical outcomes, we employed the Constant score, the American Shoulder and Elbow Surgeons score (ASES), a strength score, and a visual analog scale (VAS). Tendon healing was evaluated at 12 months using the Sugaya MRI classification. ResultsA total of 63 patients were included in the study. The mean age at the time of the surgery was 33.6 years (range, 21 to 40) and minimum follow up duration was 5 years. Thirty-nine patients reported occasional sports, eight were professional athletes, while sixteen reported no sportive activity prior to first symptoms. Mean follow-up duration was 66.8 months (range, 62.4 to 88.6 months). A significant improvement was observed in both the Constant score and ASES score. The mean Constant score increased significantly from 39.8 (range, 29 to 52) points to 88.9 (range, 34 to 100) points postoperatively (p < 0.001). Similarly, the ASES score improved significantly from 41.8 (range, 30 to 64) to 90.2 (range, 35 to 100, p < 0.001). Anterior flexion mean improved from 86° (range: 60° to 110°) to 137° (range, 90° to 180°, p < 0.001). Pain significantly decreased after the surgery, VAS descending from 6.3 (range, 3 to 10) to 1.3 (range, 0 to 9) postoperatively. The overall rate of return to previous activities in the cohort was 84% at an average of 10.1 months (range, 6 to 12 months) after surgery. Among the included patients, 85% engaging in occasional sports activities and 67% of elite athletes similarly returned to their pre-injury sports levels after 9.8 (range, 6 to 12) months and 10.7 months (range, 6 to 12) respectively. Two patients (3%) experienced cuff re-rupture, and tendon non-healing (Sugaya stage 3 and stage 4) was observed in five (7%) patients. At the final follow-up, 95% of patients were very satisfied or satisfied with their functional results. ConclusionThe use of a suture bridge technique in arthroscopic RCT repair for patients under 40 years of age resulted in excellent long-term outcomes, including successful tendon healing, pain relief, improved shoulder function, and high patient satisfaction.