Building momentum through networks: Bioimaging across the Americas.

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

Characterization of lamina propria remodeling in pediatric eosinophilic esophagitis using second harmonic generation microscopy.

Eosinophilic esophagitis (EoE) is a chronic inflammatory condition characterized by an intense infiltration of eosinophils into the esophageal epithelium. When not adequately controlled, eosinophilic inflammation can lead to changes in components of the extracellular matrix (ECM) of the lamina propria. Particularly, alterations to the collagen fiber matrix can lead to lamina propria fibrosis (LPF), which plays an important role in the fibrostenotic complications of EoE. Current approaches to assess LPF in EoE are prone to inter-observer inconsistencies and provide limited insight into the structural remodeling of the ECM. An objective approach to quantify LPF can eliminate inter-observer inconsistencies and provide novel insights into the fibrotic transformation of the lamina propria in EoE. Second harmonic generation (SHG) microscopy is a powerful modality for objectively quantifying disease associated alterations in ECM collagen structure that is finding increasing use for clinical research. We used SHG with morphometric analysis (SHG-MA) to characterize lamina propria collagen fibers and ECM porosity in esophageal biopsies collected from children with active EoE (n = 11), inactive EoE (n = 11), and non-EoE (n = 11). The collagen fiber width quantified by SHG-MA correlated positively with peak eosinophil count (r = 0.65, p < 0.005) and histopathologist scoring of LPF (r = 0.52, p < 0.005) in the esophageal biopsies. Patients with active EoE had a significant enlargement of ECM pores compared to inactive EoE and non-EoE (p < 0.005), with the mean pore area correlating positively with EoE activity (r = 0.76, p < 0.005) and LPF severity (r = 0.65, p < 0.005). These results indicate that SHG-MA can be utilized to objectively characterize and provide novel insights into lamina propria ECM structural remodeling in children with EoE, which could aid in monitoring disease progression.

Ultrasound Modulates Calcium Activity in Cultured Neurons, Glial Cells, Endothelial Cells and Pericytes.

OBJECTIVE: Ultrasound is being researched as a method to modulate the brain. Studies of the interaction of sound with neurons support the hypothesis that mechanosensitive ion channels play an important role in ultrasound neuromodulation. The response of cells other than neurons (e.g., astrocytes, pericytes and endothelial cells) have not been fully characterized, despite playing an important role in brain function. METHODS: To address this gap in knowledge, we examined cultured murine primary cortical neurons, astrocytes, endothelial cells and pericytes in an in vitro widefield microscopy setup during application of a 500 ms burst of 250 kHz focused ultrasound over a pressure range known to elicit neuromodulation. We examined cell membrane health in response to a range of pulses and used optical calcium indicators in conjunction with pharmacological antagonists to selectively block different groups of thermo- and mechanosensitive ion channels known to be responsive to ultrasound. RESULTS: All cell types experienced an increase in calcium fluorescence in response to ultrasound. Gadolinium (Gad), 2-aminoethoxydiphenyl borate (2-APB) and ruthenium red (RR) reduced the percentage of responding neurons and magnitude of response. The percentage of astrocytes responding was significantly lowered only by Gad, whereas both 2-APB and Gad decreased the amplitude of the fluorescence response. 2-APB decreased the percentage of responding endothelial cells, whereas only Gad reduced the magnitude of responses. Pericytes exposed to RR or Gad were less likely to respond to stimulation. RR had no detectable effect on the magnitude of the pericyte responses while 2-APB and Gad significantly decreased the fluorescence intensity, despite not affecting the percentage responding. CONCLUSION: Our study highlights the role of non-neuronal cells during FUS neuromodulation. All of the investigated cell types are sensitive to mechanical ultrasound stimulation and rely on mechanosensitive ion channels to undergo ultrasound neuromodulation.

In Vivo Raman Spectroscopy Reveals Biochemical Composition of the Esophageal Tissue in Pediatric Eosinophilic Esophagitis.

INTRODUCTION: Biochemical alterations in the esophagus of patients with eosinophilic esophagitis (EoE) are poorly understood. We used Raman spectroscopy through a pediatric endoscope to identify key Raman features reflective of the esophageal biochemical composition to differentiate between children with EoE from non-EoE controls and between children with active (aEoE) and inactive EoE (iEoE). METHODS: Spectral measurements were obtained using a customized pediatric endoscope-compatible fiber-optic Raman probe in real time during an esophagogastroduodenoscopy. Chemometric analysis was performed to identify key Raman features associated with EoE. Pearson correlation analysis was used to assess relationship between the key Raman features and EoE activity indices. Their diagnostic utility was ascertained using the receiver operator characteristic curve analysis. RESULTS: Forty-three children were included in the study (EoE = 32 [74%] and non-EoE control = 11 [26%]; aEoE = 20 [63%] and iEoE = 12 [37%]). Raman intensities assigned to lipids, proteins, and glycogen:protein ratio accurately distinguished children with EoE from those without EoE and aEoE from iEoE. They significantly correlated with EoE activity indices. The Raman peak ratio for lipids had 90.6% sensitivity, 100% specificity, and an area under the curve of 0.95 to differentiate children with EoE from non-EoE controls. The glycogen:protein ratio had 70% sensitivity, 91.7% specificity, and an area under the curve of 0.75 to distinguish children with aEoE from iEoE. DISCUSSION: Real-time intraendoscopy Raman spectroscopy is an effective method for identifying spectral markers reflective of the esophageal biochemical composition in children with EoE. This technique may aid in the diagnosis and monitoring of EoE and help to elucidate EoE pathogenesis.

Comparing laser speckle contrast imaging and indocyanine green angiography for assessment of parathyroid perfusion.

Accurate intraoperative assessment of parathyroid blood flow is crucial to preserve function postoperatively. Indocyanine green (ICG) angiography has been successfully employed, however its conventional application has limitations. A label-free method overcomes these limitations, and laser speckle contrast imaging (LSCI) is one such method that can accurately detect and quantify differences in parathyroid perfusion. In this study, twenty-one patients undergoing thyroidectomy or parathyroidectomy were recruited to compare LSCI and ICG fluorescence intraoperatively. An experimental imaging device was used to image a total of 37 parathyroid glands. Scores of 0, 1 or 2 were assigned for ICG fluorescence by three observers based on perceived intensity: 0 for little to no fluorescence, 1 for moderate or patchy fluorescence, and 2 for strong fluorescence. Speckle contrast values were grouped according to these scores. Analyses of variance were performed to detect significant differences between groups. Lastly, ICG fluorescence intensity was calculated for each parathyroid gland and compared with speckle contrast in a linear regression. Results showed significant differences in speckle contrast between groups such that parathyroids with ICG score 0 had higher speckle contrast than those assigned ICG score 1, which in turn had higher speckle contrast than those assigned ICG score 2. This was further supported by a correlation coefficient of -0.81 between mean-normalized ICG fluorescence intensity and speckle contrast. This suggests that ICG angiography and LSCI detect similar differences in blood flow to parathyroid glands. Laser speckle contrast imaging shows promise as a label-free alternative that overcomes current limitations of ICG angiography for parathyroid assessment.

Development of a Low-Cost Paper-Based Platform for Coffee Ring-Assisted SERS.

The need for highly sensitive, low-cost, and timely diagnostic technologies at the point of care is increasing. Surface-enhanced Raman spectroscopy (SERS) is a vibrational spectroscopic technique that is an advantageous technique to address this need, as it can rapidly detect analytes in small or dilute samples with improved sensitivity compared to conventional Raman spectroscopy. Despite the many advantages of SERS, one drawback of the technique is poor reproducibility due to variable interactions between nanoparticles and target analytes. To overcome this limitation, coupling SERS with the coffee ring effect has been implemented to concentrate and localize analyte-nanoparticle conjugates for improved signal reproducibility. However, current coffee ring platforms require laborious fabrication steps. Herein, we present a low-cost, two-step fabrication process for coffee ring-assisted SERS, utilizing wax-printed nitrocellulose paper. The platform was designed to produce a highly hydrophobic paper substrate that supports the coffee ring effect and tested using gold nanoparticles for SERS sensing. The nanoparticle concentration and solvent were varied to determine the effect of solution composition on ring formation and center clearance. The SERS signal was validated using 4-mercaptobenzoic acid (MBA) and tested with Moraxella catarrhalis bacteria to ensure functionality for chemical and biological applications. The limit of detection using MBA is 41.56 nM, and the biochemical components of the bacterial cell wall were enhanced with low spectral variability. The developed platform is advantageous due to ease of fabrication and use, representing the next step toward implementing low-cost coffee ring-assisted SERS for point-of-care sensing.

Sensitivity of the amide I band to matrix manipulation in bone: a Raman micro-spectroscopy and spatially offset Raman spectroscopy study.

The fracture resistance of bone arises from the hierarchical arrangement of minerals, collagen fibrils (i.e., cross-linked triple helices of α1 and α2 collagen I chains), non-collagenous proteins, and water. Raman spectroscopy (RS) is not only sensitive to the relative fractions of these constituents, but also to the secondary structure of bone proteins. To assess the ability of RS to detect differences in the protein structure, we quantified the effect of sequentially autoclaving (AC) human cortical bone at 100 °C (∼34.47 kPa) and then at 120 °C (∼117.21 kPa) on the amide I band using a commercial Raman micro-spectroscopy (µRS) instrument and custom spatially offset RS (SORS) instrument in which rings of collection fiber optics are offset from the central excitation fiber optics within a hand-held, cylindrical probe. Being clinically viable, measurements by SORS involved collecting Raman spectra of cadaveric femur mid-shafts (5 male & 5 female donors) through layers of a tissue mimic. Otherwise, µRS and SORS measurements were acquired directly from each bone. AC-related changes in the helical status of collagen I were assessed using amide I sub-peak ratios (intensity, I, at ∼1670 cm-1 relative to intensities at ∼1610 cm-1 and ∼1640 cm-1). The autoclaving manipulation significantly decreased the selected amide I sub-peak ratios as well as shifted peaks at ∼1605 cm-1 (µRS), ∼1636 cm-1 (SORS) and ∼1667 cm-1 in both µRS and SORS. Compared to µRS, SORS detected more significant differences in the amide I sub-peak ratios when the fiber optic probe was directly applied to bone. SORS also detected AC-related decreases in I1670/I1610 and I1670/I1640 when spectra were acquired through layers of the tissue mimic with a thickness ≤2 mm by the 7 mm offset ring, but not with the 5 mm or 6 mm offset ring. Overall, the SORS instrument was more sensitive than the conventional µRS instrument to pressure- and temperature-related changes in the organic matrix that affect the fracture resistance of bone, but SORS analysis of the amide I band is limited to an overlying thickness layer of 2 mm.

Multiparameter interferometric polarization-enhanced imaging differentiates carcinoma in situ from inflammation of the bladder: an ex vivo study.

Significance: Successful differentiation of carcinoma in situ (CIS) from inflammation in the bladder is key to preventing unnecessary biopsies and enabling accurate therapeutic decisions. Current standard-of-care diagnostic imaging techniques lack the specificity needed to differentiate these states, leading to false positives. Aim: We introduce multiparameter interferometric polarization-enhanced (MultiPIPE) imaging as a promising technology to improve the specificity of detection for better biopsy guidance and clinical outcomes. Approach: In this ex vivo study, we extract tissue attenuation-coefficient-based and birefringence-based parameters from MultiPIPE imaging data, collected with a bench-top system, to develop a classifier for the differentiation of benign and CIS tissues. We also analyze morphological features from second harmonic generation imaging and histology slides and perform imaging-to-morphology correlation analysis. Results: MultiPIPE enhances specificity to differentiate CIS from benign tissues by nearly 20% and reduces the false-positive rate by more than four-fold over clinical standards. We also show that the MultiPIPE measurements correlate well with changes in morphological features in histological assessments. Conclusions: The results of our study show the promise of MultiPIPE imaging to be used for better differentiation of bladder inflammation from flat tumors, leading to a fewer number of unnecessary procedures and shorter operating room (OR) time.

Assessment of spatially offset Raman spectroscopy to detect differences in bone matrix quality.

Since spatially offset Raman spectroscopy (SORS) can acquire biochemical measurements of tissue quality through light scattering materials, we investigated the feasibility of this technique to acquire Raman bands related to the fracture resistance of bone. Designed to maximize signals at different offsets, a SORS probe was used to acquire spectra from cadaveric bone with and without skin-like tissue phantoms attenuating the light. Autoclaving the lateral side of femur mid-shafts from 5 female and 5 male donors at 100 °C and again at 120 °C reduced the yield stress of cortical beams subjected to three-point bending. It did not affect the volumetric bone mineral density or porosity. Without tissue phantoms, autoclaving affected more Raman characteristics of the organic matrix when determined by peak intensity ratios, but fewer matrix properties depended on the three offsets (5 mm, 6 mm, and 7 mm) when determined by band area ratios. The cut-off in the thickness of the tissue phantom layers was ∼4 mm for most properties, irrespective of offset. Matching trends when spectra were acquired without phantom layers between bone and the probe, ν1PO43-/Amide III and ν1PO43-/(proline + OH-proline) were higher and lower in the non-treated bone than in the autoclaved bone, respectively, when the thickness of tissue phantom layers was 4 mm. The layers, however, caused a loss of sensitivity to autoclaving-related changes in ν3CO3/ν1PO43- and crystallinity. Without advanced post-processing of Raman spectra, SORS acquisition through turbid layers can detect changes in Raman properties of bone that accompany a loss in bone strength.

Performance assessment of probe-based Raman spectroscopy systems for biomedical analysis.

We present a methodology for evaluating the performance of probe-based Raman spectroscopy systems for biomedical analysis. This procedure uses a biological standard sample and data analysis approach to circumvent many of the issues related to accurately measuring and comparing the signal quality of Raman spectra between systems. Dairy milk is selected as the biological standard due to its similarity to tissue spectral properties and because its homogeneity eliminates the dependence of probe orientation on the measured spectrum. A spectral dataset is first collected from milk for each system configuration, followed by a model-based correction step to remove photobleaching artifacts and accurately calculate SNR. Results demonstrate that the proposed strategy, unlike current methods, produces an experimental SNR that agrees with the theoretical value. Four preconfigured imaging spectrographs that share similar manufacturer specifications were compared, showing that their capabilities to detect biological Raman spectra widely differ in terms of throughput and stray light rejection. While the methodology is used to compare spectrographs in this case, it can be adapted for other purposes, such as optimizing the design of a custom-built Raman spectrometer, evaluating inter-probe variability, or examining how altering system subcomponents affects signal quality.

Measurement of rat and human tissue optical properties for improving the optical detection and visualization of peripheral nerves.

Peripheral nerve damage frequently occurs in challenging surgical cases resulting in high costs and morbidity. Various optical techniques have proven effective in detecting and visually enhancing nerves, demonstrating their translational potential for assisting in nerve-sparing medical procedures. However, there is limited data characterizing the optical properties of nerves in comparison to surrounding tissues, thus limiting the optimization of optical nerve detection systems. To address this gap, the absorption and scattering properties of rat and human nerve, muscle, fat, and tendon were determined from 352-2500 nm. The optical properties highlighted an ideal region in the shortwave infrared for detecting embedded nerves, which remains a significant challenge for optical approaches. A 1000-1700 nm hyperspectral diffuse reflectance imaging system was used to confirm these results and identify optimal wavelengths for nerve imaging contrast in an in vivo rat model. Optimal nerve visualization contrast was achieved using 1190/1100 nm ratiometric imaging and was sustained for nerves embedded under ≥600 µm of fat and muscle. Overall, the results provide valuable insights for optimizing the optical contrast of nerves, including those embedded in tissue, which could lead to improved surgical guidance and nerve-sparing outcomes.

Label-free intraoperative nerve detection and visualization using ratiometric diffuse reflectance spectroscopy.

Iatrogenic nerve injuries contribute significantly to postoperative morbidity across various surgical disciplines and occur in approximately 500,000 cases annually in the US alone. Currently, there are no clinically adopted means to intraoperatively visualize nerves beyond the surgeon's visual assessment. Here, we report a label-free method for nerve detection using diffuse reflectance spectroscopy (DRS). Starting with an in vivo rat model, fiber- and imaging-based DRS independently identified similar wavelengths that provided optimal contrast for nerve identification with an accuracy of 92%. Optical property measurements of rat and human cadaver tissues verify that the source of contrast between nerve and surrounding tissues is largely due to higher scattering in nerve and differences in oxygenated hemoglobin content. Clinical feasibility was demonstrated in patients undergoing thyroidectomies using both probe-based and imaging-based approaches where the nerve were identified with 91% accuracy. Based on our preliminary results, DRS has the potential to both provide surgeons with a label-free, intraoperative means of nerve visualization and reduce the incidence of iatrogenic nerve injuries along with its detrimental complications.

Proceduralist criteria for evaluating interface utility of novel imaging modalities in early phase clinical trials: evaluating the need for standardized criteria.

Accelerating innovation in the space of fluorescence imaging for surgical applications has increased interest in safely and expediently advancing these technologies to clinic through Food and Drug Administration-(FDA-) compliant trials. Conventional metrics for early phase trials include drug safety, tolerability, dosing, and pharmacokinetics. Most procedural imaging technologies rely on administration of an exogenous fluorophore and concurrent use of an imaging system; both of which must receive FDA approval to proceed to clinic. Because fluorophores are classified as medical imaging agents, criteria for establishing dose are different, and arguably more complicated, than therapeutic drugs. Since no therapeutic effect is desired, medical imaging agents are ideally administered at the lowest dose that achieves adequate target differentiation. Because procedural imaging modalities are intended to enhance and/or ease proceduralists' identification or assessment of tissues, beneficial effects of these technologies may manifest in the form of qualitative endpoints such as: 1) confidence; 2) decision-making; and 3) satisfaction with the specified procedure. Due to the rapid expansion of medical imaging technologies, we believe that our field requires standardized criteria to evaluate existing and emerging technologies objectively so that both quantitative and qualitative aspects of their use may be measured and useful comparisons to assess their relative value may occur. Here, we present a 15-item consensus-based survey instrument to assess the utility of novel imaging technologies from the proceduralist's standpoint.

Identifying Parathyroids in Pediatric Thyroid/Parathyroid Surgery by Near Infrared Autofluorescence.

OBJECTIVES: Compared to adult patients undergoing thyroid surgery, pediatric patients have higher rates of hypoparathyroidism often related to parathyroid gland (PG) inadvertent injury or devascularization. Previous studies have shown that near-infrared-autofluorescence (NIRAF) can be reliably used intraoperatively for label-free parathyroid identification, but all prior studies have been performed in adult patients. In this study, we assess the utility and accuracy of NIRAF with a fiber-optic probe-based system to identify PGs in pediatric patients undergoing thyroidectomy or parathyroidectomy. METHODS: All pediatric patients (under 18 years of age) undergoing thyroidectomy or parathyroidectomy were enrolled in this IRB-approved study. The surgeon's visual assessment of tissues was first noted and the surgeon's confidence level in the tissue identified was recorded. A fiber-optic probe was then used to illuminate tissues-of-interest with a wavelength of 785 nm and resulting NIRAF intensities from these tissues were measured while the surgeon was blinded to results. RESULTS: NIRAF intensities were measured intraoperatively in 19 pediatric patients. Normalized NIRAF intensities for PGs (3.63 ± 2.47) were significantly higher than that of thyroid (0.99 ± 0.36, p < 0.001) and other surrounding soft tissues (0.86 ± 0.40, p < 0.001). Based on the PG identification ratio threshold of 1.2, NIRAF yielded a detection rate of 95.8% (46/48 pediatric PGs). CONCLUSION: Our findings indicate that NIRAF detection can potentially be a valuable and non-invasive technique to identify PGs during neck operations in the pediatric population. To our knowledge, this is the first study in children to assess the accuracy of probe-based NIRAF detection for intraoperative parathyroid identification. LEVEL OF EVIDENCE: Level 4 Laryngoscope, 133:3208-3215, 2023.

Infrared neural stimulation markedly enhances nerve functionality assessment during nerve monitoring.

In surgical procedures where the risk of accidental nerve damage is prevalent, surgeons commonly use electrical stimulation (ES) during intraoperative nerve monitoring (IONM) to assess a nerve's functional integrity. ES, however, is subject to off-target stimulation and stimulation artifacts disguising the true functionality of the specific target and complicating interpretation. Lacking a stimulation artifact and having a higher degree of spatial specificity, infrared neural stimulation (INS) has the potential to improve upon clinical ES for IONM. Here, we present a direct comparison between clinical ES and INS for IONM performance in an in vivo rat model. The sensitivity of INS surpasses that of ES in detecting partial forms of damage while maintaining a comparable specificity and sensitivity to more complete forms. Without loss in performance, INS is readily compatible with existing clinical nerve monitoring systems. These findings underscore the clinical potential of INS to improve IONM and surgical outcomes.

Enhanced characterization of breast cancer phenotypes using Raman micro-spectroscopy on stainless steel substrate.

Biochemical insights into varying breast cancer (BC) phenotypes can provide a fundamental understanding of BC pathogenesis, while identifying novel therapeutic targets. Raman spectroscopy (RS) can gauge these biochemical differences with high specificity. For routine RS, cells are traditionally seeded onto calcium fluoride (CaF2) substrates that are costly and fragile, limiting its widespread adoption. Stainless steel has been interrogated previously as a less expensive alternative to CaF2 substrates, while reporting increased Raman signal intensity than the latter. We sought to further investigate and compare the Raman signal quality measured from stainless steel versus CaF2 substrates by characterizing different BC phenotypes with altered human epidermal growth factor receptor 2 (HER2) expression. Raman spectra were obtained on stainless steel and CaF2 substrates for HER2 negative cells - MDA-MB-231, MDA-MB-468 and HER2 overexpressing cells - AU565, SKBr3. Upon analyzing signal-to-noise ratios (SNR), stainless steel provided a stronger Raman signal, improving SNR by 119% at 1450 cm-1 and 122% at 2925 cm-1 on average compared to the CaF2 substrate. Utilizing only 22% of laser power on sample relative to the CaF2 substrate, stainless steel still yielded improved spectral characterization over CaF2, achieving 96.0% versus 89.8% accuracy in BC phenotype discrimination and equivalent 100.0% accuracy in HER2 status classification. Spectral analysis further highlighted increased lipogenesis and altered metabolism in HER2 overexpressing cells, which was subsequently visualized with coherent anti-Stokes Raman scattering microscopy. Our findings demonstrate that stainless steel substrates deliver improved Raman signal and enhanced spectral characterization, underscoring its potential as a cost-effective alternative to CaF2 for non-invasively monitoring cellular biochemical dynamics in translational cancer research.

Emerging Imaging Technologies for Parathyroid Gland Identification and Vascular Assessment in Thyroid Surgery: A Review From the American Head and Neck Society Endocrine Surgery Section.

Importance: Identification and preservation of parathyroid glands (PGs) remain challenging despite advances in surgical techniques. Considerable morbidity and even mortality result from hypoparathyroidism caused by devascularization or inadvertent removal of PGs. Emerging imaging technologies hold promise to improve identification and preservation of PGs during thyroid surgery. Observation: This narrative review (1) comprehensively reviews PG identification and vascular assessment using near-infrared autofluorescence (NIRAF)-both label free and in combination with indocyanine green-based on a comprehensive literature review and (2) offers a manual for possible implementation these emerging technologies in thyroid surgery. Conclusions and Relevance: Emerging technologies hold promise to improve PG identification and preservation during thyroidectomy. Future research should address variables affecting the degree of fluorescence in NIRAF, standardization of signal quantification, definitions and standardization of parameters of indocyanine green injection that correlate with postoperative PG function, the financial effect of these emerging technologies on near-term and longer-term costs, the adoption learning curve and effect on surgical training, and long-term outcomes of key quality metrics in adequately powered randomized clinical trials evaluating PG preservation.

Engineering functional 3-dimensional patient-derived endocrine organoids for broad multiplatform applications.

BACKGROUND: Recent advancements in 3-dimensional patient-derived organoid models have revolutionized the field of cancer biology. There is an urgent need for development of endocrine tumor organoid models for medullary thyroid carcinoma, adrenocortical carcinoma, papillary thyroid carcinoma, and a spectrum of benign hyperfunctioning parathyroid and adrenal neoplasms. We aimed to engineer functionally intact 3-dimensional endocrine patient-derived organoids to expand the in vitro and translational applications for the advancement of endocrine research. METHODS: Using our recently developed fine needle aspiration-based methodology, we established patient-derived 3-dimensional endocrine organoid models using prospectively collected human papillary thyroid carcinoma (n = 6), medullary thyroid carcinoma (n = 3), adrenocortical carcinoma (n = 3), and parathyroid (n = 5). and adrenal (n = 5) neoplasms. Multiplatform analyses of endocrine patient-derived organoids and applications in oncoimmunology, near-infrared autofluorescence, and radiosensitization studies under 3-dimensional in vitro conditions were performed. RESULTS: We have successfully modeled and analyzed the complex endocrine microenvironment for a spectrum of endocrine neoplasms in 3-dimensional culture. The endocrine patient-derived organoids recapitulated complex tumor microenvironment of endocrine neoplasms morphologically and functionally and maintained cytokine production and near-infrared autofluorescence properties. CONCLUSION: Our novel engineered endocrine patient-derived organoid models of thyroid, parathyroid and adrenal neoplasms represent an exciting and elegant alternative to current limited 2-dimensional systems and afford future broad multiplatform in vitro and translational applications, including in endocrine oncoimmunology.

Multimodal Raman spectroscopy and optical coherence tomography for biomedical analysis.

Optical techniques hold great potential to detect and monitor disease states as they are a fast, non-invasive toolkit. Raman spectroscopy (RS) in particular is a powerful label-free method capable of quantifying the biomolecular content of tissues. Still, spontaneous Raman scattering lacks information about tissue morphology due to its inability to rapidly assess a large field of view. Optical Coherence Tomography (OCT) is an interferometric optical method capable of fast, depth-resolved imaging of tissue morphology, but lacks detailed molecular contrast. In many cases, pairing label-free techniques into multimodal systems allows for a more diverse field of applications. Integrating RS and OCT into a single instrument allows for both structural imaging and biochemical interrogation of tissues and therefore offers a more comprehensive means for clinical diagnosis. This review summarizes the efforts made to date toward combining spontaneous RS-OCT instrumentation for biomedical analysis, including insights into primary design considerations and data interpretation.

Label-Free Enhancement of Adrenal Gland Visualization Using Near-Infrared Autofluorescence for Surgical Guidance.

BACKGROUND: During adrenalectomy, surgeons have traditionally relied on their subjective visual skills to distinguish adrenal glands (AGs) from retroperitoneal fat and surrounding structures, while ultrasound and exogenous contrast agents have been employed for intraoperative AG visualization, all of which have their limitations. We present a novel label-free approach that uses near-infrared autofluorescence (NIRAF) detection, which demonstrates potential for enhanced intraoperative AG visualization and efficient tumor resection during adrenalectomies. METHODS: Patients undergoing adrenalectomy or nephrectomy were enrolled for this feasibility study. NIRAF emitted beyond 800 nm was detected in vivo from AGs and surrounding tissues during open adrenalectomies or nephrectomies. NIRAF was also measured ex vivo in excised AGs following robotic adrenalectomies. NIRAF images of tissues were captured using near-infrared (NIR) camera systems, whereas NIRAF intensities were recorded concurrently using fiber-optic probe-based NIR devices. Normalized NIRAF intensities (expressed as mean ± standard error) were analyzed and compared. RESULTS: Among the 55 enrolled patients, NIRAF intensity was elevated significantly for AGs versus retroperitoneal fat and other structures. NIR images of AGs also revealed a distinct demarcation of NIRAF between adrenal cortex and other periadrenal structures. NIRAF intensity in AGs was decreased markedly in malignant adrenal tumors, while benign adrenal cortical tumors and healthy adrenal cortex exhibited the strongest NIRAF levels. CONCLUSIONS: Our preliminary findings indicate that NIRAF detection could be a promising label-free technology to enhance intraoperative AG visualization and holds immense potential for effective tumor demarcation during cortical-sparing adrenalectomies or adrenal-conserving surgeries.