Journal of Biophotonics

Cardiac amyloidosis is caused by deposits of misfolded proteins in the heart and can lead to heart failure. Histological confirmation typically involves staining tissue sections with Congo red and examining them under polarized light for apple-green birefringence; however, achieving an interpretable, label-free, and observer-independent assessment remains challenging. Here we show that Pearson kurtosis of channel intensities within the amide I window provides an interpretable, fitting-free contrast for cardiac amyloid in unstained human cardiac tissue sections from ten participants imaged by ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS). In a discovery participant, high-kurtosis regions corresponded with Congo red-positive tissue in two CARS-histology pairs of adjacent sections (descriptive spatial AUCs, 0.79 and 0.73). Across 15 fields from nine additional participants, field-level kurtosis values did not overlap between severe-deposition and Congo red-negative tissue fields. Among the descriptors examined, kurtosis showed the largest standardized participant-level separation. Exploratory multivariate curve resolution yielded a deposit-associated component with aromatic-residue features shared with reagent transthyretin. In one field from the discovery participant, the second harmonic generation (SHG) signal, acquired simultaneously with CARS, showed a broadly inverse spatial pattern relative to the amide I kurtosis map, revealing distinct tissue-architectural contrast. Together, these findings support a potential label-free approach to detecting cardiac amyloid deposits that combines amide I kurtosis derived from broadband CARS with SHG-derived architectural contrast.

Abstract Background Advanced in vitro models of the human heart have become promising tools for studying cardiac diseases and drug discovery. Three-dimensional (3D) engineered heart tissues (EHTs) provide more adult cardiac tissue-like structural organization compared to traditional two-dimensional (2D) in vitro models with straightforward contractile readouts. However, the assessment of the architecture of EHTs, especially the quantification of cellular and tissue structural components, has remained poorly studied. Here we demonstrate the use of light-sheet microscopy for structural imaging of intact EHTs and provide quantification of the EHT cellular and subcellular structures. Methods We developed an in-house inverted selective plane illumination microscopy (iSPIM) platform and combined it with iterative indirect immunofluorescence (IT-IF) staining for better signal acquisition. We developed an image-processing and analysis pipeline dedicated to volumetric EHT analysis. Results With this integrated platform, we achieved volumetric imaging of intact EHTs and quantification of 3D tissue architecture, revealing aligned cardiomyocytes within the imaged volume and increasing cardiac nuclei content towards the core of the tissue. The developed methods allowed the detection of heterogeneities between tissue sections, demonstrating the sensitivity required for future disease modeling or maturation studies. Conclusions The developed integrated platform opens new possibilities for volumetric analysis and characterization of 3D tissue structure using iSPIM, providing new insights into EHT architecture and the use of EHTs as disease modeling tools.

Objective: To develop and validate a dual-probe Diffuse Correlation Spectroscopy (DCS) system for non-invasive and simultaneous, monitoring of cerebral blood flow (CBF) in the bilateral Middle Cerebral Artery (MCA) territories, and expanding the utility of conventional DCS limited to cortical-volume-based CBF measurements to vessel-specific cerebral perfusion monitoring. Methods: A dual-probe DCS system was designed for non-invasive monitoring of MCA-specific perfusion. Probe placement and protocol optimization study has been performed using anatomical landmarks, motor and speech activation tasks in healthy volunteers. System stability and repeatability were further evaluated in a pilot cohort of 30 healthy (age, 25{+/-}7 years) participants using optimized probe position and protocol. A bilateral MCA ischemic Lacunar Infract stroke case report also validated the feasibility of the system in clinical settings. Results: Measurements demonstrated superior sensitivity towards MCA-territory perfusion at targeted probe locations compared to off-MCA positions. In pilot cohort, significant increase of 30.34 {+/-} 21.56% and 36.48 {+/-} 21.22% in rCBF corresponding to hand squeeze and speech task respectively showed reproducible physiological responsiveness of the system (p<0.001). Measurement done on a patient with bilateral MCA ischemic Lacunar Infract stroke showed a significant change of 30% during speech for both the MCAs but no significant change is observed for hand squeeze tasks. Conclusion: The custom built dual-probe DCS system enables non-invasive, operator-independent, targeted and continuous monitoring of rCBF within bilateral MCA territories. Significance: This approach enables the potential use of DCS system for bilateral and vessel-specific monitoring of cerebral perfusion in the MCA territories.

Background and Objective: High-speed and high-resolution retinal imaging using adaptive optics line confocal ophthalmoscopy (AOLCO) resolves individual red blood cells (erythrocytes) flowing through the smallest retinal capillaries, but quantifying their velocity first requires removing linear and nonlinear distortions caused by eye motion that may result in a displacement of tens to hundreds of pixels between frames. We present a computational pipeline that stabilizes AOLCO capillary video and measures erythrocyte velocity in true physical units, delivered as an interactive 3D Slicer extension and a batch command-line tool sharing one analysis core. Methods: Our video analysis pipeline has four stages: preprocessing, registration, postprocessing, and velocity measurement. During preprocessing, illumination is corrected, and closed-eye frames are identified. During registration, each frame is aligned to a reference frame using affine and, when needed, B-Spline registration, initialized by our robust and fast (GPU-based) translation-registration algorithm to accommodate large eye movements (e.g., microsaccades). During postprocessing, adjacent registered frames are differenced to suppress stationary structures, and a 2D projection of the frame-difference video reveals the capillary network. During velocity measurement, an operator manually identifies capillary segments on the 2D projection image. Spatiotemporal images are then generated along these segments from the frame-difference video, and blood-flow velocity curves are obtained from these images using the Radon transform. Results: We validate the velocimetry of the pipeline against synthetic ground-truth flow data, and the experiments show that the velocimetry recovers the speed within an error of 4% The GPU-based translation registration algorithm aligns the large eye motions (~90 um per frame on average) and runs about 3.6 x (100 Hz) to 38 x (400 Hz) faster than the baseline method. Across the full cohort (2344 videos, 57 subjects, 30 Hz to 400 Hz), our registration approach improves structural similarity (SSIM) of the video frames on every acquisition from 0.73 to 0.86. The recovered blood flow velocities are physiologically plausible, of the same order as published AO measurements. Conclusions: Our computational pipeline enables AOLCO velocimetry on the capillary-level.

This study presents a systematic investigation of the brain's own microwave emissions in the 0.85-5.0 GHz range, aiming to validate microwave encephalography (MWEG) as a novel tool for cognitive state assessment. Early experiments (2016-2019) using comparisons with background noise, a control phantom, and different antenna types confirmed the endogenous origin of the recorded signals. In a 2021 experiment (118 measurements on one subject, right and left occipital areas), we observed a significant functional hemispheric asymmetry: the number of "substantial spikes" was significantly higher in darkness for the right hemisphere (p<0.001) and higher in light for the left hemisphere (p<0.001). A 2025 group study (three subjects) confirmed the asymmetry (p<0.05 for right, p<0.01 for left). We describe the signal processing pipeline, including spike detection and statistical analysis. Two complementary mathematical models are presented: an energy-based model linking optical input to microwave emission via neural activity modes, and an information-based model interpreting spectral changes in terms of structural pattern overlap with memory engrams. An estimation of the physical dimensions of possible brain resonators (2-22 mm) is provided. The results are discussed in the context of hemispheric specialization and the information commutation theory. This work opens new perspectives for developing low cost, contactless brain monitoring systems.

Point-of-care (POC) molecular detection has enabled rapid and accessible testing at or near the site of need, facilitating timely decision-making in clinical, field, and industrial settings. Despite these advancements, molecular detection platforms remain limited in their ability to prepare DNA from complex tissue matrices, restricting their applicability to a narrow range of sample types. To address sample preparation inefficiencies, focused ultrasound extraction (FUSE) has been developed for the rapid release of DNA from robust tissue matrices. FUSE disintegrates target tissue and releases DNA by delivering high pressure ultrasound pulses that generate a cavitation bubble cloud. In prior work, FUSE was performed using large, laboratory-based equipment. Here, a miniaturized FUSE device was designed and tested to enable POC DNA sample preparation. Design constraints were defined based on prior FUSE experiments, and based on these metrics, a 750 kHz cylindrical transducer was fabricated. The pressure output was measured, and high-speed optical imaging demonstrated that the transducer generated sustained cavitation within the defined focal region. The feasibility of DNA extraction from piscine and timber tissues was evaluated, and results show that the device can release DNA with quality suitable for PCR-based detection from both tissue types. These results demonstrate the potential of this device to rapidly extract DNA from complex samples and expand the applicability of molecular detection platforms.

Gas-bubble-enhanced focused ultrasound (FUSGB) is advancing clinically across a myriad of applications, including targeted drug delivery through transient blood-brain barrier opening (BBBO). Its broader translation requires a better understanding of the bioeffects that enable reproducible enhancement of barrier permeability while avoiding vascular injury, underscoring the need to clarify the relationship between acoustic exposure, bubble activity, and the biological response central to treatment optimization. These interactions are difficult to isolate in vivo, and acoustic reflections, geometry, and field distortion in conventional cultureware hinder quantitative in vitro studies. Herein, we present a theoretically and experimentally characterized ultrasound-transparent organ-on-chip device with >99.9% US transparency that enables quantitative in vitro modeling of the FUSGB procedure and study of BBBO. Furthermore, submicron bubbles (sMB, ~800nm) were fabricated and characterized. Acoustic dose-dependent subharmonic and broadband cavitation dose curves (SCD and BCD), as well as temporal responses, were studied. A 3-day accelerated Caco--2 surrogate barrier with a high TEER (~1200xcm2) was used to demonstrate US dose-dependent targeted barrier opening, visualized using an in vitro Evans Blue assay. Together, this study and its findings establish a foundation for quantitative in vitro modeling of FUS BBBO, as well as other US interactions in microphysiological systems, supporting systematic evaluation of procedural regimens, large-parameter-space optimization, acoustic safety window, and exposure conditions for brain drug delivery, and additionally enabling mechanistic insight into FUSGB BBBO.

Optical coherence tomography angiography (OCTA) has been extensively explored for the detection of pathological retinal changes arising in various ophthalmological diseases. Due to phase instability of conventional systems, most OCTA signal processing methods to date have relied on intensity-based averaging, despite the resulting lower contrast and dynamic range compared to coherent averaging approaches. In this work, we (1) present a novel computation pipeline that enables fully-coherent processing in any phase-stable or phase-unstable system, and (2) demonstrate the first angiography metric with integrated signal-to-noise ratio (SNR) correction and coherent spatiotemporal ensemble averaging effective in any OCT system. Our method does not rely on 2D en face projections or binarization to produce high-contrast en face views and it significantly enhances the contrast-to-noise ratio of computed OCTA volumes, particularly in regions with reduced SNR levels. Our OCTA volumes also have high cross-sectional quality, enabling the use of single-depth en face views and the localization of capillary paths in the axial dimension. We can now identify capillaries below the retinal pigment epithelium, near the lamina cribrosa in the optic cup, which was not previously possible due to low SNR. We also conducted an analysis of strategies for bias reduction in OCTA and developed the optimal method utilizing coherent spatiotemporal ensemble averaging. Because our method leverages coherent averaging across all dimensions, it provides flexibility in shaping the averaging kernel to target specific workflows. The value of this additional degree of freedom is illustrated in our vessel tracing investigations, in which the depth resolution was maximized by limiting coherent ensemble averaging to the two lateral dimensions even in presence of strong phase noise, enabling the identification of 3D features such as overlapping capillary paths.

Background: Understanding fetal brain responses to hypoxia-ischemia is of growing interest, as it remains unclear to what extent these responses differ from those of newborns and whether developmental adaptations during gestation contribute to such differences. Materials and methods: Using a combination of broadband near-infrared spectroscopy (bNIRS) and diffuse correlation spectroscopy (DCS), we measured brain microvascular oxygen saturation (StO2), total hemoglobin concentration (THC), relative cerebral blood flow (rCBF), and the oxidation state of cytochrome c oxidase (oxCCO) during an acute ischemic challenge in the brains of five rabbit fetuses in utero. Simultaneous measurements were acquired from four control fetuses located in the contralateral uterine horn. Results: During the hypoxia-ischemia phase, we observed a rapid reduction of CBF (47% +/- 29%) and StO2, (from 64.7% +/- 1.5% at baseline to 40.6% +/- 2.0%) together with an increase of oxCCO (3.7 uM +/- 0.5 uM). Conclusion: The simultaneous increase in oxCCO and decrease in rCBF and StO2 during hypoxia-ischemia reveals a cerebral response pattern that differs from that commonly reported in the newborn brain. These findings support the hypothesis that unique developmental adaptations influence fetal cerebral metabolism during hypoxic-ischemic stress.

In magnetic particle imaging (MPI), signal is generated from the non-linear magnetic response of superparamagnetic iron oxide (SPIO) nanoparticles. The optimization of SPIOs for MPI is an active area of investigation. Tracer performance can be evaluated by magnetic particle relaxometry (MPR) and image-based metrics. This study characterized and compared the performance of ten commercially available tracers, spanning a broad range of magnetic core sizes, hydrodynamic diameters and surface coatings, using the peak signal intensity measured from MPR, and the total signal and maximum signal intensity measured from 2D MPI images. Synomag-D tracers exhibited the highest MPR peak signal intensities and highest maximum signal per {micro}g iron. ProMag, a micron-sized iron oxide particle (MPIO), yielded a low MPR peak signal intensity but had the highest total MPI signal per {micro}g of iron. A strong correlation was observed between MPR peak signal intensity and MPI maximum signal intensity (R2=0.94). In contrast, there was a weak correlation between MPR peak signal intensity and MPI total signal intensity (R2=0.38), though this correlation improved when MPIOs were excluded from the analysis (R2=0.91). While MPR generally predicts the tracer performance, it does not completely replicate the complex imaging conditions. As such, comprehensive tracer evaluation requires combining both MPR and image-based metrics.

Microlasers based on whispering-gallery modes (WGMs) provide bright, spectrally narrow emission, making them ideal optical probes for high-resolution cellular tagging, single-cell tracking, and biosensing. To reproducibly apply WGM microlasers in biomedical or tissue-engineering contexts, control over the number of lasers in a cell population is highly desirable. Here we use a commercial instrument for flow cytometry-based cell sorting to obtain HEK293T cell populations with a controlled number of polystyrene laser particles (LPs). By modifying the particle surface with poly(ethyleneimine) (PEI) or lipofectamine, we enhance LP-association to cells. We demonstrate that our sorting procedure yields viable cells showing unchanged proliferation rates. Manufacturer-dependent differences in particle-cell interactions were observed and investigated by scanning and transmission electron microscopy (SEM/TEM) and flow cytometry. Finally, we show that the same sorting approach enables the generation of multi-cellular spheroids with a defined ratio of LP-containing and LP-free cells. This work lays the foundation for larger tissue engineering studies with defined LP populations and control over LP-placement with respect to the cell.

Vasculogenic mimicry (VM) describes the ability for tumor cells to mimic endothelial cells and form tumor-lined blood vessels. Although VM is associated with poor prognosis, resistance to therapeutics and metastasis, its presence, relevance and functionality have been debated, largely due to the techniques employed to determine its presence. Dual CD31 and periodic acid-Schiff (PAS) histological staining, the method widely used to detect VM within tumor tissue, is hampered by issues with artifacts of 2D sections and does not assess active blood flow through vessels and hence cannot confirm vessel functionality. Here, we present a 3D imaging pipeline to detect functionally perfused VM and host vessels within mouse tumor models. Mice tumor models are intravenously injected with fluorescently-labelled tomato lectin which binds to the lumen of functional blood vessels. These tumors are thickly sliced, optically-cleared, stained with endothelial markers and imaged using light-sheet microscopy. We apply this imaging modality to image tumors from six breast cancer patient-derived tissue xenograft models, an orthotopic mammary cancer model and two small cell lung cancer circulating-tumor cell explant models, where VM vessels, mosaic vessels and host-VM vessel junctions can be detected. To our knowledge, this is the largest collection of 3D VM vessel images to date, providing convincing evidence of the presence and functionality of VM within tumors and defining the level of evidence necessary to detect VM in vivo.

Significance: Fluorescence imaging remains largely qualitative and device-specific, limiting reproducibility and intersystem comparisons. SI-traceable radiometric characterization requires calibrated sources suitable for transferring known radiance to imaging systems. Aim: Establish and evaluate a compact, calibrated solid-state radiometric emitter target (RET) for SI-traceable radiance transfer, and assess methods for evaluating radiance invariance, imager responsivity, and extension of radiance transfer to digitally defined regions of interest (ROIs). Approach: The radiance invariance of an LED-based RET was evaluated by measuring its radiance across varying source-to-aperture distances and collection apertures. A previously described radiance-transfer formalism based on calibrated source radiance and collection geometry was implemented to determine imager responsivity (R{lambda}), which was evaluated across varying working distances and lens apertures. Grubbs and MAD outlier tests and one-way ANOVA were used to assess radiance and responsivity invariance. Finally, responsivity obtained using digitally defined ROIs was compared with matched physical apertures to assess ROI-based radiance transfer. Results: The measured RET radiance remained invariant across the tested source-to-aperture distances and collection apertures, with no significant differences observed across distance (p = 0.88) or aperture (p = 0.15). Imager responsivity remained stable across working distances for both tested lens configurations (p = 0.48 and p > 0.99). Responsivity was also invariant across lens apertures, except for deviations at f/11 that were consistent with practical aperture tolerances. Digitally defined ROIs reproduced responsivity values obtained using matched physical apertures, demonstrating that the radiance-transfer approach can be extended to image-defined regions. Conclusions: The presented methods provide an approach for evaluating compact LED-based sources for invariant-radiance emission and suitability for SI-traceable radiance transfer. The characterized RET supported stable imager responsivity across changes in imaging geometry and extension to digitally defined ROIs. These methods can be adapted to other LED-based sources, with further angular characterization and uncertainty analysis needed to establish broader applicability.

Image scanning microscopy (ISM) is emerging as the natural evolution of confocal laser-scanning microscopy, combining optical sectioning and a two-fold improvement in lateral resolution with high signal-to-noise ratio. Single-photon avalanche diode (SPAD) array detectors enable an effective implementation of ISM by recording a micro-image at each scan position with single-photon sensitivity, negligible readout noise, and without the fixed frame-readout constraints of conventional cameras. Nevertheless, SPAD array-based ISM remains largely confined to laboratories with expertise in microscope development. Here, we present BrightEyes-Scope, a reference implementation of a SPAD array-based ISM system. We provide step-by-step guidelines for optical design, system construction, alignment, calibration, data acquisition, and image reconstruction. Presented as a complete ISM microscope, BrightEyes-Scope also serves as a reference for upgrading existing laser-scanning microscopes. We anticipate that it will accelerate the adoption of ISM for quantitative super-resolution imaging.

The chick embryo is a widely used vertebrate model for developmental biology, cardiovascular, and bioengineering research. We present an automated laser speckle contrast imaging (LSCI) platform for non-invasive, longitudinal, label-free imaging of the developing heart and extraembryonic vasculature of the chick embryo through the intact eggshell. The platform can detect blood vessels as small as 70 +/- 17 um in diameter and enable repeated imaging from day 0 to day 8 of incubation without disturbing normal embryonic development. It provides both structural (a map of growing extraembryonic blood vessels) and functional (blood flow dynamics and heart rate) information. Using this system, we generated the first continuous, non-invasive, time-lapse recordings of the early functioning chick heart and its extraembryonic vessels. Longitudinal imaging was performed on 15 embryos from different chicken breeds with eggshell colors white, brown and green. Analysis revealed slight variability in early cardiac and vascular morphogenesis across breeds and shell types and shows its potential for automated developmental staging. Finally, for the first time, the growing vasculature of the yolk sac and the chorioallantoic (CAM) membrane was imaged from day 4 to 8. The heart rate and growth of the extraembryonic blood vessels were quantified automatically during imaging.

Sepsis is life-threatening, yet diagnosis remains a major global challenge because existing approaches lack speed and accuracy. Here we show that label-free imaging cytometry of 10 L whole-blood samples by Remoscope rapidly distinguishes patients with sepsis from those with non-infectious critical illness and healthy controls. In a prospective study of 29 critically ill adults, multi-physician adjudication identified sepsis (n=13), non-infectious critical illness (n=12), and indeterminate status (n=4); these patients were compared to healthy controls (n=22). A multiple instance learning (MIL) model pooled unbiased features from thousands of blood images per patient. Optimized for rule-out, the three-class model detected sepsis with 100% sensitivity and 85.3% specificity. Class activation heatmaps, unsupervised single cell embeddings, and morphometrics converged on abnormal leukocyte and erythrocyte morphology, including immature neutrophil-like cells. Label-free whole-blood imaging with MIL detects sepsis-associated hematologic signatures without stains or cold chain, establishing a foundation for rapid critical care diagnostics.

The development of NIR fluorescent dyes with favorable optical properties is of great importance especially for applications within the field of optical imaging. In this study, we report the design, synthesis and photophysical characterization of novel BODIPY-Xanthene hybrid dyes obtained via Knoevenagel condensation between an asymmetrical BODIPY core and the functionalized xanthene aldehydes intermediates. The synthesized dyes ex-hibit absorption (667 nm) and emission bands (720-722 nm) within the NIR region. Detailed UV-Vis and fluo-rescence studies were performed and molar extinction coefficients and stokes shifts also calculated. Moreover, findings highlighting the influence of structural modifications on the optical behavior of the hybrid dyes and their potential utility in optical imaging applications are reported.

The Transport-of-Intensity Equation (TIE) and polarization differential phase contrast (pDPC) exhibit transfer characteristics that limit their use as standalone quantitative phase imaging modalities. TIE provides stable recovery of low spatial frequencies but attenuates fine structure, while pDPC enhances phase gradients but is intrinsically insensitive near zero frequency. We present a hybrid reconstruction framework that combines a single-shot pDPC measurement with an in-focus-defocused TIE image pair and fuses the resulting phase estimates in the Fourier domain using frequency-selective weighting. The illumination geometry is co-designed to enforce angular separation between modalities, enabling high-frequency phase-gradient acquisition in a single exposure while preserving low-frequency sensitivity through low-numerical-aperture symmetric illumination. The proposed method requires three intensity measurements without requiring interferometric stability or sequential asymmetric illumination. Experimental results demonstrate improved phase fidelity across spatial frequencies, reduced halo artifacts, and enhanced recovery of both smooth and fine structural features. This approach provides a compact and robust method that moves towards extended spatial-frequency phase imaging for dynamic and resource-constrained applications.

Studying algae requires specialized equipment to characterize culture growth and physiological performance and non-invasive monitoring of these cultures typically relies on optical density (OD) measurements. Currently, OD is often measured using standard spectrometers that suffer from detector saturation at high cell densities, which requires time consuming sample dilutions and lacks compatibility with flat-walled cell culture flasks like Roux bottles. Here we describe a 3D-printable device to measure optically dense algal cultures directly within these cultivation vessels. This instrument combines light attenuation and scattering, resulting in a dual-sensor system that utilizes near-infrared light (950 nm) to precisely quantify biomass without interference from changing photosynthetic pigments. Cultures that reach high cell densities can be assumed to be accurately quantified in situ using the scattered signal, preserving the integrity of the culture and eliminating the need for subsampling. This method is adaptable to the most common geometries of Roux bottles (T-25, T-75, and T-175) and could be used to monitor small-scale batch cultures without contamination risks. A cyanobacteria algal species (Limnospira platensis) and three flask sizes were analysed on light attenuation linearity and scattering detection limits at different cell concentration, demonstrating that the light attenuation module can reliably measure biomass up to an OD of 3.1 (equivalent to >1.5 g/L) without requiring dilution.

Background: The Central Brain Tumor Registry of the United States (CBTRUS) uses a histology grouping model based on the World Health Organization (WHO) to classify cancer registry records for clinically relevant statistical reporting. Methods: CBTRUS, together with its consulting neuropathologists, reviewed and aligned its histology groupings with the 2021 WHO Classification update. Obsolete histology nomenclature along with their International Classification of Diseases for Oncology 3rd edition (ICD-O-3) codes were identified, currently included histologies were reclassified as appropriate, and new codes found in 2021 WHO were incorporated. An evaluation was conducted of the frequency of histology codes affected in the reclassification process, as well as incidence and survival pre- and post- reclassification of CBTRUS Tumor Type Groupings. Results: After review of the CBTRUS grouping scheme, 205 codes were reclassified and 2 new codes were incorporated. Tumor type groups most affected were those classified as glioma. Reclassification resulted in 167,038 of 491,804 (34%) cases having a grouping reclassification or reporting change, indicating that the 2021 revision of the WHO Classification has significantly impacted the collection and reporting of primary b CNS tumors. Conclusion: This work demonstrates the need to be responsive to changes in classification and coding for statistics to reflect real-world clinical use. This will require collaboration from all parties to have the ability to reconcile clinical practices and surveillance requirements.