Biology subjects

Ruiz, A. J.

Publications and source records attributed to Ruiz, A. J..

3 recordsLinked to original sources

bioRxiv2026

Radiance-Invariance Evaluation of a Compact LED-Based Source for Imager Radiance Transfer

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.

bioengineering↗

bioRxiv2025

Indocyanine green excitation-emission matrix characterization: spectral shifts and application-specific spectra

SignificanceIndocyanine green (ICG) is the most widely used fluorophore in fluorescence-guided surgery (FGS), yet its spectral response depends on microenvironment, with implications for system design, inter-system comparisons, and phantom development. AimTo characterize ICG with excitation-emission matrices (EEMs) in the microenvironments of dimethyl sulfoxide (DMSO), bovine serum albumin (BSA) solutions, and 3D-printed (3DP) resin, and assess excitation-dependent emission, including red-edge excitation shifts (REES) and departures from Kashas rule of excitation-independent emission. ApproachEEMs and absorbance spectra were acquired with extracted excitation spectra, emission spectra, emission peaks, centroids, and integrated emission areas under the curve (AUCs). Concentration-dependent behavior was examined in DMSO, and albumin concentration dependence was assessed from 5-100 mg/mL. Data processing employed robust local regression to mitigate excitation scattering artifacts. ResultsICG in DMSO exhibited excitation-independent emission consistent with Kasha-Vavilov behavior. In contrast, ICG in BSA solution and 3DP resin displayed excitation-dependent emission with pronounced REES and additional non-linear departures from Kashas rule. To our knowledge, this represents the first documentation of REES and broader anti-Kasha effects for ICG or any FGS fluorophore. Within the excitation range most relevant to ICG-FGS ([~]760-805 nm), emission spectra of the BSA solution and 3DP resin overlapped closely, with similar AUC-based comparisons, suggesting that ICG in 3DP resin can serve as a suitable surrogate reference for albumin-bound ICG. ConclusionsThe EEM characterization shows that excitation-dependent behavior is a defining feature of ICG in biologically relevant environments, demonstrating that emission cannot be assumed to follow classical Kasha-Vavilov behavior. Reliable comparisons and imaging system design therefore require spectra acquired at defined excitation wavelengths with AUC integration within the emission detection band. Excitation-specific spectra from EEMs establish a consistent framework for inter-system comparisons and phantom standards, while the resulting datasets provide a practical reference for addressing excitation-dependent behavior in ICG sensing applications.

biochemistry↗

bioRxiv2025

A Phantom for Fluorescence Uniformity and Distortion Assessment of Near-Infrared Fluorescence Guided Surgery Systems

SignificanceThe expanding use of fluorescence in surgery necessitates standardized characterization methods to facilitate reproducibility and regulatory review of imaging devices. Current guidelines suggest the use of optical phantoms as tools to quantify optical system performance, yet measurements of uniformity and spatial accuracy or distortion remain challenging and are performed in an ad hoc manner or not collected at all. AimThis work introduces a photostable solid phantom, the Reference Uniformity and Distortion (RUD) phantom, and accompanying analysis code for characterizing fluorescence uniformity and geometric distortion. Additionally, the concept of fluorescence flat-field correction is explored using this phantom. ApproachThe RUD phantom was imaged on a custom fluorescence imaging device, as well as five commercial systems. The analysis code characterized uniformity and distortion in these systems. Flat-field correction was explored on the custom device by imaging solid fluorescent reference phantoms at different locations within the field of view. ResultsSuccessful characterization of the imaging systems uniformity and geometric distortion was achieved. Flat-fielding experiments showed that while it qualitatively improves the appearance of images, it could negatively impact quantitative analyses. ConclusionsThe RUD addresses the need for standardized characterization of fluorescence uniformity and geometric distortion. While fluorescence flat-field correction qualitatively enhances image uniformity, caution is advised as it may adversely affect quantitative accuracy.

bioengineering↗