Littler, E. A. | bioRxiv Science

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

Quantitative Fluorescence Imaging with SI-Traceable Radiance: Radiometric Fluorescence Characterization with a Calibrated Solid-State Emitter

SignificanceFluorescence imaging remains largely qualitative and device-specific, limiting reproducibility and intersystem comparisons. Advancing toward quantitative imaging requires a radiometric characterization framework that provides SI-traceable units while explicitly addressing the interdependent factors that govern image formation. AimEstablish and evaluate a radiometric characterization framework that converts device-native counts to SI-traceable imaged radiance ({micro}W{middle dot}cm-2{middle dot}sr-1) and aggregate fluorescence yield (sr-1) while accounting for interdependent factors that influence fluorescence image formation. ApproachA calibrated Lambertian solid-state radiometric emitter target (RET) was combined with a three-step radiometric framework consisting of the Radiance Transfer Curve (RTC), the Radiance Imaging Transform (RIT), and the Fluorescence Imaging Transform (FIT). The RTC establishes system responsivity as a function of radiance; the RIT applies this calibration at the pixel level to map digital counts to SI-traceable radiance; and the FIT performs pixel-wise excitation normalization of the RIT image to produce an aggregate fluorescence yield image. The framework was tested through distance and aperture invariance, digital-vs-physical ROI analyses, RTC acquisition, and application of the RIT and FIT to an ICG concentration target and a breast lumpectomy phantom. ResultsThe RET exhibited Lambertian behavior, with no significant dependence of the measured radiance on distance or aperture; imager responsivity (R{lambda}) also remained invariant within uncertainty across working distances and f-numbers. Digitally masked ROIs reproduced R{lambda} obtained with matched physical apertures, enabling ROI and pixel-level radiance transfer. RTCs acquired over 49 radiances captured sensor and processing nonlinearities. The RIT provided a per-pixel mapping from counts to radiance ({micro}W{middle dot}cm-2{middle dot}sr-1). Applying pipeline-specific RTCs, the RIT and FIT reconciled large discrepancies across RAW, 8-bit, and log10 image processing pipelines, yielding closely aligned radiance-concentration curves and improved SBR/SNR/CNR agreement. In a breast lumpectomy phantom, FIT produced SI-traceable aggregate fluorescence yield (sr-1) images and absolute contrast metrics in an anthropomorphic geometry. ConclusionsThe combined framework converts device-native counts into SI-traceable radiance and aggregate fluorescence yield at the pixel level, providing a practical basis for reproducible quantitative fluorescence imaging. The feasibility results across distance/aperture tests, ROI analyses, image pipelines, and phantom imaging indicate readiness for broader evaluation. Future work will establish formal uncertainty budgets and assess robustness across devices, geometries, and excitation conditions to support adoption as a quantitative reporting standard.

bioengineering↗