Biomedical Optics Express

Three-photon microscopy enables deep-tissue imaging but is highly sensitive to excitation pulse quality, since three-photon excitation efficiency scales with the cube of instantaneous intensity. Group-delay dispersion (GDD) and third-order dispersion (TOD) accumulated through the laser and microscope optics can substantially reduce peak intensity at the focal plane, yet these quantities are rarely measured where imaging occurs. Here, we use third-harmonic generation (THG) interferometric autocorrelation, together with Dispersion Look-Up-Table Estimation (D-LUTE) and a joint two-measurement fitting procedure validated on synthetic data, to estimate baseline GDD and TOD directly at the objective focal plane, requiring only a compact autocorrelator module added to the microscope beam path. Applying this method at 1300 nm and 1600 nm excitation across two microscope systems equipped with different units of the same laser model, we find pulse durations 1.4- to 1.7-fold longer than the transform limit at every condition, driven predominantly by TOD, which varied by roughly 2.7-fold between the two systems. Using the endogenous THG signal from myelinated fibers, we further demonstrate in vivo pulse characterization in the mouse brain, finding no measurable broadening between the tissue surface and a depth of nearly a millimeter. This low-cost, easily implemented approach enables routine, in situ pulse monitoring across multiphoton microscopy platforms.

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 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.

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.

Fluorescence microscopy applications often require specialized instruments that are optimized for different experimental goals. Here, we present a reconfigurable microscopy module that integrates highly inclined swept tile (HIST) illumination for high-sensitivity single-molecule imaging and line-scanning confocal microscopy for rapid and optically sectioned volumetric acquisition. The system shares major hardware components, including lasers, scanning optics, and detection hardware, while employing unique beam shaping pathways to enable rapid switching between modalities without realignment. We characterize the module performance by measuring the excitation beam profiles, the point spread functions (PSF), and the optical transfer functions (OTF) across 40x, 60x, and 100x magnifications and demonstrate imaging applications including diffraction-limited fixed and live-cell volumetric imaging, fluorescence recovery after photobleaching, and super-resolution DNA-PAINT and single particle tracking (SPT). We also demonstrate the capability to execute multimodal imaging workflows by performing confocal imaging for chromatin density classification correlated with SPT data of nuclear proteins with diverse functions. Together, these results demonstrate a versatile imaging platform capable of supporting complementary fluorescence imaging modalities within a single instrument.

Mechanical stretchers are used to physically expand biological samples for microscopic studies of mechanobiology. Existing stretchers suffer from limited strain capacity and non-quantifiable forces, along with focus drift and feature-tracking limitations during microscopy. We develop an iris-based cell stretcher that applies isotropic equibiaxial force to achieve aerial strain of 1664% that corresponds to 4.2x linear expansion for Tensile Expansion Microscopy (TExM), a super-resolution method which increases sample size above the diffraction limit of light. The TExM stretcher uses 3D-printed, printed circuit board (PCB) and cost-effective parts, is portable and automated. We verify the performance of the TExM cell stretcher using image analysis, achieving ~90% mechanical precision, a maximum of four degrees of arm angular deviation, precise speed control down to 0.01 cm/sec, and an average expansion resolution of 3.98 x 10^-3 x. Integrated strain gauge sensors confirm equal application of force by each arm of the stretcher throughout expansion and finite-element simulation and planar-polariscope photoelastic imaging characterize the uniform substrate stress distribution while indicating high stress at the substrate gripping area. An AutoTracking software in communication with the stretcher hardware and microscope enables continuous autofocus and feature tracking during TExM and fiducial markers verify uniform equibiaxial stretch. The stretcher is demonstrated with fixed NIH 3T3 fibroblasts and live HeLa cells, observing ~4x cellular expansion of fixed cell size and separation of live cell clusters, highlighting the potential of TExM cell stretcher for biological imaging.

Diffuse optical measurements of blood flow, blood volume, and blood oxygenation often use separate devices and photon populations. Combining these contrasts within a common photon population would enable their direct co-registration while retaining time-of-flight-based depth discrimination. We introduce dual-wavelength time-of-flight interferometric speckle-contrast optical spectroscopy (TOF-iSCOS), built on an interferometric near-infrared spectroscopy (iNIRS) architecture that temporally multiplexes 780 and 852 nm swept lasers through a shared detection channel. It recovers co-registered flow-sensitive field dynamics and wavelength-resolved attenuation from the same complex-field acquisition. Three 50 ps TOF gates sample increasingly late-arriving photons. Within each gate, g_1 was calculated directly as the normalized temporal autocorrelation of the reconstructed complex field. {kappa}2 was reconstructed by finite-time integration of measured g_1 and finite-integration fits yielded decorrelation rates. No camera speckle-variance contrast or Siegert substitution was used. Gate-integrated TPSF intensities yielded optical-density changes {delta}OD, and division by the baseline-TPSF-weighted mean photon path length gave effective {delta}_a for two-wavelength hemoglobin inversion. In 12 forearm cuff-occlusion recordings from 11 adults, TOF-iSCOS detected flow suppression during occlusion and reactive hyperemia after release at both wavelengths. At 780 nm, the Late-minus-Early reactive-hyperemia amplitude in the relative blood-flow index (rBFI) was +0.647 (95% CI 0.194 to 1.124), whereas the Early-minus-Late cuff {delta}_a contrast was +0.196 cm^{-1} (95% CI 0.123 to 0.280). In the photon-limited 852 nm Late gate, the accepted-frame fraction was 0.994 for {kappa}^2-based fitting versus 0.831 for g_1-based fitting. Exploratory phase-resolved analysis suggested that the initial hemoglobin response was concentrated in early-arriving photons, while later responses differed in magnitude and direction. Wavelength, field dynamics, and photon time-of-flight thus provide complementary physiological information; the largest response need not identify the phase of interest.

While light-sheet microscopy is emerging as a robust method for volumetric imaging with improved axial resolution, its capability regarding two-dimensional, surface-level mapping is often hindered by limitations in data redundancy and reconstruction efficiency stemming from volumetric registration methods. We demonstrate that a multiview imaging approach in an axially-swept, dithered light-sheet microscope paired with computational image reconstruction of view projections is able to address these trade-offs to enable large-scale mapping of surface structural features, leveraging the advantages of multiview light-sheet in scalable field of view, working distance, and near isotropic resolution across the entire imaging depth. To aid in the acquisition and analysis of two-dimensional surface structures, we present a tailored surface mapping workflow and a Fiji plugin for computational reconstruction, promoting robust and comprehensive visualization of surface features of uncleared volumetric samples. Our strategy, termed projection reconstruction for imaging surface morphology (PRISM), integrates axially swept dithered light-sheet microscopy and post-processing software for multiview imaging. The imaging hardware enables near-isotropic resolution across its entire field of view, while the software implementation leverages rigid and affine transformations to align two-dimensional projections of multiview samples. It is designed to work with the BigStitcher pipeline, leveraging its robust algorithm to provide support for two-dimensional image alignment and stitching. We demonstrate the capability of PRISM in studies of lymphatic network mapping in the epicardial layer of intact mouse hearts, as well as surface profiles of FaDu spheroids labeled with antibody-nanodiamond conjugates. This method allows us to quantify cardiac lymphatic branch numbers, diameters, and lengths of a Prox1-tdTomato mouse cardiac model, as well as cluster number and diameters of epidermal growth factor receptor within a FaDu spheroid labeled with a nanodiamond-antibody conjugate, with a significant reduction of post-processing data size. PRISM leverages multiview image projections to promote studies of cardiac lymphatics in mouse models and surface receptor distributions within spheroid models, enabling efficient surface mapping of large, intact, and uncleared biological samples across a variety of scales.

Augmented reality glasses typically have poor optical efficiency (<1%), which is detrimental for high power applications, such as prosthetic vision. Maxwellian view optics are a candidate for increasing efficiency, but suffer from a very limited exit pupil and thus field of view. Many approaches to extend the eyebox inherently decrease the optical power. We present a design that uses a high-density VCSEL array and eye tracking to create an extended eyebox without sacrificing the advantages of a Maxwellian view display. Our system achieves a 20 degree field of view, a resolution 48.8 LP/mm (10 m), and an end-to-end optical efficiency of 83%

Bone adapts to its mechanical and physiological environment through the coordinated actions of osteocytes and marrow-resident cell populations. Because these processes depend on complex interactions within native tissue microenvironments, intravital imaging offers a unique opportunity to reveal cellular behaviors that cannot be fully captured ex vivo. However, the highly scattering nature of mineralized bone limits imaging depth and direct observation of cells within intact tissue. Two-photon (2P) microscopy has enabled important advances in bone biology, while three-photon (3P) microscopy has been proven to extend imaging depth and image quality in skeletal tissues. Here, we directly compared the performance of 2P and 3P microscopy in ex vivo mouse long bones by quantifying laser attenuation, signal-to-noise ratio, signal-to-background ratio, and spatial resolution, as well as assessed the impact of wave-front correction. We found that 2P and 3P microscopy generated comparable image quality through approximately 50 {micro}m of cortical bone. Beyond this depth, however, 3P microscopy provided superior brightness, contrast, and resolution, enabling improved visualization of structures deep within the cortex and at the cortical-marrow interface. To assess compatibility with intravital imaging, we evaluated endogenous markers of cellular stress during 3P imaging. Although prolonged continuous imaging decreased osteocyte spontaneous calcium signaling magnitude and increased autofluorescence, short intermittent imaging bouts produced negligible evidence of cellular damage compared to controls. Leveraging the enhanced penetration depth of 3P microscopy, we further show visualization of immune cell migration within the marrow cavity through intact cortical bone in both the third metatarsal (MT3) and tibia. Together, these findings affirm 3P microscopy as a powerful tool for studying cellular dynamics in living bone. By extending imaging beyond superficial cortical regions and enabling direct visualization of marrow-resident cells through intact bone, 3P microscopy expands opportunities to investigate osteocyte biology, marrow niche function, and skeletal adaptation 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.

Spectroscopic single-molecule localization microscopy (sSMLM) enables multiplexed super-resolution imaging by simultaneously acquiring the spatial position and spectral information of individual fluorophores. Dual-wedge prism (DWP)-based implementations provide a compact, alignment-stable approach to spectral dispersion, but trade-offs between localization precision, spectral precision, and experimental complexity remain. We systematically compare five DWP-based sSMLM configurations, including two-dimensional (2D) and three-dimensional (3D) implementations using single DWP (DWP-sSMLM) and symmetrically-dispersed DWP (SDDWP-sSMLM). We evaluate lateral precision, spectral precision, and ease of use. SDDWP configurations acquire spectral images in both channels and utilize both for spatial localization, yielding the highest lateral and spectral precision. However, for applications that do not require axial information, 2D-DWP provides a simple, plug-and-play solution with robust performance. This work offers a guideline for selecting DWP configurations based on experimental needs.

Significance: Continuous, non-invasive monitoring of cardiovascular physiology provides critical information for clinical decision-making and patient care. Emerging optical sensors enable non-contact measurement of physiological parameters such as heart rate and respiratory rate, but current methods are limited in their ability to capture spatially resolved physiological waveforms. Aim: We aimed to extend the capabilities of non-contact cardiovascular monitoring by using an imaging-based approach from which spatially resolved physiological waveforms can be extracted and cardiovascular biomarkers can be derived in vivo. Approach: We implemented a digital holographic imaging sensor to continuously measure calibrated tissue motion for in vivo assessment of cardiovascular biomarkers in six adult male Sprague-Dawley rats, with validation against electrocardiographic and invasive arterial blood pressure measurements. Results: Heart rate and pulse arrival time-derived pulse wave velocity calculated from digital holographic imaging demonstrate strong agreement with reference-derived metrics (concordance correlation coefficient [&ge;] 0.98). Heart rate variability shows moderate agreement with reference-derived metrics (concordance correlation coefficient [&ge;] 0.59).

Interactions between light and biological tissue could reveal disease-related changes that could be advantageous for rapid diagnosis when applied to whole limbs. Here, we tested the extent to which visible/near-infra-red hyperspectral imaging (HSI) deployed in the emergency department could classify cellulitis, non-cellulitis edema, and healthy lower-leg tissue across skin pigmentation types. We collected HSI data of the lower leg from 83 emergency-room patients and analyzed the calibrated normalized spectra (400-1000 nm) using a machine-learning classification algorithm, evaluated by cross-validation and benchmarked against standard spectral indices (oxygenation, hemoglobin, water, near-infrared perfusion). Classification at patient-level and pixel-level resolution performed comparably well using whole-spectrum and indices, for cellulitis versus healthy (AUC: 0.93 vs. 0.96) and non-cellulitis edema versus cellulitis (AUC: 0.92 vs. 0.89). Full-spectrum analysis substantially improved non-cellulitis edema versus healthy classification (patient-level AUC: 0.72 vs. 0.41; pixel-level AUC: 0.78 vs. 0.50), with no significant effect of skin type on accuracy.

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.

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.

Intracellular dynamics span a broad range of time scales and biomolecular processes, offering insights into cell health, functional state, phenotype, and response to external perturbations. Several label-free optical imaging approaches have been used to capture intracellular dynamics but are limited by spatiotemporal resolution and biomolecular specificity required to distinguish unique subcellular and metabolic processes. In this work, we demonstrate deep-ultraviolet (UV) microscopy as a powerful, label-free, high-resolution approach for quantifying multiscale intracellular dynamics with biomolecular specificity. By leveraging power spectral analysis and phasor analysis, we capture multiscale intracellular dynamics and analyze their UV wavelength-dependent behavior predicated by the absorption of different endogenous biomolecules. We apply this technique to prostate epithelial cell lines of increasing malignancy and reveal quantitative differences in dynamic intracellular activity that correlate with increased metabolic and organelle activity between phenotypes. Furthermore, we elucidate the molecular identities of structures and activity measured via UV dynamics with broad-band coherent anti-Stokes Raman scattering spectroscopy and fluorescence microscopy. We identify lipid-specific structures, and mitochondrial-specific dynamics, among other biomolecular-specific dynamic behaviors. Together, this study demonstrates deep-UV microscopy as a powerful imaging platform for probing spatial and temporally variant intracellular dynamics with biomolecular specificity, with broad implications for cell phenotyping, tissue pathology, and studying new dynamic subcellular processes.

Background Tumor hypoxia and abnormal vasculature are closely associated with aggressiveness in solid tumors. Therefore, noninvasive assessment of these features in primary breast cancer is needed. Photoacoustic (PA) imaging is an emerging modality that enables real-time visualization of vascular architecture and hemoglobin oxygenation. Methods Breast PA imaging was performed in patients with primary breast cancer using a bed-type PA imaging system equipped with a hemispherical sensor and a flat specimen holder enabling mild breast compression. Three independent evaluators assessed predefined characteristics of tumor-associated vasculature: centripetal/disrupted vessels and intratumoral vessel-like signals. Oxygenation (S-factor) of tumor-associated vessels was estimated using dual-wavelength laser irradiation at 756 and 797 nm. Results PA imaging was performed in 9 tumors from 8 patients. Eight tumors were evaluable, after the exclusion of 1 tumor with segmental bloody discharge. Centripetal/disrupted vessels were identified in 7 tumors (87.5%). Intratumoral vessel-like signals were observed in all tumors (100%), with higher signal density than in surrounding tissue in 5 lesions (62.5%). Increased intratumoral signal density was associated with a higher Ki67-labeling index (two-sided P = .01). Mean intratumoral S-factor level (76.9% {+/-} 9.1%) was significantly lower than that of peritumoral vessels at 5 mm (86.4% {+/-} 5.9%) and 20 mm (88.5% {+/-} 4.9%) from the tumor margin (two-sided P < .01). Conclusion PA imaging with a flat specimen holder enables noninvasive visualization of tumor-associated vasculature with reduced oxygenation in primary breast cancer. This approach may provide a novel imaging platform for the early detection and functional assessment of breast cancer.

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.

Advances in single-photon sensitive detectors are rapidly expanding the adoption of photon-counting fluorescence microscopy. Under the Poisson photon-counting statistics, iterative Richardson-Lucy (RL) -type algorithms are statistically optimal for image deconvolution but suffers from a fundamental semi-convergent behaviour: prolonged iterations inevitably amplify noise, requiring heuristic early stopping or regularisation typically based on assumptions about object morphology. Here we present a morphology-agnostic regularisation framework for RL deconvolution that exploits independent noise realisations instead of structural object priors. Preserving the Poisson statistics of the acquisition process, we formulate the Regularized by Noise (RbN): a regularized variational framework and its corresponding iterative minimization algorithm that exploit the statistical consistency of independent noise realisations to distinguish reproducible image features from stochastic noise. The regularisation strength is selected automatically using the Poisson residual whiteness principle, resulting in a fully data-driven reconstruction without heuristic parameter tuning. Photon-timing-resolved systems naturally provide the independent noise realisations exploited by our framework, whereas computational photon splitting provides a statistically equivalent implementation for photon-counting systems. We validate the approach experimentally using photon-timing-resolved confocal microscopy and image scanning microscopy across eight morphologically distinct subcellular targets, and further demonstrate its applicability to photon-counting microscopes through computational photon splitting. Across imaging modalities, detector technologies, biological structures and signal-to-noise regimes, our method eliminates RL semi-convergence, removes sensitivity to the stopping criterion, and consistently outperforms conventional RL while preserving fine structural detail. More broadly, our results establish independent noise realisations as a general source of morphology-agnostic regularisation. Although demonstrated here for SPAD-based laser-scanning microscopy and Poisson statistics, the underlying principle could be extended to other imaging modalities and, more generally, to statistical inverse problems through appropriate noise-specific formulations.