Journal of Biomedical Optics

We investigate the effects of skin pigmentation and light source characteristics on the performance of reflective Pulse oximetry (PO) devices used in healthcare and well-being applications. We use Monte Carlo (MC) simulations to compare ideal monochromatic and realistic LED spectral emission profiles and tolerance-related wavelength shifts. The simulation covers photon transport in skin models with melanin concentrations (2.55% to 30.5%) and arterial oxygen saturations SaO2 (70% to 100%.) Accuracy was assessed by SpO2 error, root-mean-square error RMSE (Arms), and percentile tail-errors (P90, P95, and P99). Monochromatic spectral emission yielded the lowest SpO2 error (RMSE = 1.32), while LED spectral emission profiles increased errors (RMSE = 2.10). Infrared wavelength tolerances increased SpO2 RMSE by 1.1 {+/-} 0.3. SpO2 error increased with melanin concentration, from underestimation (-1.8 {+/-} 0.1%) at 2.55% melanin concentration to overestimation (+3.9 {+/-} 1.2%) at 30.5% for low SaO2 (70%) and LED spectral emission profiles. At 30.5% melanin concentration, P95 and P99 exceeded FDA and DIN EN ISO 80601-2-61 thresholds, in particular at low SaO2 (70%). Clipping SpO2 estimates at 100% resulted in an apparent RMSE decrease of up to 3%, reflecting error masking rather than real error reduction. In conclusion, LED spectral emission profiles and wavelength tolerances can amplify melanin-related bias in SpO2 estimates. Monochromatic emission and tighter wavelength control can reduce SpO2 error and should be considered in device design and regulation. Regulatory standards should discourage clipping SpO2 estimates at 100% and mandate additional metrics as RMSE fails to reflect clinically critical percentile error thresholds, i.e. P95 and P99.

Cerebral blood volume (CBV) and blood flow (CBF) constitute key metrics for cerebrovascular monitoring, enabling assessment of stroke severity and risk-prediction, aging-related changes, and neurological diseases. CBF and CBV monitoring are key aspects in diagnosis, treatment triage, and clinical outcome of ischemic and hemorrhagic strokes. In recent years, there have been ongoing efforts toward the development of optical devices for noninvasive monitoring of CBV and CBF. Speckle contrast optical spectroscopy (SCOS) has recently emerged as a strong candidate for clinical translation in monitoring CBF and CBV, due to its affordability, compact and wearable design, and noninvasive nature. However, experimental demonstrations that SCOS can effectively monitor brain hemodynamics remain sparse. This is primarily due to challenges in design experiments that isolate cerebral blood dynamics from those in the scalp and skull. In this paper, we report experiments using SCOS to monitor cerebral hemodynamics in rats during intracerebral blood flow modulation. To modify cerebral blood dynamics, a surgical procedure was performed to insert a catheter for direct injection of flow modulation fluids into the brain. Using the SCOS device, we monitored changes in CBV during deliberate CBF interventions into the brains of five rats. A saline solution was also injected as a sham control of the flow intervention. The results show a significant decrease in CBV during injection, followed by a return to baseline. This behavior is consistent with physiological expectations, as the injected fluids dilute the blood, leading to a transient reduction in blood volume. Notably, the CBV decrease induced by the flow modulation fluid solution required more than twice as long to recover to baseline compared with the saline solution, which is consistent with the delayed clearance of the flow modulation fluid by design. These experimental results demonstrate the effectiveness of SCOS for monitoring cerebral hemodynamics in animal models and highlight its potential for translation to human studies. Moreover, this work paves the way for the testing and characterization of cerebral therapeutic agents intended for blood flow modulation in animal models.

Objective and RationaleBrain biomechanics is a rapidly evolving field, with mechanical properties influencing both normal development and pathological conditions such as cancer. Brillouin microscopy, a non-contact optical technique, offers a promising approach for studying the biomechanics of fresh brain tumors and organoids at subcellular resolution. However, challenges such as tissue heterogeneity and signal attenuation necessitate an in-depth evaluation of measurement strategies and potential confounding factors. MethodsFresh human brain tumor samples and tumor organoids were analyzed using Brillouin microscopy with 780 nm excitation. Measurements in the form of maps of various size were performed, and the impact of focal position, tissue heterogeneity and blood contamination on Brillouin data was assessed. Complementary Raman spectroscopy was performed as reference for tissue composition. ResultsBrillouin signal intensity decreased exponentially with depth, with valid measurements achievable up to 80 {micro}m. Low signal intensities at greater depths compromised data reliability due to fitting algorithm limitations. Structural heterogeneity, including different cell types, differentially affected signal attenuation. Blood contamination was identified as a major confounder, leading to erroneous biomechanical readings. Brillouin intensity maps provided essential quality control for accurate data interpretation. Raman spectroscopy identified the presence of blood and tissue-specific biochemical signatures, reinforcing the importance of multimodal analysis. ConclusionsBrillouin microscopy can effectively probe biomechanical properties of fresh brain tumors but is influenced by tissue heterogeneity and contaminants. Proper sample preparation, strategic focal positioning, and complementary techniques like Raman spectroscopy are critical for ensuring reliable data. These findings contribute to refining Brillouin microscopy protocols for neuro-oncological research and potential future clinical applications.

Airway epithelium plays a major role as the primary interface between human body and the external environment, acting both as a physical and functional barrier. In vitro airway models that reproduce the epithelium architecture are therefore a valuable tool for studying infection, inflammation, and transport processes. In this work, we present a label-free, non-invasive method to visualize and measure mucociliary transport in air-liquid human models using third-harmonic generation (THG) microscopy with an optical parametric amplifier laser source at 1300 nm. By exploiting the intrinsic nonlinear contrast at optical heterogeneities, THG provides high-resolution images of both epithelial structures and of the overlying mucus layer without the need for fluorescence staining or sample processing. Time-lapse THG imaging reveals depth-dependent transport dynamics within the mucus, offering new insights into mucociliary transport mechanism. Our approach offers a physiologically relevant way to assess mucociliary function in vitro and could support studies on respiratory diseases, drug delivery and efficacy, and epithelial remodeling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/717621v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@62e8acorg.highwire.dtl.DTLVardef@199a8b7org.highwire.dtl.DTLVardef@113bb84org.highwire.dtl.DTLVardef@7be3f8_HPS_FORMAT_FIGEXP M_FIG For Table of Contents Only C_FIG

Collagen remodeling in the uterine cervix is a vital process in pregnancy that allows for timely fetal delivery, yet its spatio-temporal details are still not fully understood. In this study, we measured collagen reorganization at different stages of murine gestation and at various cervical depths. We used polarization-resolved Second Harmonic Generation microscopy to specifically detect fibrillar collagen and assess its orientation with sub-micrometer resolution. We imaged large cervical areas using automated mosaicking and implemented an analysis pipeline that showed significant region-dependent changes in collagen quantity, porosity, and orientation disorder. Notably, we found that collagen disorganization begins in the lower cervix at gestation day 12 and extends throughout the entire cervix by day 15. Additionally, we demonstrated that the temporal dynamics of disorganization, without spatial sensitivity, can also be tracked using Mueller Matrix imaging, which is a clinically deployable method. These findings should improve understanding and diagnosis of gestation-related issues such as premature birth.

SignificanceQuantifying lipid droplet (LD) remodeling in 3D hepatic organoids is often limited to endpoint staining or phototoxic live fluorescence imaging, thereby obscuring droplet-level kinetics. AimWe aimed to develop a label-free method to track LD dynamics in living hepatic organoids under different fatty-acid loads. ApproachTime-lapse 3D refractive-index tomograms were acquired using holotomography and analyzed with a depth-adaptive, multi-threshold segmentation pipeline to quantify LD number, volume, sphericity, and refractive-index-derived concentration and dry mass at single-droplet resolution. ResultsOleic acid and linoleic acid induced LD accumulation while preserving organoid integrity, whereas palmitic acid triggered rapid structural collapse. Despite increases in total LD burden under both oleic acid and linoleic acid, droplet-level dynamics diverged: oleic acid produced volume-dominated accumulation via enlargement of fewer LDs and increased size heterogeneity, whereas linoleic acid produced number-dominated accumulation via sustained increases in LD number, yielding a more uniform population of small droplets. ConclusionsLabel-free holotomography with depth-adaptive analysis enables non-invasive, longitudinal, and multi-scale quantification of LD dynamics in intact organoids and reveals fatty-acid- dependent temporal modes of lipid storage. Statement of DiscoveryWe developed a label-free, longitudinal 3D holotomography framework with depth-adaptive lipid droplet segmentation that quantifies single-droplet dynamics in living mouse hepatic organoids. Using this platform, we found that oleic acid and linoleic acid induce LD accumulation via distinct strategies--oleic acid via droplet enlargement and linoleic acid via sustained increases in droplet number--while palmitic acid rapidly compromises organoid integrity.

Time domain Dynamic full-field optical coherence tomography (D-FFOCT) is a powerful label-free imaging modality that enables functional visualization of cellular activity in living tissues with subcellular resolution. However, its sensitivity remains a major limitation for imaging highly scattering three-dimensional (3D) biological models such as retinal organoids, where incoherent background and inefficient optical flux distribution reduce dynamic contrast and limit imaging depth. In this work, we introduce a ratio-free optical configuration for time-domain D-FFOCT that enables continuous tuning of the sample-to-reference field ratio while minimizing photon losses and suppressing parasitic reflections. This polarization-based architecture allows optimal redistribution of optical flux according to sample scattering conditions and improves sensitivity under both power-limited and dose-limited conditions. Compared with conventional non-polarizing beam splitter configurations, the proposed approach provides a [Formula]-fold (3 dB) sensitivity improvement through optical optimization alone. In addition, we investigate for the first time the use of partial field illumination (PFI) in time-domain D-FFOCT to reduce incoherent background arising from multiple scattering. In retinal organoids imaged at 120 {micro}m depth, PFI yields up to a 14.5-fold (23.2 dB) increase in dynamic signal sensitivity, while preserving functional contrast. When combined, ratio-free detection and PFI provide a cumulative sensitivity improvement of 20.5-fold (26.2 dB). These gains enable improved visualization of photoreceptor precursor organization, rosette structures, and Muller glial cell dynamics in both 3D retinal organoids and 2D cell cultures. This work establishes a practical framework for sensitivity optimization in D-FFOCT and expands its potential for functional imaging, disease modelling, and live-cell monitoring in complex biological systems. O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/719402v1_ufig1.gif" ALT="Figure 1"> View larger version (123K): org.highwire.dtl.DTLVardef@1651e7org.highwire.dtl.DTLVardef@15b42e5org.highwire.dtl.DTLVardef@850180org.highwire.dtl.DTLVardef@25a3cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tissue-clearing techniques have transformed optical imaging of fixed specimens, yet their application to living systems remains limited by toxicity and removal of key tissue components. We recently demonstrated that absorbing molecules such as tartrazine can reversibly render live mouse skin transparent. Subsequently, it was reported that isotonic protein solutions can achieve ex vivo and in vivo cellular clearing. However, discrepancies remain regarding the optimal refractive index (RI) for live-cell clearing and the impact of elevated osmolality on cell viability. Here, using cultured mammalian cells, we systematically examine the dependence of optical contrast on medium RI and the effects of hyperosmolality. We find that, contrary to the recent report of an optimal RI of 1.36[~]1.37 for suspended cells, densely-packed adherent cells exhibit a monotonic decrease in phase contrast up to an RI of 1.41 with tartrazine. Moreover, even under highly hyperosmotic conditions ([~]1200 mOsm/kg), cultured cells exhibit minimal deformation and negligible loss of viability for up to 30 min in the clearing solution. These results demonstrate that tartrazine enables effective live-cell clearing at RI up to 1.41 while preserving viability under elevated osmolality, and motivate future studies to define optimal conditions for in vivo optical clearing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/717314v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ec9fbforg.highwire.dtl.DTLVardef@1ebe9e6org.highwire.dtl.DTLVardef@1492c3corg.highwire.dtl.DTLVardef@f75559_HPS_FORMAT_FIGEXP M_FIG C_FIG

Stimulated emission depletion (STED) microscopy is a super-resolution fluorescence imaging technique that achieves high spatial and temporal resolution by exploiting stimulated emission to induce fluorescence depletion (FD) and is expected to have substantial utility for imaging applications using fluorescent proteins. However, the compatibility of fluorescent proteins with STED microscopy systems has been understood primarily through empirical observations, and there is no established methodology for the rational selection of fluorescent proteins for STED microscopy. In this study, we systematically evaluated the compatibility of commonly used fluorescent proteins with STED microscopy systems by measuring FD properties using transient absorption spectroscopy and fluorescence dip spectroscopy, both of which are classified as two-color spectroscopy (TCS). Fluorescent proteins identified as compatible with the STED microscopy system based on the TCS measurements were employed for three-dimensional STED imaging of cellular samples expressing each protein. In all samples, three-dimensional spatial resolution was improved relative to confocal laser microscopy, with particularly marked improvements in z-axis resolution. These findings demonstrate that measurements of FD properties via TCS provide a robust approach for evaluating the compatibility of fluorescent proteins with the STED microscopy system and for selecting suitable fluorescent proteins for STED imaging.

Cardiac rhythm is a critical clinical indicator for cardiac arrhythmias and adverse events during drug toxicity studies. In vivo, cardiomyocyte responses to pharmacological agents occur within minutes and are strongly influenced by dynamic drug delivery through blood flow. However, conventional 2D and 3D static culture systems fail to replicate these fluid flow kinetics, limiting their physiological relevance for assessing beat rate responses. Here, we present Mera, an advanced microphysiological system (MPS) developed by Hooke Bio, designed for high-throughput, long-term culture and functional analysis of 3D cardiac spheroids composed of human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts. Mera enables dynamic perfusion, allowing investigation of cardiomyocyte beat rates under physiologically relevant flow conditions. The platform supports up to 640 spheroids per run and integrates automated imaging, fluid handling, and user-friendly software, operating under controlled physiological conditions (37{degrees}C, 5% CO2). Flow rates are tunable between 0 and 12.5 mL/min to mimic in vivo environments. Pharmacological testing with verapamil, isoproterenol, calcium chloride, and propranolol demonstrated real-time, reversible modulation of beat rate under flow, including recovery following drug-induced suppression. System variability was comparable to a temperature-controlled reference platform, supporting robust statistical analysis. Dose-response studies yielded IC values consistent with literature, confirming physiological relevance. Collectively, these results demonstrate that Mera provides a reproducible, scalable, and human-relevant platform for cardiac drug testing. By enabling dynamic drug exposure and automated analysis, Mera represents a powerful new approach methodology (NAM) for improving the predictive assessment of cardiac safety and beat-rate modulation drug responses.

AimsTo characterize subtype-associated heterogeneity in type 2 diabetes mellitus (T2DM), particularly normal-weight diabetes, using extracellular vesicle (EV)-associated molecular features in a clinically stratified cohort. MethodsEVs were isolated from plasma using ExoTIC and validated by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry, and Western blotting. EVs from Asian normal-weight (A-NWD), Asian overweight (A-OWD), Non-Hispanic White normal-weight (W-NWD), and Non-Hispanic White overweight (W-OWD) T2DM patients were analyzed by multimodal surface-enhanced Raman spectroscopy (SERS; n=65) and EV-RNA sequencing (n=39). ResultsSERS identified subgroup-associated spectral fingerprints that distinguished the four BMI- and race/ethnicity-defined groups in this cohort. EV-RNA sequencing revealed differential microRNA expression across subgroups, with higher miR-208a and miR-132 in A-OWD and higher miR-484 in A-NWD. Unsupervised analyses also showed partially overlapping EV-associated molecular features between A-NWD and W-OWD, suggesting that BMI-based subgrouping alone may not fully capture shared metabolic states. ConclusionsMultimodal EV profiling identified subgroup-associated spectral and miRNA features in clinically stratified T2DM and provides a framework for studying diabetes heterogeneity, including molecular patterns associated with normal-weight diabetes.

SignificanceDiffuse in vivo flow cytometry (DiFC) is an emerging technique for enumerating rare, fluorescentlylabeled circulating tumor cells (CTCs) in small animals without drawing blood samples. DiFC uses detection of transient fluorescent peaks in time-series data. Previously, we used a simple amplitude threshold-based method for identifying peak candidates, but it ignores potentially useful information in peak shape that could reduce false-positive detections from instrument noise and increase detection efficiency of lower-amplitude peaks. AimTo develop a machine learning (ML)-integrated signal processing approach for improved CTC enumeration using DiFC by distinguishing CTC peaks from artifacts. ApproachWe developed an ML-integrated approach that incorporates a convolutional neural network (CNN) classifier. The CNN was trained to distinguish CTC peaks from artifacts by analyzing peak amplitude and temporal shape characteristics. Performance was validated on in-silico, control, and CTC-bearing mouse datasets. ResultsThe CNN classifier achieved accuracy, precision, sensitivity, and specificity exceeding 98% on test data. Compared with our previously published threshold-based approach, the ML-integrated method increased the number of correctly identified CTCs and their flow direction while reducing false detections across validation datasets. ConclusionsThe ML-integrated approach significantly improves DiFC CTC enumeration, enabling robustness against artifacts in noisy conditions.

Invertebrate vision relies on bistable visual pigments flipping upon photon absorption between rhodopsin and metarhodopsin states. In living butterflies, the UV-VIS absorption spectra of rhodopsin and metarhodopsin, respectively with 11-cis and all-trans isomers of 3-hydroxy-retinal (A3) chromophore, can be conveniently recorded from the eyeshine, the light reflected from the compound eye after passing twice through the light-guiding rhabdoms. * Here, a microscope coupled with a broadband LED source and a microspectrometer was used to record photorelaxations reported in eyeshine reflection spectra. Fitting temporal exponential relaxations to log-reflectance arrays yielded transient and baseline spectra that are analogous to absorbance difference and sum, respectively. Both types of spectra were subjected to singular value decomposition and to fitting of templated visual pigment absorption spectra. * The compound eye of the high brown fritillary Fabriciana adippe was exposed to a series of second-long broadband light pulses, causing photorelaxations with time constants between 40 and 120 ms that led to 80% metarhodopsin in equilibrium. The transient and baseline spectra were fitted with pigment templates, estimating the alpha peak wavelength 547-552 nm for rhodopsin and 496-501 nm for metarhodopsin. The metarhodopsin to rhodopsin alpha peak absorbance ratio 1.25-1.35 is consistent with the isosbestic wavelength at 530 nm. The second isosbestic wavelength indicates that rhodopsin beta (UV) peak absorbs more strongly than metarhodopsin below 405 nm. * Baseline spectra, which were not explicitly analysed in previous studies, enable concatenation of exposures, monitor long-term changes of pigment, and enhance the estimation of beta peak parameters. * The method can be directly used in many butterflies and could be adapted to other insects, particularly fruitflies, facilitating studies of the relation between the visual pigment spectra and the opsin sequences. Spectroscopic results can be complemented with physiologically measured photoreceptor spectral sensitivity datasets and analysed with the same global fitting procedure.

1.We report a repetition-controllable gain-managed nonlinear fiber amplifier (GMNA) that delivers near-infrared 50-fs pulses with pulse energies up to 150 nJ and a widely tunable repetition rate from 1-20 MHz, while maintaining stable pulse quality across the full range. Using this source, we demonstrate label-free multiphoton imaging--including metabolic autofluorescence (2PF/3PF), second/third-harmonic generation, and Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy imaging--across live cells, human lung spheroids, and hard tissues. We further assess the impact of laser repetition rate on photodamage at fixed pulse energy, supported by preliminary measurements indicating lower damage at lower repetition rate. Collectively, the compact architecture and repetition-rate agility of the GMNA enable real-time optimization of imaging speed, depth, and sample safety for advanced biological microscopy.

BackgroundPhotobiomodulation (PBM) therapy has demonstrated therapeutic potential in promoting cellular repair, modulating inflammation, and enhancing mitochondrial function. Platelet-rich plasma (PRP) is widely used in regenerative medicine due to its concentration of growth factors and cytokines. Very small embryonic-like stem cells (VSELs), a rare population of pluripotent stem cells present in adult tissues, have emerged as a potential contributor to tissue regeneration. While PBM and PRP are used in combination, how VSELs or Multi-lineage stress enduring (MUSE) cells are at play, and the biological mechanisms underlying their synergistic effects remain incompletely characterized. ObjectiveThis exploratory pilot study aimed to evaluate whether application of the MD Biophysics laser to autologous PRP is associated with measurable changes in VSEL-related antibody marker expression, and to identify directional trends to inform future controlled studies. MethodsPRP samples were collected from participants across seven test dates (July 2024 to February 2025), yielding 18 participant-session datasets. Samples were analyzed before (Pre) and after (Post) laser application using flow cytometry conducted at a UCLA Flow Cytometry Laboratory. Four VSEL-associated antibody markers were assessed: CD45-CD34+, CXCR4+, CD133+, and SSEA-4+. Analyses were descriptive and focused on paired differences and directional trends due to the exploratory design and absence of a control group. ResultsThree of four VSEL-associated markers (CXCR4+, CD133+, and SSEA-4+) demonstrated a group-level increase in median paired differences following laser application. Directional increases were observed in 12/18 sessions for CXCR4+, 10/18 for CD133+, and 9/18 for SSEA-4+. CD45-CD34+ showed a near-equal distribution of increases and decreases. Ki-67 positivity indicated the presence of viable, proliferative cells. While no findings reached statistical significance due to limited sample size, consistent directional trends were observed across multiple markers. ConclusionApplication of PBM to autologous PRP was associated with directional increases in multiple VSEL-associated antibody markers, suggesting a potential role for stem cell activation or mobilization in the mechanism of action. Although preliminary and not statistically powered, these findings provide hypothesis-generating evidence supporting further investigation. The observed trends informed iterative protocol refinement and establish a foundation for future controlled, adequately powered studies to evaluate clinical efficacy and underlying biological mechanisms.

This study investigates a novel light-activated drug delivery system designed to produce on-demand drug release. The light-activated system was developed by incorporating a photostable photothermal agent, croconium dye, into liposomes to enable thermally triggered drug release. The drug release from the liposomes was determined at three powers of 210, 295, and 380 mW under 0-, 1-, and 2-minute light irradiation. A continuous wave 808 nm laser was used as the light source. Dexamethasone sodium phosphate (DSP) released from the liposomes was tunable depending on the power and irradiation time with a range of 1 -19 g released depending on irradiation power and time. For local temperature measurement during the photothermal activation, polymerized 10, 12 - Pentacosadiynoic acid (PCDA) was incorporated in the lipid bilayer. Under heating polymerized PCDA undergoes a transition into a red phase from a blue phase. Utilizing the spectrum changes under known temperatures a regression model was developed to calculate the local temperature of the liposomes under irradiation. The ability of the liposomes to release DSP under irradiation in the presence of a phantom tissue was tested under different attenuation coefficients to match various common biological tissues. The liposomes were still able to release DSP in the presence of tissue phantoms for a certain thickness of the tissue. Finally, the cytotoxicity of the liposomes with the croconium dye for chemical and thermal toxicity was determined. The liposomes displayed good biocompatibility with Human Microvascular Endothelial Cell line-1 (HMEC-1). The results support the use of croconium dye as a potential alternative to commonly photothermal agents used in drug delivery such as metal nanoparticles. Future work will focus on optimization of absorbance spectrum for drug release, and in vivo studies for efficacy and safety.

Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture (NA) objective for both illuminating the sample and collecting emission fluorescence from a tilted plane within the specimen. OPM has become indispensable in biological and biomedical research, providing rapid, high-resolution volumetric fluorescence imaging of live cells and tissues while minimising phototoxicity and photobleaching. It also overcomes the sample mounting challenges associated with conventional light sheet microscopes that require two orthogonally placed objectives. However, the application of OPM has been limited by the complex design and the intricate optical alignment and characterisation needed, particularly with the remote-refocusing system (RFS) in the emission path. This protocol offers a detailed, step-by-step guide for constructing an OPM setup using commercially available components and for characterising its performance to ensure optimal imaging quality. We aim to deliver the unique merits of OPM to researchers in life science and medicine, enabling them to visualise the spatiotemporal organisation of key biomolecules, structures, and cells in 3D at high resolutions.

A wide variety of protocols have been proposed for optical clearing of tissues, whole-mount organs, and other bulky specimens to enable their volumetric fluorescence imaging. However, quantitative comparisons of tissue clearing protocols that take into account the fluorescence of the final specimens remain rare. Here, we propose a volumetric fluorescence image-based workflow for evaluating tissue clearing and fluorescence staining protocols. The workflow calculates depth-dependent fluorescence attenuation coefficients using data from entire 3D images, thereby avoiding spatial sampling bias and eliminating reliance on simple readouts, such as light transmittance, to predict fluorescence image quality. By combining autofluorescence signal with the signal from a specific fluorescence label, we independently evaluated transparency and the quality of fluorescence staining in cleared specimens. Using the proposed workflow, we systematically compared clearing and staining performance of three CUBIC-based protocols in murine liver, kidney, spleen, thymus, and intestine, and revealed differences in final fluorescence image quality across protocol-organ combinations.

It is increasingly necessary to both study biology in 3D and obtain quantitative measurements. Not all 3D-reconstructions are created equal, particularly when using the anatomical model as a basis for force calculations, i.e. computational modeling. Here, we compare 3D anatomical reconstructions from two emerging imaging modalities: 4D ultrasound (4DUS) and light sheet fluorescent microscopy (LSFM) against our previous nano-computed tomography (nanoCT) cohort data, using the tortuous highly intricate pharyngeal arch artery system of the chick embryo as a test bed. We highlight modality-specific morphological image acquisition discrepancies and their influence on subsequent computational fluid dynamics results. Overall, LSFM accurately captured quantitative volumetric measurements of small rapidly-changing vascular morphologies while 4DUS systematically inflated small tortuous vessels. Differences in image-based morphology changes led to significant changes in computationally-obtained force magnitudes and flow patterns linked to vessel angle and tortuosity. This validates LSFM as a comparative preclinical vascular quantitative imaging tool and suggests that 4DUS needs extensive 3D anatomical validation for non cardiac chamber vessels.

Epithelial ovarian cancer remains one of the most lethal malignancies among women, with late-stage diagnoses yielding 5-year survival rates below 30%. The metabolic heterogeneity of the tumor microenvironment (TME) highlights the need for methods capable of rapid, chemically specific phenotyping. Stimulated Raman scattering (SRS) microscopy when combined with deuterium labeled metabolites enables the non-invasive high contrast interrogation of cellular metabolic pathways. In this study, we used SRS microscopy to profile fatty acid and glycogen metabolism in epithelial ovarian cancer (SKOV-3) and cervical cancer (HeLa) cell models. Deuterium labeled glucose revealed striking differences in glycogen synthesis and intracellular distribution, with SKOV-3 cells exhibiting markedly greater single-cell heterogeneity than HeLa. Complementary measurements of lipid droplet (LD) synthesis and turnover under nutrient starvation further revealed cell-line-specific metabolic strategies, identifying LD and glycogen dynamics as a potential diagnostic marker of cancer metabolic phenotypes. These results demonstrate that SRS microscopy in the Raman silent region, paired with metabolic labeling, can sensitively resolve metabolic diversity across cancer cell subpopulations. Such metabolic phenotyping may inform both early diagnostic strategies and therapeutic approaches that combine cytotoxic treatment with targeted metabolic disruption.