Biomedical optics express最新文献

We propose FPM2Stain Net, a jointly optimized end-to-end computational pipeline that integrates physics-guided super-resolution with deep learning-based virtual staining to enable high-resolution, multi-modal digital histopathology. The first stage, bidirectional physics-based Fourier ptychographic microscopy (BiP-FPM), reconstructs high-resolution amplitude and phase maps from a multi-LED FPM image stack using a self-supervised ResNet-U-Net guided by a differentiable point spread function (PSF) model with learnable pupil correction and bidirectional physics-consistent constraints. The second stage employs a multi-task conditional generative adversarial network (cGAN) enhanced with wavelet-based spatial-frequency fusion and perceptual supervision to synthesize clinically relevant staining modalities, including H&E, DAPI, LAP2, and panCK. Extensive experiments on both simulated and real tissue datasets demonstrate that FPM2Stain Net outperforms conventional FPM, GAN-based methods, and diffusion-based models in both reconstruction fidelity and staining accuracy. The synthesized virtual stains not only preserve fine structural details but also enable downstream analysis, such as cell segmentation and biomarker quantification, with higher accuracy than using images acquired with a conventional 40× objective microscope. These results confirm that FPM2Stain Net achieves a greater-than-10× pixel-level upsampling factor relative to the low-magnification (4×) input image, and provides a fast, scalable, and cost-effective alternative to chemical staining in digital pathology, multiplex imaging, and point-of-care diagnostics.

Bioreactors are used for the industrial-scale culture of cells to obtain valuable products such as pharmaceuticals, enzymes, and biofuels; however, monitoring the growth conditions within the vessels is challenging and is often dependent on single-point ex-situ measurements. Further, spatial heterogeneities are known to exist within these environments, thereby creating regions of low growth or incomplete reactions, reducing yield, and importantly, reducing the applicability of single-point measurement methods. Optical imaging is an attractive method for remote spatially-resolved measurement platforms; however, the strong optical scattering within cell cultures makes imaging almost impossible. Here, we utilise this parasitic scattering effect and present a spatially resolved optical method for monitoring cell density within a bioreactor, using optical measurement of local scattering parameters as a proxy measurement for cell density. Our method is non-invasive and does not require the removal of any cell material from within the vessel. We propose that our optical measurement method can be incorporated into process-control feedback systems, providing insightful information on cell growth that can be used to deliver higher spatial homogeneity and increased yields.

The blue appearance of veins has been addressed in previous studies; however, an experimental demonstration that reproduces this phenomenon with comprehensible optical properties has not yet been achieved. To address this gap, silicone-based skin phantoms were fabricated to replicate the optical properties of human skin. Cylindrical vessel inclusions of varying depths and diameters were incorporated into the phantoms and filled with silicone mimicking blood at different oxygenation states. The vessels' appearance was quantitatively evaluated using calibrated photography and hyperspectral imaging. Monte Carlo light transport simulations were employed to support the experimental findings. The phantoms with venous inclusions exhibited the characteristic blue hue of veins, thus demonstrating that vein blueness does not arise solely from blood's intrinsic absorption but from the complex interplay of tissue optics and visual perception. These experimental findings support our previously proposed theory.

Characterization of myelin degradation in the white matter (WM) is important for understanding neurodegeneration. We demonstrate label-free in vivo imaging of myelin structure in the WM of mice, through intact cortex, using third harmonic generation (THG) microscopy at 1320-nm excitation. Longitudinal THG imaging of the same axons in the cuprizone mouse model of multiple sclerosis revealed dynamics of myelin blistering. Further, we measured intranodal distance at nodes of Ranvier in vivo and developed a novel metric of myelin structural change based on spatial concentration of the brightest THG signal. We also demonstrated compatibility with three-photon excited fluorescence microscopy by imaging GFP-labeled microglia in the WM in parallel with THG microscopy, thereby enabling detailed tracking of subcortical myelin and other cellular dynamics in neurodegenerative disease models.

In light-sheet fluorescence microscopy (LSFM), the axial resolution is governed by the illumination beam profile, motivating the development of advanced beam-shaping techniques to enhance imaging performance. Two-photon LSFM (2P-LSFM), in particular, improves the signal-to-background ratio by reducing laser scattering and distortion in biological specimens. However, we report a potentially detrimental thermal effect in 2P-LSFM: the high laser powers required for two-photon excitation induce localized heating, which alters the refractive index of the medium and effectively forms a divergent thermal lens in water. At 500 mW, the light-sheet waist broadens by 25% and shifts by 300 μm before stabilizing several seconds after the laser shutter is opened. Both experiments and simulations reveal that this thermal lensing effect scales with laser power and the path length the beam travels through water. The resulting degradation in resolution and signal-to-noise ratio may compromise imaging applications that require high laser powers for rapid volumetric imaging of large specimens or functional brain imaging. This limitation is particularly critical in dynamic sample environments, such as during stepwise repositioning or flow-based delivery of chemical or hydrodynamic sensory stimuli, where changes occur on timescales comparable to the thermal settling time.

Here we present the design of telecentric model eyes for measuring image distortion in adaptive optics ophthalmoscopes, using pairs of achromatic or near-achromatic commercial off-the-shelf (COTS) and/or custom doublet lenses. The proposed model eyes can operate over visible or visible plus near infrared wavelength ranges, across a 15-diopter focus range, with an 8 mm entrance pupil diameter. COTS lens selection was implemented as a systematic evaluation of catalog lenses after optimization of axial distances. Custom lens optimization was initially guided by wavefront aberration constraints derived from object-shift 3^rd order aberration theory, before traditional variation of surfaces and distances to minimize a performance metric. When a wavefront corrector compensates for field-constant aberrations, the model eyes achieve nominal wavefront RMS below λ/20 across a ∼6.9° circular full field of view for COTS and hybrid lens pairs, limited by vignetting from a self-imposed 27.9 mm clear aperture. For custom lens pairs, the field of view is about 10.2°, limited by aberrations. A tolerance study indicates that a COTS lens pair for 450-1100 nm can achieve as-built diffraction-limited wavefront error of 0.044 λ @450 nm at 95% production yield. Model eyes with this lens pair yielded telecentricity quantified as image size changed per diopter of 0.1%/D when assembled with a camera and 0.01%/D when assembled with a power meter. The model eye was tested with an optical setup that emulates a fundus camera with Badal focus correction.

Age-related macular degeneration (AMD) is a major cause of global blindness that affects millions worldwide. Certain forms of AMD cause neovascularisations (NVs), which form retinal-choroidal anastomoses. This disrupts healthy haemodynamic patterns, and early detection and treatment are crucial for preserving vision. Here, we employ a custom-built optical coherence tomography (OCT) imaging system to investigate these NVs in a very low-density lipoprotein receptor (VLDLR) knockout mouse model. Mice were imaged before, during, and after contrast agent injection, aiming to enhance our understanding of the NV haemodynamics. Doppler signal analysis techniques were employed to calculate flow velocities within individual NVs. Flow rates pre- and post-injection were determined based on these velocity measurements. Particle tracking was performed on two NVs for a comparative analysis with the Doppler velocity measurements. Both methods of measuring flow velocities showed good agreement post-contrast injection. The analysis of post-injection flow rates from the NVs revealed diverse behaviours. Some NVs exhibited stable flow rates over time, while others showed signs of instability, with flow rates changing substantially or even changing flow direction at different time points. Additionally, it was observed at multiple time points that flow from certain NVs moved from the choroid to the retina at the same time that other NVs displayed flow in the opposite direction. These observations suggest complex interactions between choroidal and retinal vascular networks in diseases like AMD. Further characterisation using contrast-enhanced Doppler OCT may improve our understanding of neovascular haemodynamics.

Osteoporosis and osteopenia remain vastly underdiagnosed. Current clinical screening relies almost exclusively on dual-energy X-ray absorptiometry (DXA), which measures bone mineral density (BMD) but fails to capture the compositional changes that lead to BMD loss. We investigated whether spatially offset Raman spectroscopy (SORS) applied to excised finger bones can assess subsurface biochemical markers associated with bone health and estimate wrist DXA T-scores. Raman spectra were acquired ex vivo on the mid-shaft of the proximal phalanx of the second digit from 25 female cadavers spanning the three T-score categories (n = 8 normal, n = 6 osteopenic, and n = 11 osteoporotic) at spatial offsets of 0, 3, and 6 mm from a laser irradiation spot. After normalizing spectra to the PO₄ ^3- peak, group-averaged spectra of the three categories, measured at 3-mm offset, showed clear differences in the CO₃ ^2-, Amide III, CH₂, and Amide I bands. Quantitatively, four out of five mineral-to-matrix ratios discriminated (p ≤ 0.05) osteopenia from both normal and osteoporotic bone, and all five ratios showed significant differences between normal and osteoporotic bone. In contrast, the 0-mm offset showed reduced biochemical contrast in univariate analyses, while the 6-mm offset did not provide additional discrimination relative to 3-mm. Multivariate partial-least-squares regression (PLSR) models using 0-mm and 3-mm spectra predicted distal radius T-scores with comparable accuracy (Pearson r ≈ 0.90, RMSE_CV ≈ 0.8), but the 3-mm offset required fewer latent variables (1 vs. 9), consistent with the univariate analysis trends favoring 3-mm offset. These excised-bone findings justify future studies extending this approach to transcutaneous fingers for non-ionizing assessment of bone health.

The inherent fluorescent properties of anti-leukemic drugs offer unique advantages for real-time therapeutic tracking and optimization. In this study, we systematically screened the absorption and emission spectra of 82 leukemia-related compounds, identifying 28 autofluorescent drugs suitable for fluorescence-based optical concentration monitoring. Excitation and emission parameters were evaluated across various solvents (DMSO, fetal bovine serum, and culture media), revealing solvent-dependent spectral changes, intensity variations, and effect on detection limits. These 28 compounds were further assessed for cytotoxicity screening in case of drug-naive and drug-resistant K562 leukemia lymphoblast cells. By correlating their spectral properties with cytotoxic responses, our study establishes a robust framework for fluorescence-assisted drug profiling, enabling pharmacokinetic insights, resistance prediction, and informed therapeutic adjustments. These findings underscore the translational potential of fluorescence-based methodologies in supporting precision medicine for leukemia treatment.

A method for estimating the absolute concentrations of chromophores in highly scattering tissues using near-infrared spectra is presented, which also yields estimates of the differential pathlength factor (DPF) and the power-dependency of scattering on wavelength. It involves measuring the attenuation spectral gradient and comparing it with an expression derived from diffusion theory. The validity of the approach is first explored using a diffusion model of light propagation in a homogenous slab, which is used to simulate measurements of diffuse reflectance in the adult forearm muscle during a vascular occlusion. Thereafter, the method is applied to experimental measurements performed on the forearms of five volunteers. The simulation results suggest that accuracy is significantly enhanced if some derived parameters are constrained to ranges which are physiologically realistic, and that the absolute concentrations of oxy- and deoxy-hemoglobin are estimated to within 40% and 20% of the true values, respectively. Furthermore, the wavelength-averaged DPF can be estimated to within around 10%. The measurements on volunteers revealed broadly consistent concentrations of the hemoglobins in the range 2-105 µM, and differential pathlength factors in the range 2.2-5.1.