Extended depth-of-field reflected-light microscopy with a low-cost robotic microscope and Fiji focus stacking: Workflow integration and baseline evaluation.
Aug 2026· Journal of Microscopy· 0 citations· 14 references
Medicine
Abstract
Optical reflected-light microscopy is widely used in mechanical and tribological testing laboratories, but its limited depth of field (DOF) restricts imaging of non-planar post-test surfaces when high-NA objectives are needed to resolve fine features. Here we present a low-cost robotic microscopy system (bill of materials around USD 300) that extends depth of field through controlled axial scanning and computational focus stacking. Built on the OpenFlexure platform and using open-source software tools, the system captures z-stacks and reconstructs all-in-focus micrographs in Fiji (ImageJ distribution) using extended-depth-of-field (EDOF) fusion engines. The workflow is demonstrated on mechanically tested surfaces (scratches and indentations), providing all-in-focus visualisation of features such as scratch lips, pile-up, indentation boundaries and microcracks on reflective textured surfaces, with Scanning Electron Microscopy used for cross-checking of feature detectability. Baseline characterisation includes operational DOF and resolving-power measurements, a geometric-distortion assessment using a micrometric grid target, and a time-cost analysis of stack acquisition. Comparative evaluation of three widely used fusion methods (Sobel, variance and complex wavelets) using image-quality metrics and visual inspection indicates that complex-wavelet fusion yields sharper reconstructions and improved edge preservation relative to Sobel and variance, at the expense of higher computation time. Finally, we summarise practical constraints and observed failure modes under representative conditions, providing baseline guidance for routine use, including deployment close to mechanical test stations. LAY DESCRIPTION: Low-cost reflected-light microscopy has shallow depth of field when high-NA objectives image rough surfaces. We demonstrate a fully open (∼USD 300) OpenFlexure-based robotic microscope with an end-to-end Fiji focus-stacking workflow that produces all-in-focus micrographs of scratches and indentations, cross-checked against SEM. Baseline characterisation covers DOF, resolving power, geometric distortion, and acquisition-time effects. Complex-wavelet fusion outperforms Sobel and variance in sharpness at higher computation time. Practical constraints are identified for post-test failure analysis.
High-speed three-dimensional imaging in scattering tissues remains challenging because volumetric microscopy generally requires scanning, whereas snapshot light-field approaches divide limited detector pixels among multiple views. This constraint is particularly severe in the second near-infrared window (NIR-II), where commonly used InGaAs cameras typically have relatively small sensor formats and high detector noise. Here we introduce NIR-II squeezed light-field microscopy (NIR-II SLIM), which optically rotates and compresses multiple perspective views before detection, allowing efficient use of camera pixels while retaining complementary spatial information for three-dimensional reconstruction. NIR-II SLIM acquires volumes at up to 600 volumes s-1 with a reconstructed lateral sampling grid of 512 × 512 pixels. We use the method for label-free four-dimensional imaging of cardiac dynamics in pigmented late-larval zebrafish, resolving chamber deformation and millisecond-scale atrioventricular-valve motion, and for NIR-II fluorescence imaging of vascular and lymphatic transport in mice. NIR-II SLIM provides a detector-efficient approach for high-speed volumetric imaging of rapid biological dynamics in scattering tissues.
Do Young Kim, Zihan Zang, Eric Y. Lin et al.· bioRxiv· 0 citations
Compact Open-Source Multimodal Illumination Cluster ('COSMIC') is a condenser-free illumination module that enables bright field, dark field, diascopic fluorescence and differential phase contrast (DPC) imaging on both commercial and open-source microscope platforms. COSMIC is constructed using low-cost, widely available components and is controlled through µManager, facilitating straightforward integration into existing imaging systems. We demonstrate multimodal transmitted-light imaging of live cell cocultures and living Caenorhabditis elegans specimens, including quantitative phase imaging (QPI) derived from DPC measurements. We further validate and calibrate quantitative DPC (qDPC) using a quantitative phase target on a range of open-source and commercial systems and introduce a practical method for optimising the axial position of the illuminator without access to the objective's back focal plane. COSMIC provides an effortless route to multimodal and quantitative phase imaging on any optical microscopy system, using minimal optical hardware and supporting both research-grade and resource constrained microscopy environments.
Ewan Drever-Smith, F. Pascut, George I Wadsworth et al.· Journal of Microscopy· 0 citations
Beam engineering effectively overcomes the resolution-depth of focus trade-off in high-NA optical coherence microscopy (OCM). Conventional OCM is limited by this constraint, hindering stable volumetric acquisition. To overcome this, we present an intrinsically registered imaging platform based on Bessel beam–enabled extended-depth-of-focus OCM for quantitative, label-free volumetric phenotyping of three-dimensional cell models. Annular illumination extends the focal range to 193 µm, enabling stable scattering contrast in dense spheroids. By exploiting structural scattering signatures associated with cellular disintegration, we demonstrate volumetric viability mapping with 94.57 ± 2.69% aggregate viability concordance relative to fluorescence references. The platform differentiates drug-specific responses and captures pathological transitions in spheroids and organoids, establishing a quantitative paradigm for volumetric biological phenotyping.
Hyunji Lee, Jeong Bin Kim, Ahreum Beak et al.· Biomedical Optics Express· 0 citations
Three-dimensional (3D) microscopic imaging is indispensable for fundamental scientific research and clinical medical diagnosis. Given that conventional widefield optical microscopy and standard confocal microscopy fail to realize high-magnification, full-color, real-time stereoscopic imaging simultaneously, we herein propose a single-optical-path 3D microscopic framework enabled by liquid crystal polarization gratings (LCPGs). The LCPG integrated at the sample plane performs polarization-dependent beam splitting to generate paired left and right viewing channels. These two disparity-bearing view channels share a unified imaging optical path compatible with commercial upright microscopes, wherein an active liquid crystal cell modulates temporal view switching for sequential camera acquisition. We further construct a white-light microscopic platform supporting integrated reflection and transmission imaging modes. Two customized LCPGs with lattice periods of 72.6 μm and 56.9 μm are fabricated, offering angular view separations of 0.84° and 1.07°, respectively. Both gratings achieve ±1st-order diffraction efficiencies above 97% with polarization crosstalk not exceeding 0.8%. The developed system acquires paired left-right images with valid binocular disparity, which can be reconstructed into intuitive stereoscopic perceptions via a 3D display monitor. This LCPG-based optical architecture upgrades standard upright microscopes to compact dual-view stereoscopic imaging systems, while fully inheriting the native merits of white-light illumination and high-magnification microscopic observation.
Jiaoyang Li, Chenhao Li, Zi-Hao Tan et al.· Nanomaterials· 0 citations
Three-dimensional (3D) tracking of microscopic objects is essential for probing dynamic processes across biological, microfluidic, and nanoscale systems. In-line digital holographic microscopy (DHM) provides a powerful framework for this purpose by encoding volumetric information into a single intensity measurement, enabling scan-free reconstruction through numerical wave propagation. However, despite its conceptual simplicity, the practical implementation of in-line DHM remains fundamentally constrained by its reliance on bulky relay optics, which limit system compactness, alignment robustness, and integration with emerging miniaturized platforms. Here, we rearchitect in-line DHM using planar meta-optics by replacing conventional refractive relay optics with metalenses, thereby establishing a compact single-path holographic imaging platform. This architecture preserves the intrinsic advantages of in-line holography, including single-shot volumetric encoding and numerically propagated reconstruction, while substantially reducing optical complexity and system footprint. We validate the metasurface-enabled in-line DHM experimentally using resolution targets, microspheres, and live microorganisms and demonstrate scan-free 3D tracking in a compact optical configuration. Our results establish planar meta-optics not only as a substitute for refractive components but also as an effective system-level strategy for miniaturizing holographic microscopy and advancing integrable volumetric imaging platforms.
Cheng Hung Chu, Chen-Ming Tsai, S. Vyas et al.· ACS Photonics· 0 citations
Light-field microscopy enables snapshot volumetric imaging, but its information rate is constrained by both optical encoding and detector readout architecture. Here we develop a task-dependent Fisher-information framework that evaluates optical encoders relative to the detector resource limiting acquisition throughput. We compare full Fourier light-field microscopy (FLFM), squeezed light-field microscopy (SLIM), and frame-rate-matched FLFM under a common optical geometry, photon budget, and row-limited camera model. Sparse scenes are analyzed using a 3D point-emitter Fisher matrix, and dense scenes using Fourier-mode information on tilted spectral slices. At s=0.25, SLIM provides 2.40x higher axial Fisher information per camera bandwidth and 1.89x higher 3D D-optimal position information than frame-rate-matched FLFM. For dense scenes, it provides 1.85x higher integrated Fourier-mode Fisher information per bandwidth, 4x greater axial-frequency extent, and approximately 11x larger projected lateral hard-support area. Sweeps over compression factor and view tilt show that these advantages reflect a general detector-allocation principle rather than a specific operating point. More broadly, the framework can be adapted to other camera architectures by incorporating architecture-specific measurement models and detector-throughput costs, providing a general basis for co-designing optical encoding, scene statistics, and camera readout.