Differential Interference Contrast (DIC) microscopy is a label-free technique crucial for visualizing transparent biological specimens. In this study, we introduce a novel DIC interferometry imaging technique based on calcite beam displacer (CBD), significantly enhancing resolution and contrast. Our system, requires only two polarizers and a calcite beam displacer, offers a practical and straightforward implementation on standard microscopes. We demonstrate its efficacy by imaging human red blood cells (RBCs) and bacteria, retrieving differential phase information, and recording the dynamics of U2OS cells over an hour at 15 frames per second (fps). This advancement in DIC microscopy holds promise for broader applications in high-resolution imaging of transparent specimens in cell biology research.
With applications in quantitative metabolomics and label-free digital pathology, Quantitative Phase Microscopy (QPM) measures refractive index maps of thin transparent specimens like live cells or tissue sections. In QPM, refractive index maps are usually reconstructed from interference measurements of the object’s light field with respect to a reference field. To this end, many previous works focused on designing stable full-field interferometers from the bottom up. In this work, we present an alternative strategy to design a QPM system top-down, starting from the desired measurement outcomes, with no explicit knowledge about interferometry. We call our inverse design strategy Differentiable Microcopy. To this end, our Differentiable Microcopy approach designed a range of Fourier-filter-based QPM systems that do not require computational post-reconstruction. Our designs are superior to existing similar designs in numerical benchmarks. We also experimentally validated one design using a spatial light modulator. Finally, to fabricate these custom designs in the future, we also propose a new fabrication-aware Differentiable Microcopy pipeline.
The ubiquitous nature of microplastics in marine environments poses a significant threat to Atlantic salmon aquaculture, and fish in general. Mucosal surfaces are continuously exposed to microparticles – such as the skin epithelial layer with highly migrating keratocyte cells. This study applied the capabilities of Linnik-based Quantitative Phase Microscopy (QPM) equipped with a pseudo-thermal light source (PTLS) to examine the interactions between polystyrene microplastics (MPs) and Atlantic salmon keratocytes. The PTLS enables the QPM system to achieve high spatial phase sensitivity, allowing for the observation of precise morphological changes within the cell and migration in real time. Our study shows that cells that were exposed to polystyrene MPs exhibited dynamic fluctuations of the lamellipodia and provide evidence for early-stage phase membrane invaginations around the beads indicative of phagocytic activity. Implementation of QPM based on PTLS is a high-speed, non-invasive, label-free approach that provides vital insights into the morphological dynamics of MP exposure. This research not only contributes to the understanding of cellular morphological responses to microplastics in Atlantic salmon but also highlights the utility of advanced QPM imaging techniques from an environmental toxicology perspective.
Quantitative phase microscope (QPM) is used for the quantitative information and dynamic phase imaging of biological specimen, which provides wide application in biomedical sciences. High temporal phase stability of the QPM system is the primary requirement for accurate phase measurement. We have developed a common-path QPM geometry based on beam displacer and pinhole unit to achieve high temporal stability. The convenient adjustment of reference and object beams makes optical system compact and low-cost. The membrane fluctuations and qualitative phase are measured to demonstrate the capability and applicability of the system.
Quantitative phase microscopy (QPM) has recently become indispensable technology for label-free quantitative analysis of various biological cells and tissues, such as, sperm cells, liver sinusoidal cells, cancerous cells, red blood cells etc. The key parameters controlling measurement accuracy and capability of QPM system depends on its spatial and temporal phase sensitivity. The spatial phase sensitivity of QPM is governed by coherence properties of light source and temporal stability depends on optical interferometric configuration. Most of the QPM techniques utilize highly coherent light sources like lasers benefited by their high spatial and temporal coherence, and brightness. But high spatio-temporal coherence leads to occurrence of speckle noise and spurious fringes leading to inhomogeneous illumination and poor spatial phase sensitivity. We have developed QPM systems using partially spatially coherent monochromatic (PSCM) light sources which guarantees high contrast interferograms over large field-of-view to increase space-bandwidth product of QPM system by ten-times and demonstrated ten-fold improvement in spatial-phase sensitivity and phase measurement accuracy compared to coherent laser light. By means of using PSCM with common path configuration we could also achieve ten-fold temporal phase stability. We have demonstrated advantages of PSCM based QPM in various industrial and bio-imaging applications. Experimental results of reduced speckle noise, free-from spurious fringes, spatial phase sensitivity using industrial objects are demonstrated and compared with highly coherent light using single mode fiber. Finally, phase map of biological samples is also presented with high accuracy in phase measurement. Thus, the use of PSCM light in phase microscopy, holography of realistic objects, i.e., industrial and biological samples leads to high accuracy in the measurement of quantitative information.
We have investigated the enhanced Raman spectra of AMR bacteria strains of E. coli using silver coated silicon nanowires SERS assay. Three different E. coli strains, E. coli CCUG17620, NCTC 13441, and A239, were detected using two different excitation laser wavelengths. We found stable and enhanced SERS spectrum using 785 nm laser as opposed to 532 nm. Future development of SERS-chip could offer a reliable platform for direct identification of the pathogen in bio-fluid samples at strains level.
Optical coherence tomography (OCT) is low temporal coherence gated cross-sectional imaging modality which is noncontact, non-invasive and in vivo technology. In conventional OCT mainly a broad-band light source is used to achieve high axial resolution which is inversely proportional to spectral bandwidth of the light source. The most preferred light source in OCT is super-luminescent diode (SLD) or a supercontinuum light source, but they are bulky and costly. In addition, use of broad band light in OCT systems require dispersion compensation system because most of biological tissues are birefringence, therefore, it further adds complexity to the system. Further, use of highly spatially coherent light leads to speckle noise which degrades the image quality.
We report longitudinal spatial coherence (LSC) gated line-field optical coherence tomography (LF-OCT) of multilayer structures using partially spatially coherent monochromatic light with speckle-free and reduced crosstalk. The LSC properties of low-cost diode laser source was engineered to significantly reduce LSC length to achieve high axial-resolution. The OCT system was operated both in full-field and line-field mode and it was found that line-field version has better contrast and less crosstalk. Experimental results of LSC gated OCT of multi-layer samples both biological and industrial objects along with speckle contrast, interferometric fringe contrast and LSC length measurement will be presented. The use of a low-cost diode laser in the wavelength range 800 nm for high resolution OCT is important for many biological applications. In addition, the system does not require a dispersion compensation mechanism.
We report high-speed and highly sensitive quantitative phase microscopy (QPM) using dynamic speckle illumination (DSI). The DSI-QPM is used for real-time analyses of highly motile human spermatozoa. The DSI-QPM supports high-speed and high spatial phase sensitivity, that are crucial for imaging tail (nanoscale) of living spermatozoa during motion. The scalable FoV and high temporal coherence offered by DSI-QPM is harnessed for histopathology and marine biology. Further, by integrating the single molecule localization microscopy (SMLM) with QPM, nanoscale imaging and quantification in lateral (via SMLM) and axial (via QPM) directions was achieved on liver cells.
With applications ranging from metabolomics to histopathology, quantitative phase microscopy (QPM) is a powerful label-free imaging modality. However, the speed of current QPM systems is limited by electronic hardware. To improve throughput further, here we propose differentiable optical-electronic quantitative phase microscopy (∂μ) that acquires images in a compressed form such that more information can be transferred beyond the electronic hardware bottleneck. The proposed microscopy uses optical feature extractors as image compressors. The resultant intensity distribution is then decompressed into QPM images by a reconstruction network. By optimizing optical-electronic parameters in an end-to-end manner, our method can improve the QPI throughput from Hours to Seconds (more than an order of magnitude).
Histology is a well-known examination technique to study the biological cell and tissue structures. For histological assessment, imaging throughput, contrast, resolution, and quantification of morphology are crucial parameters. Although, there are techniques available which can scan the whole slide, but they lack specificity and quantification. In present study, we introduce a photonic chip based platform for multimodal imaging of FFPE tissue sections. Here, the photonic chip platform was integrated with Linnik type QPM module, which enables high contrast TIRF imaging and optical thickness of the specimen over scalable FOV. The proposed system has been used as a high throughput microscopy platform to study the functional and morphological features of FFPE human placenta tissue sections. The investigation of the tissue sections facilitates the identification and diagnosis of the various diseases, which can provide direction for treatment and can assist the prognosis of clinical outcome.
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