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Significance: Three-dimensional photoacoustic imaging (PAM) has emerged as a promising technique for non-invasive label-free visualization and characterization of biological tissues with high spatial resolution and functional contrast., Aim: The application of PAM and ultrasound as a microscopy technique of study for Atlantic salmon skin is presented here., Approach: A custom ultrasound and photoacoustic experimental setup was used for conducting this experiment with a sample preparation method where the salmon skin is embedded in agarose and lifted from the bottom of the petridish., Results: The results of C-scan, B-scan, and overlayed images of ultrasound and photoacoustic are presented. The results are then analyzed for understanding the pigment map and its relation to salmon behavior to external stimuli. The photoacoustic images are compared with the optical images and analyzed further. A custom colormap and alpha map is designed and the matrices responsible for PAM and ultrasound are inserted together to overlay the ultrasound image and PAM image on top of each other., Conclusions: In this study, we propose an approach that combines scanning acoustic microscopy (SAM) images with PAM images for providing a comprehensive understanding of the salmon skin tissue. Overlaying acoustic and photoacoustic images enabled unique visualization of tissue morphology, with respect to identification of structural features in the context of their pigment distribution., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ranjan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Current low coherence quantitative phase microscopy (LC-QPM) systems suffer from either reduced field of view (FoV) or reduced temporal resolution due to the short temporal coherence (TC) length of the light source. Here, we propose a hybrid, experimental and numerical approach to address this core problem associated with LC-QPM. We demonstrate high spatial resolution and high phase sensitivity in LC-QPM at high temporal resolution. High space-time bandwidth product is achieved by employing incoherent light source for sample illumination in QPM to increase the spatial resolution and single-shot Hilbert spiral transform (HST) based phase recovery algorithm to enhance the temporal resolution without sacrificing spatial resolution during the reconstruction steps. The high spatial phase sensitivity comes by default due to the use of incoherent light source in QPM which has low temporal coherence length and does not generate speckle noise and coherent noise. The spatial resolution achieved by the HST is slightly inferior to the temporal phase-shifting (TPS) method when tested on a specimen but surpasses that of the single-shot Fourier transform (FT) based phase recovery method. Contrary to HST method, FT method requires high density fringes for lossless phase recovery, which is difficult to achieve in LC-QPM over entire FoV. Consequently, integration of HST algorithm with LC-QPM system makes an attractive route. Here, we demonstrate scalable FoV and resolution in single-shot LC-QPM and experimentally corroborate it on a test object and on both live and fixed biological specimen such as MEF, U2OS and human red blood cells (RBCs). LC-QPM system with HST reconstruction offer high-speed single-shot QPM imaging at high phase sensitivity and high spatial resolution enabling us to study sub-cellular dynamic inside U2OS for extended duration (3 h) and observe high-speed (50 fps) dynamics of human RBCs. The experimental results validate the effectiveness of the present approach and will open new avenues in the domain of biomedical imaging in the future., (© 2024. The Author(s).)

Programmable nanoscale carriers, such as liposomes and DNA, are readily being explored for personalized medicine or disease prediction and diagnostics. The characterization of these nanocarriers is limited and challenging due to their complex chemical composition. Here, we demonstrate the utilization of surface-enhanced Raman spectroscopy (SERS), which provides a unique molecular fingerprint of the analytes while reducing the detection limit. In this paper, we utilize a silver coated nano-bowl shaped polydimethylsiloxane (PDMS) SERS substrate. The utilization of nano-bowl surface topology enabled the passive trapping of particles by reducing mobility, which results in reproducible SERS signal enhancement. The biological nanoparticles' dwell time in the nano-trap was in the order of minutes, thus allowing SERS spectra to remain in their natural aqueous medium without the need for drying. First, the geometry of the nano-traps was designed considering nanosized bioparticles of 50-150 nm diameter. Further, the systematic investigation of maximum SERS activity was performed using rhodamine 6 G as a probe molecule. The potential of the optimized SERS nano-bowl is shown through distinct spectral features following surface- (polyethylene glycol) and bilayer- (cholesterol) modification of empty liposomes of around 140 nm diameter. Apart from liposomes, the characterization of the highly crosslinked DNA specimens of only 60 nm in diameter was performed. The modification of DNA gel by liposome coating exhibited unique signatures for nitrogenous bases, sugar, and phosphate groups. Further, the unique sensitivity of the proposed SERS substrate displayed distinct spectral signatures for DNA micelles and drug-loaded DNA micelles, carrying valuable information to monitor drug release. In conclusion, the findings of the spectral signatures of a wide range of molecular complexes and chemical morphology of intra-membranes in their natural state highlight the possibilities of using SERS as a sensitive and instantaneous characterization alternative., Competing Interests: The authors declare no conflicts of interest., (© 2024 Optica Publishing Group.)

With applications ranging from metabolomics to histopathology, quantitative phase microscopy (QPM) is a powerful label-free imaging modality. Despite significant advances in fast multiplexed imaging sensors and deep-learning-based inverse solvers, the throughput of QPM is currently limited by the pixel-rate of the image sensors. Complementarily, to improve throughput further, here we propose to acquire images in a compressed form so that more information can be transferred beyond the existing hardware bottleneck of the image sensor. To this end, we present a numerical simulation of a learnable optical compression-decompression framework that learns content-specific features. The proposed differentiable quantitative phase microscopy (∂-QPM) first uses learnable optical processors as image compressors. The intensity representations produced by these optical processors are then captured by the imaging sensor. Finally, a reconstruction network running on a computer decompresses the QPM images post aquisition. In numerical experiments, the proposed system achieves compression of × 64 while maintaining the SSIM of ∼0.90 and PSNR of ∼30 dB on cells. The results demonstrated by our experiments open up a new pathway to QPM systems that may provide unprecedented throughput improvements., Competing Interests: The authors declare that there are no conflicts of interest related to this article, (© 2024 Optica Publishing Group.)

Computational methods have been established as cornerstones in optical imaging and holography in recent years. Every year, the dependence of optical imaging and holography on computational methods is increasing significantly to the extent that optical methods and components are being completely and efficiently replaced with computational methods at low cost. This roadmap reviews the current scenario in four major areas namely incoherent digital holography, quantitative phase imaging, imaging through scattering layers, and super-resolution imaging. In addition to registering the perspectives of the modern-day architects of the above research areas, the roadmap also reports some of the latest studies on the topic. Computational codes and pseudocodes are presented for computational methods in a plug-and-play fashion for readers to not only read and understand but also practice the latest algorithms with their data. We believe that this roadmap will be a valuable tool for analyzing the current trends in computational methods to predict and prepare the future of computational methods in optical imaging and holography., Supplementary Information: The online version contains supplementary material available at 10.1007/s00340-024-08280-3., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2024.)

Xanthines such as caffeine and theobromine are among the most consumed psychoactive stimulants in the world, either as natural components of coffee, tea and chocolate, or as added ingredients. The present study assessed if xanthines affect liver sinusoidal endothelial cells (LSEC). Cultured primary rat LSEC were challenged with xanthines at concentrations typically obtained from normal consumption of xanthine-containing beverages, food or medicines; and at higher concentrations below the in vitro toxic limit. The fenestrated morphology of LSEC were examined with scanning electron and structured illumination microscopy. All xanthine challenges had no toxic effects on LSEC ultrastructure as judged by LSEC fenestration morphology, or function as determined by endocytosis studies. All xanthines in high concentrations (150 μg/mL) increased fenestration frequency but at physiologically relevant concentrations, only theobromine (8 μg/mL) showed an effect. LSEC porosity was influenced only by high caffeine doses which also shifted the fenestration distribution towards smaller pores. Moreover, a dose-dependent increase in fenestration number was observed after caffeine treatment. If these compounds induce similar changes in vivo, age-related reduction of LSEC porosity can be reversed by oral treatment with theobromine or with other xanthines using targeted delivery., (© 2023. Springer Nature Limited.)

Antimicrobial resistance (AMR) is a major health threat worldwide and the culture-based bacterial detection methods are slow. Surface-enhanced Raman spectroscopy (SERS) can be used to identify target analytes in real time with sensitivity down to the single-molecule level, providing a promising solution for the culture-free bacterial detection. We report the fabrication of SERS substrates having tightly packed silver (Ag) nanoparticles loaded onto long silicon nanowires (Si NWs) grown by the metal-assisted chemical etching (MACE) method for the detection of bacteria. The optimized SERS chips exhibited sensitivity down to 10 -12 M concentration of R6G molecules and detected reproducible Raman spectra of bacteria down to a concentration of 100 colony forming units (CFU)/mL, which is a thousand times lower than the clinical threshold of bacterial infections like UTI (10 5 CFU/mL). A Siamese neural network model was used to classify SERS spectra from bacteria specimens. The trained model identified 12 different bacterial species, including those which are causative agents for tuberculosis and urinary tract infection (UTI). Next, the SERS chips and another Siamese neural network model were used to differentiate AMR strains from susceptible strains of Escherichia coli ( E. coli ). The enhancement offered by SERS chip-enabled acquisitions of Raman spectra of bacteria directly in the synthetic urine by spiking the sample with only 10 3 CFU/mL E. coli . Thus, the present study lays the ground for the identification and quantification of bacteria on SERS chips, thereby offering a potential future use for rapid, reproducible, label-free, and low limit detection of clinical pathogens.

In 1934, Frits Zernike demonstrated that it is possible to exploit the sample's refractive index to obtain superior contrast images of biological cells. The refractive index contrast of a cell surrounded by media yields a change in the phase and intensity of the transmitted light wave. This change can be due to either scattering or absorption caused by the sample. Most cells are transparent at visible wavelengths, which means the imaginary component of their complex refractive index, also known as extinction coefficient k, is close to zero. Here, we explore the use of c-band ultra-violet (UVC) light for high-contrast high-resolution label-free microscopy, as k is naturally substantially higher in the UVC than at visible wavelengths. Using differential phase contrast illumination and associated processing, we achieve a 7- to 300-fold improvement in contrast compared to visible-wavelength and UVA differential interference contrast microscopy or holotomography, and quantify the extinction coefficient distribution within liver sinusoidal endothelial cells. With a resolution down to 215 nm, we are, for the first time in a far-field label-free method, able to image individual fenestrations within their sieve plates which normally requires electron or fluorescence superresolution microscopy. UVC illumination also matches the excitation peak of intrinsically fluorescent proteins and amino acids and thus allows us to utilize autofluorescence as an independent imaging modality on the same setup., (© 2023. The Author(s).)

This study aims to demonstrate the generation and detection of Scholte waves inside polystyrene microparticles. This was proven using both experimental analysis and COMSOL simulation. Microspheres of different sizes were excited optically with a pulsed laser (532 nm), and the acoustic signals were detected using a transducer (40 MHz). On analyzing the laser-generated ultrasound signals, the results obtained experimentally and from COMSOL are in close agreement both in the time and frequency domain. A simplified analysis of Scholte wave generation by laser irradiation for homogeneous, isotropic microspheres is presented. The theoretical wave velocity of the Scholte wave was calculated and found close to our experimental results. A representation of pressure wave motion showing the Scholte wave generation is presented at different times.

We present experimental demonstration of tilt-mirror assisted transmission structured illumination microscopy (tSIM) that offers a large field of view super resolution imaging. An assembly of custom-designed tilt-mirrors are employed as the illumination module where the sample is excited with the interference of two beams reflected from the opposite pair of mirror facets. Tunable frequency structured patterns are generated by changing the mirror-tilt angle and the hexagonal-symmetric arrangement is considered for the isotropic resolution in three orientations. Utilizing high numerical aperture (NA) objective in standard SIM provides super-resolution compromising with the field-of-view (FOV). Employing low NA (20X/0.4) objective lens detection, we experimentally demonstrate [Formula: see text] (0.56 mm[Formula: see text]0.35 mm) size single FOV image with [Formula: see text]1.7- and [Formula: see text]2.4-fold resolution improvement (exploiting various illumination by tuning tilt-mirrors) over the diffraction limit. The results are verified both for the fluorescent beads as well as biological samples. The tSIM geometry decouples the illumination and the collection light paths consequently enabling free change of the imaging objective lens without influencing the spatial frequency of the illumination pattern that are defined by the tilt-mirrors. The large and scalable FOV supported by tSIM will find usage for applications where scanning large areas are necessary as in pathology and applications where images must be correlated both in space and time., (© 2023. The Author(s).)

Quantitative phase microscopy (QPM) is often based on recording an object-reference interference pattern and its further phase demodulation. We propose pseudo Hilbert phase microscopy (PHPM) where we combine pseudo thermal light source illumination and Hilbert spiral transform (HST) phase demodulation to achieve hybrid hardware-software-driven noise robustness and an increase in resolution of single-shot coherent QPM. Those advantageous features stem from physically altering the laser spatial coherence and numerically restoring spectrally overlapped object spatial frequencies. The capabilities of PHPM are demonstrated by analyzing calibrated phase targets and live HeLa cells in comparison with laser illumination and phase demodulation via temporal phase shifting (TPS) and Fourier transform (FT) techniques. The performed studies verified the unique ability of PHPM to combine single-shot imaging, noise minimization, and preservation of phase details.

Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations-after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.

Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in terms of phase sensitivity, and spatial and temporal resolution compared to conventional light source illumination. To date, despite conspicuous advantages, it has not been extensively implemented in the field of phase imaging due to inadequate understanding of interference fringe formation, which is challenging to obtain in dynamic speckle illumination interference microscopy (DSI-IM). The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference microscopy systems such as quantitative phase microscopy, digital holographic microscopy and optical coherence tomography. Using the developed understanding of DSI, we demonstrated its capabilities which enables the use of non-identical objective lenses in both arms of the interferometer and opens the flexibility to use user-defined microscope objective lens for scalable field of view and resolution phase imaging. It is contrary to the present understanding which forces us to use identical objective lenses in conventional IM system and limits the applicability of the system for fixed objective lens. In addition, it is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light source based IM system. The theory and explanation developed here would enable wider penetration of DSI-IM for applications in biology and material sciences., (© 2022. The Author(s).)

The article elucidates the physical mechanism behind the generation of superior-contrast and high-resolution label-free images using an optical waveguide. Imaging is realized by employing a high index contrast multi-moded waveguide as a partially coherent light source. The modes provide near-field illumination of unlabeled samples, thereby repositioning the higher spatial frequencies of the sample into the far-field. These modes coherently scatter off the sample with different phases and are engineered to have random spatial distributions within the integration time of the camera. This mitigates the coherent speckle noise and enhances the contrast (2-10) × as opposed to other imaging techniques. Besides, the coherent scattering of the different modes gives rise to fluctuations in intensity. The technique demonstrated here is named chip-based Evanescent Light Scattering (cELS). The concepts introduced through this work are described mathematically and the high-contrast image generation process using a multi-moded waveguide as the light source is explained. The article then explores the feasibility of utilizing fluctuations in the captured images along with fluorescence-based techniques, like intensity-fluctuation algorithms, to mitigate poor-contrast and diffraction-limited resolution in the coherent imaging regime. Furthermore, a straight waveguide is demonstrated to have limited angular diversity between its multiple modes and therefore, for isotropic sample illumination, a multiple-arms waveguide geometry is used. The concepts introduced are validated experimentally via high-contrast label-free imaging of weakly scattering nanosized specimens such as extra-cellular vesicles (EVs), liposomes, nanobeads and biological cells such as fixed and live HeLa cells., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

The point spread function of a fixed fluorophore with its dipole axis colinear to the optical axis appears donut-shaped when seen through a microscope, and its light distribution in the pupil plane is radially polarized. Yet other techniques, such as photolithography, report that this same light distribution in the pupil plane appears as a solid spot. How can this same distribution lead to a spot in one case but a donut in the other? Here, we show how the tube lens of the system plays a critical role in determining this shape. Using a vectorial treatment of image formation, we simulate the relative contributions of both longitudinal and radial components to the image of a dipole emitter and thus show how the donut (typically reported for z-polarized single molecule fluorescence microscopy) transforms into a solid spot (as commonly reported for photolithography) as the numerical aperture of the tube lens increases. We find that the transition point occurs around 0.7 NA, which is significantly higher than used for most microscopy systems and lower than for common photolithography systems, thus resolving the seeming paradox of dipole shape., (© 2022 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

This three-dimensional structured illumination microscopy (3DSIM) dataset was generated to highlight the suitability of 3DSIM to investigate mitochondria-derived vesicles (MDVs) in H9c2 cardiomyoblasts in living or fixed cells. MDVs act as a mitochondria quality control mechanism. The cells were stably expressing the tandem-tag eGFP-mCherry-OMP25-TM (outer mitochondrial membrane) which can be used as a sensor for acidity. A part of the dataset is showing correlative imaging of lysosomes labeled using LysoTracker in fixed and living cells. The cells were cultivated in either normal or glucose-deprived medium containing galactose. The resulting 3DSIM data were of high quality and can be used to undertake a variety of studies. Interestingly, many dynamic tubules derived from mitochondria are visible in the 3DSIM videos under both glucose and galactose-adapted growth conditions. As the raw 3DSIM data, optical parameters, and reconstructed 3DSIM images are provided, the data is especially suitable for use in the development of SIM reconstruction algorithms, bioimage analysis methods, and for biological studies of mitochondria., (© 2022. The Author(s).)

Histology involves the observation of structural features in tissues using a microscope. While diffraction-limited optical microscopes are commonly used in histological investigations, their resolving capabilities are insufficient to visualize details at subcellular level. Although a novel set of super-resolution optical microscopy techniques can fulfill the resolution demands in such cases, the system complexity, high operating cost, lack of multi-modality, and low-throughput imaging of these methods limit their wide adoption for histological analysis. In this study, we introduce the photonic chip as a feasible high-throughput microscopy platform for super-resolution imaging of histological samples. Using cryopreserved ultrathin tissue sections of human placenta, mouse kidney, pig heart, and zebrafish eye retina prepared by the Tokuyasu method, we demonstrate diverse imaging capabilities of the photonic chip including total internal reflection fluorescence microscopy, intensity fluctuation-based optical nanoscopy, single-molecule localization microscopy, and correlative light-electron microscopy. Our results validate the photonic chip as a feasible imaging platform for tissue sections and pave the way for the adoption of super-resolution high-throughput multimodal analysis of cryopreserved tissue samples both in research and clinical settings., (© 2022. The Author(s).)

Mitochondria are essential energy-providing organelles of particular importance in energy-demanding tissue such as the heart. The production of mitochondria-derived vesicles (MDVs) is a cellular mechanism by which cells ensure a healthy pool of mitochondria. These vesicles are small and fast-moving objects not easily captured by imaging. In this work, we have tested the ability of the optical super-resolution technique 3DSIM to capture high-resolution images of MDVs. We optimized the imaging conditions both for high-speed video microscopy and fixed-cell imaging and analysis. From the 3DSIM videos, we observed an abundance of MDVs and many dynamic mitochondrial tubules. The density of MDVs in cells was compared for cells under normal growth conditions and cells during metabolic perturbation. Our results indicate a higher abundance of MDVs in H9c2 cells during glucose deprivation compared with cells under normal growth conditions. Furthermore, the results reveal a large untapped potential of 3DSIM in MDV research., (© 2021 The Authors. Journal of Biophotonics published by Wiley-VCH GmbH.)

Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging., Competing Interests: Competing interest statement: B.S.A. has applied for patent GB1606268.9 for chip-based optical nanoscopy. B.S.A. is a cofounder of the company Chip NanoImaging AS, which commercializes on-chip superresolution microscopy systems., (Copyright © 2021 the Author(s). Published by PNAS.)

Contrast in fluorescence microscopy images allows for the differentiation between different structures by their difference in intensities. However, factors such as point-spread function and noise may reduce it, affecting its interpretability. We identified that fluctuation of emitters in a stack of images can be exploited to achieve increased contrast when compared to the average and Richardson-Lucy deconvolution. We tested our methods on four increasingly challenging samples including tissue, in which case results were comparable to the ones obtained by structured illumination microscopy in terms of contrast., Competing Interests: Authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

High space-bandwidth product with high spatial phase sensitivity is indispensable for a single-shot quantitative phase microscopy (QPM) system. It opens avenue for widespread applications of QPM in the field of biomedical imaging. Temporally low coherence light sources are implemented to achieve high spatial phase sensitivity in QPM at the cost of either reduced temporal resolution or smaller field of view (FOV). In addition, such light sources have low photon degeneracy. On the contrary, high temporal coherence light sources like lasers are capable of exploiting the full FOV of the QPM systems at the expense of less spatial phase sensitivity. In the present work, we demonstrated that use of narrowband partially spatially coherent light source also called pseudo-thermal light source (PTLS) in QPM overcomes the limitations of conventional light sources. The performance of PTLS is compared with conventional light sources in terms of space bandwidth product, phase sensitivity and optical imaging quality. The capabilities of PTLS are demonstrated on both amplitude (USAF resolution chart) and phase (thin optical waveguide, height ~ 8 nm) objects. The spatial phase sensitivity of QPM using PTLS is measured to be equivalent to that for white light source and supports the FOV (18 times more) equivalent to that of laser light source. The high-speed capabilities of PTLS based QPM is demonstrated by imaging live sperm cells that is limited by the camera speed and large FOV is demonstrated by imaging histopathology human placenta tissue samples. Minimal invasive, high-throughput, spatially sensitive and single-shot QPM based on PTLS will enable wider penetration of QPM in life sciences and clinical applications., (© 2021. The Author(s).)

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy., (© 2021 Abudu et al.)

Photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM) is an emerging technology enabling a large TIRF excitation area decoupled from the detection objective. Additionally, due to the inherent multimodal nature of wide waveguides, it is a convenient platform for introducing temporal fluctuations in the illumination pattern. The fluorescence fluctuation-based nanoscopy technique multiple signal classification algorithm (MUSICAL) does not assume stochastic independence of the emitter emission and can therefore exploit fluctuations arising from other sources, as such multimodal illumination patterns. In this work, we demonstrate and verify the utilization of fluctuations in the illumination for super-resolution imaging using MUSICAL on actin in salmon keratocytes. The resolution improvement was measured to be 2.2-3.6-fold compared to the corresponding conventional images.

On-chip super-resolution optical microscopy is an emerging field relying on waveguide excitation with visible light. Here, we investigate two commonly used high-refractive index waveguide platforms, tantalum pentoxide (Ta 2 O 5 ) and silicon nitride (Si 3 N 4 ), with respect to their background with excitation in the range 488-640 nm. The background strength from these waveguides were estimated by imaging fluorescent beads. The spectral dependence of the background from these waveguide platforms was also measured. For 640 nm wavelength excitation both the materials had a weak background, but the background increases progressively for shorter wavelengths for Si 3 N 4 . We further explored the effect of the waveguide background on localization precision of single molecule localization for direct stochastic optical reconstruction microscopy (dSTORM). An increase in background for Si 3 N 4 at 488 nm is shown to reduce the localization precision and thus the resolution of the reconstructed images. The localization precision at 640nm was very similar for both the materials. Thus, for shorter wavelength applications Ta 2 O 5 is preferable. Reducing the background from Si 3 N 4 at shorter wavelengths via improved fabrication will be worth pursuing.

The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system's instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems.

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Correlative light and electron microscopy (CLEM) unifies the versatility of light microscopy (LM) with the high resolution of electron microscopy (EM), allowing one to zoom into the complex organization of cells. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) microscopy over large field of view and high precision localization of the target area of interest within EM. The photonic chips are used as a substrate to hold, to illuminate and to provide landmarking of the sample through specially designed grid-like numbering systems. Using this approach, we demonstrate its applicability for tracking the area of interest, imaging the three-dimensional (3D) structural organization of nano-sized morphological features on liver sinusoidal endothelial cells such as fenestrations (trans-cytoplasmic nanopores), and correlating specific endo-lysosomal compartments with its cargo protein upon endocytosis.

High resolution microscopy is heavily dependent on superb optical elements and superresolution microscopy even more so. Correcting unavoidable optical aberrations during post-processing is an elegant method to reduce the optical system's complexity. A prime method that promises superresolution, aberration correction, and quantitative phase imaging is Fourier ptychography. This microscopy technique combines many images of the sample, recorded at differing illumination angles akin to computed tomography and uses error minimisation between the recorded images with those generated by a forward model. The more precise knowledge of those illumination angles is available for the image formation forward model, the better the result. Therefore, illumination estimation from the raw data is an important step and supports correct phase recovery and aberration correction. Here, we derive how illumination estimation can be cast as an object detection problem that permits the use of a fast convolutional neural network (CNN) for this task. We find that faster-RCNN delivers highly robust results and outperforms classical approaches by far with an up to 3-fold reduction in estimation errors. Intriguingly, we find that conventionally beneficial smoothing and filtering of raw data is counterproductive in this type of application. We present a detailed analysis of the network's performance and provide all our developed software openly.

A novel experimental technique based on point contact and Coulomb coupling is devised and optimized for ultrasonic imaging of bulk and guided waves propagation in piezo-ceramics. The Coulomb coupling technique exploits the coupling and transfer of electric field to mechanical vibrations by excitation of phonons. The point contact excitation and detection technique facilitates the spatial-temporal imaging of ultrasonic waves. The motivation of this research is the diagnosis and localization of surface cracks in the piezoelectric sensors and actuators. The underlying principle of the detection scheme is that any discontinuity on the surface causes high localization of electric gradient. The localized electric field at the defect boundaries enables then to behave as secondary passive ultrasonic sources resulting in strong back reflections. However, due to the interference between transmitted and reflected wave components from rigid boundaries and defect, the resolution on the localization of the damage is challenging. Therefore, an algorithm based on the two-dimensional spectral decomposition is utilized for selective suppression of the transmitted wave. The algorithm includes data transformation and vectorization in polar coordinates for efficient spectral decomposition. In the spectral domain, the complex wave component (phase and amplitude) are suppressed for the transmitted wave field. The reflected wave component in the spectral domain is retained and retrieved back using inverse spectral transformation. The algorithm is successful in retaining and exemplifying only the reflected wave sources arising from the strong scattering of ultrasonic waves from the surface and sub-surface defects. In summary, a novel experimental technique based on Coulomb coupling and spectral decomposition technique has been implemented for localization of surface defect in piezo-ceramic structures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Photonic-chip based TIRF illumination has been used to demonstrate several on-chip optical nanoscopy methods. The sample is illuminated by the evanescent field generated by the electromagnetic wave modes guided inside the optical waveguide. In addition to the photokinetics of the fluorophores, the waveguide modes can be further exploited for introducing controlled intensity fluctuations for exploitation by techniques such as super-resolution optical fluctuation imaging (SOFI). However, the problem of non-uniform illumination pattern generated by the modes contribute to artifacts in the reconstructed image. To alleviate this problem, we propose to perform Haar wavelet kernel (HAWK) analysis on the original image stack prior to the application of (SOFI). HAWK produces a computational image stack with higher spatio-temporal sparsity than the original stack. In the case of multimoded non-uniform illumination patterns, HAWK processing breaks the mode pattern while introducing spatio-temporal sparsity, thereby differentially affecting the non-uniformity of the illumination. Consequently, this assists nanoscopy methods such as SOFI to better support super-resolution, which is otherwise compromised due to spatial correlation of the mode patterns in the raw image. Furthermore, applying HAWK prior to SOFI alleviates the problem of artifacts due to non-uniform illumination without degrading temporal resolution. Our experimental results demonstrate resolution enhancement as well as reduction in artifacts through the combination of HAWK and SOFI.

Quantitative phase microscopy (QPM) is a label-free technique that enables monitoring of morphological changes at the subcellular level. The performance of the QPM system in terms of spatial sensitivity and resolution depends on the coherence properties of the light source and the numerical aperture (NA) of objective lenses. Here, we propose high space-bandwidth quantitative phase imaging using partially spatially coherent digital holographic microscopy (PSC-DHM) assisted with a deep neural network. The PSC source synthesized to improve the spatial sensitivity of the reconstructed phase map from the interferometric images. Further, compatible generative adversarial network (GAN) is used and trained with paired low-resolution (LR) and high-resolution (HR) datasets acquired from the PSC-DHM system. The training of the network is performed on two different types of samples, i.e. mostly homogenous human red blood cells (RBC), and on highly heterogeneous macrophages. The performance is evaluated by predicting the HR images from the datasets captured with a low NA lens and compared with the actual HR phase images. An improvement of 9× in the space-bandwidth product is demonstrated for both RBC and macrophages datasets. We believe that the PSC-DHM + GAN approach would be applicable in single-shot label free tissue imaging, disease classification and other high-resolution tomography applications by utilizing the longitudinal spatial coherence properties of the light source.

Multiple signal classification algorithm (MUSICAL) exploits temporal fluctuations in fluorescence intensity to perform super-resolution microscopy by computing the value of a super-resolving indicator function across a fine sample grid. A key step in the algorithm is the separation of the measurements into signal and noise subspaces, based on a single user-specified parameter called the threshold. The resulting image is strongly sensitive to this parameter and the subjectivity arising from multiple practical factors makes it difficult to determine the right rule of selection. We address this issue by proposing soft thresholding schemes derived from a new generalized framework for indicator function design. We show that the new schemes significantly alleviate the subjectivity and sensitivity of hard thresholding while retaining the super-resolution ability. We also evaluate the trade-off between resolution and contrast and the out-of-focus light rejection using the various indicator functions. Through this, we create significant new insights into the use and further optimization of MUSICAL for a wide range of practical scenarios.

Significance: High temporal stability, wavelength independency, and scalable field of view (FOV) are the primary requirements of a quantitative phase microscopy (QPM) system. The high temporal stability of the system provides accurate measurement of minute membrane fluctuations of the biological cells that can be an indicator of disease diagnosis., Aim: The main aim of this work is to develop a high temporal stable technique that can accurately quantify the cell's dynamics such as membrane fluctuations of human erythrocytes. Further, the technique should be capable of acquiring scalable FOV and resolution at multiple wavelengths to make it viable for various biological applications., Approach: We developed a single-element nearly common path, wavelength-independent, and scalable resolution/FOV QPM system to obtain temporally stable holograms/interferograms of the biological specimens., Results: With the proposed system, the temporal stability is obtained ∼15  mrad without using any vibration isolation table. The capability of the proposed system is first demonstrated on USAF resolution chart and polystyrene spheres (4.5-μm diameter). Further, the system is implemented for single shot, wavelength-independent quantitative phase imaging of human red blood cells (RBCs) with scalable resolution using color CCD camera. The membrane fluctuation of healthy human RBCs is also measured and was found to be around 47 nm., Conclusions: Contrary to its optical counterparts, the present system offers an energy efficient, cost effective, and simple way of generating object and reference beam for the development of common-path QPM. The present system provides the flexibility to the user to acquire multi-wavelength quantitative phase images at scalable FOV and resolution.

Optical coherence tomography (OCT) is being increasingly adopted as a label-free and non-invasive technique for biomedical applications such as cancer and ocular disease diagnosis. Diagnostic information for these tissues is manifest in textural and geometric features of the OCT images, which are used by human expertise to interpret and triage. However, it suffers delays due to the long process of the conventional diagnostic procedure and shortage of human expertise. Here, a custom deep learning architecture, LightOCT, is proposed for the classification of OCT images into diagnostically relevant classes. LightOCT is a convolutional neural network with only two convolutional layers and a fully connected layer, but it is shown to provide excellent training and test results for diverse OCT image datasets. We show that LightOCT provides 98.9% accuracy in classifying 44 normal and 44 malignant (invasive ductal carcinoma) breast tissue volumetric OCT images. Also, >96% accuracy in classifying public datasets of ocular OCT images as normal, age-related macular degeneration and diabetic macular edema. Additionally, we show ∼96% test accuracy for classifying retinal images as belonging to choroidal neovascularization, diabetic macular edema, drusen, and normal samples on a large public dataset of more than 100,000 images. The performance of the architecture is compared with transfer learning based deep neural networks. Through this, we show that LightOCT can provide significant diagnostic support for a variety of OCT images with sufficient training and minimal hyper-parameter tuning. The trained LightOCT networks for the three-classification problem will be released online to support transfer learning on other datasets., Competing Interests: Authors declare no competing interest., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Sperm cell motility and morphology observed under the bright field microscopy are the only criteria for selecting a particular sperm cell during Intracytoplasmic Sperm Injection (ICSI) procedure of Assisted Reproductive Technology (ART). Several factors such as oxidative stress, cryopreservation, heat, smoking and alcohol consumption, are negatively associated with the quality of sperm cell and fertilization potential due to the changing of subcellular structures and functions which are overlooked. However, bright field imaging contrast is insufficient to distinguish tiniest morphological cell features that might influence the fertilizing ability of sperm cell. We developed a partially spatially coherent digital holographic microscope (PSC-DHM) for quantitative phase imaging (QPI) in order to distinguish normal sperm cells from sperm cells under different stress conditions such as cryopreservation, exposure to hydrogen peroxide and ethanol. Phase maps of total 10,163 sperm cells (2,400 control cells, 2,750 spermatozoa after cryopreservation, 2,515 and 2,498 cells under hydrogen peroxide and ethanol respectively) are reconstructed using the data acquired from the PSC-DHM system. Total of seven feedforward deep neural networks (DNN) are employed for the classification of the phase maps for normal and stress affected sperm cells. When validated against the test dataset, the DNN provided an average sensitivity, specificity and accuracy of 85.5%, 94.7% and 85.6%, respectively. The current QPI + DNN framework is applicable for further improving ICSI procedure and the diagnostic efficiency for the classification of semen quality in regard to their fertilization potential and other biomedical applications in general.

Super-resolution fluorescence microscopy is a widely employed technique in cell biology research, yet remains relatively unexplored in the field of histopathology. Here, we describe the sample preparation steps and acquisition parameters necessary to obtain fluorescent multicolor super-resolution structured illumination microscopy (SIM) images of both formalin-fixed paraffin-embedded and cryo-preserved placental tissue sections. We compare super-resolved images of chorionic villi against diffraction-limited deconvolution microscopy and show the significant contrast and resolution enhancement attainable with SIM, demonstrating the applicability of this imaging technique for both clinical diagnosis and biological research., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In pregnancy during an inflammatory condition, macrophages present at the feto-maternal junction release an increased amount of nitric oxide (NO) and pro-inflammatory cytokines such as TNF- α and INF-γ, which can disturb the trophoblast functions and pregnancy outcome. Measurement of the cellular and sub-cellular morphological modifications associated with inflammatory responses are important in order to quantify the extent of trophoblast dysfunction for clinical implication. With this motivation, we investigated morphological, cellular and sub-cellular changes in externally inflamed RAW264.7 (macrophage) and HTR-8/SVneo (trophoblast) using structured illumination microscopy (SIM) and quantitative phase microscopy (QPM). We monitored the production of NO, changes in cell membrane and mitochondrial structure of macrophages and trophoblasts when exposed to different concentrations of pro-inflammatory agents (LPS and TNF- α ). In vitro NO production by LPS-induced macrophages increased 22-fold as compared to controls, whereas no significant NO production was seen after the TNF- α challenge. Under similar conditions as with macrophages, trophoblasts did not produce NO following either LPS or the TNF- α challenge. Super-resolution SIM imaging showed changes in the morphology of mitochondria and the plasma membrane in macrophages following the LPS challenge and in trophoblasts following the TNF- α challenge. Label-free QPM showed a decrease in the optical thickness of the LPS-challenged macrophages while TNF- α having no effect. The vice-versa is observed for the trophoblasts. We further exploited machine learning approaches on a QPM dataset to detect and to classify the inflammation with an accuracy of 99.9% for LPS-challenged macrophages and 98.3% for TNF- α -challenged trophoblasts. We believe that the multi-modal advanced microscopy methodologies coupled with machine learning approach could be a potential way for early detection of inflammation., Competing Interests: Authors declare no competing interest., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1,2] and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGCs). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications., (© 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present an open-source implementation of the fluctuation-based nanoscopy method MUSICAL for ImageJ. This implementation improves the algorithm's computational efficiency and takes advantage of multi-threading to provide orders of magnitude faster reconstructions than the original MATLAB implementation. In addition, the plugin is capable of generating super-resolution videos from large stacks of time-lapse images via an interleaved reconstruction, thus enabling easy-to-use multi-color super-resolution imaging of dynamic systems., Competing Interests: The computational enhancements in the back end are a part of the invention disclosure 2019/3595 claimed by UiT The Arctic University of Norway. This part of the invention disclosure is not intended for commercialization and does not conflict with the commercialization of the remaining part. The authors declare that there are no conflicts of interest related to this article., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Phase shifting interferometric (PSI) techniques are among the most sensitive phase measurement methods. Owing to its high sensitivity, any minute phase change caused due to environmental instability results into, inaccurate phase measurement. Consequently, a well calibrated piezo electric transducer (PZT) and highly-stable environment is mandatory for measuring accurate phase map using PSI implementation. Here, we present an inverse approach, which can retrieve phase maps of the samples with negligible errors under environmental fluctuations. The method is implemented by recording a video of continuous temporally phase shifted interferograms and phase shifts were calculated between all the data frames using Fourier transform algorithm with a high accuracy ≤ 5.5 × 10 -4 π rad. To demonstrate the robustness of the proposed method, a manual translation of the stage was employed to introduce continuous temporal phase shift between data frames. The developed algorithm is first verified by performing quantitative phase imaging of optical waveguide and red blood cells using uncalibrated PZT under the influence of vibrations/air turbulence and compared with the well calibrated PZT results. Furthermore, we demonstrated the potential of the proposed approach by acquiring the quantitative phase imaging of an optical waveguide with a rib height of only 2 nm and liver sinusoidal endothelial cells (LSECs). By using 12-bit CMOS camera the height of shallow rib waveguide is measured with a height sensitivity of 4 Å without using PZT and in presence of environmental fluctuations.vn.

The advantages of quantitative phase microscopy (QPM) such as label-free imaging with high spatial sensitivity, live cell compatibility and high-speed imaging makes it viable for various biological applications. The measurement accuracy of QPM strongly relies on the shape of the recorded interferograms, whether straight or curved fringes are recorded during the data acquisition. Moreover, for a single shot phase recovery high fringe density is required. The wavefront curvature for the high-density fringes over the entire field of view is difficult to be discerned with the naked eye. As a consequence, there is a quadratic phase aberration in the recovered phase images due to curvature mismatch. In the present work, we have implemented sampling moiré method for real-time sensing of the wavefront curvature mismatch between the object and the reference wavefronts and further for its correction. By zooming out the interferogram, moiré fringes are generated which helps to easily identify the curvature of the fringes. The wavefront curvature mismatch correction accuracy of the method is tested with the help of low temporal coherent light source such as a white light (temporal coherence ∼ 1.6 µm). The proposed scheme is successfully demonstrated to remove the quadratic phase aberration caused due to wavefront mismatch from an USAF resolution target and the biological tissue samples. The phase recovery accuracy of the current scheme is further compared with and found to better than the standard method called principle component analysis. The proposed method enables recording of the corrected wavefront interferogram without needing any additional optical components or modification and also does not need any post-processing correction algorithms. The proposed method of curvature compensation paves the path for a high-throughput and accurate quantitative phase imaging.

Fringe patterns encode the information about the result of a measurement performed via widely used optical full-field testing methods, e.g., interferometry, digital holographic microscopy, moiré techniques, structured illumination etc. Affected by the optical setup, changing environment and the sample itself fringe patterns are often corrupted with substantial noise, strong and uneven background illumination and exhibit low contrast. Fringe pattern enhancement, i.e., noise minimization and background term removal, at the pre-processing stage prior to the phase map calculation (for the measurement result decoding) is therefore essential to minimize the jeopardizing effect the mentioned error sources have on the optical measurement outcome. In this contribution we propose an automatic, robust and highly effective fringe pattern enhancement method based on the novel period-guided bidimensional empirical mode decomposition algorithm (PG-BEMD). The spatial distribution of the fringe period is estimated using the novel windowed approach and then serves as an indicator for the truly adaptive decomposition with the filter size locally adjusted to the fringe pattern density. In this way the fringe term is successfully extracted in a single (first) decomposition component alleviating the cumbersome mode mixing phenomenon and greatly simplifying the automatic signal reconstruction. Hence, the fringe term is dissected without the need for modes selection nor summation. The noise removal robustness is ensured employing the block matching 3D filtering of the fringe pattern prior to its decomposition. Performance validation against previously reported modified empirical mode decomposition techniques is provided using numerical simulations and experimental data verifying the versatility and effectiveness of the proposed approach.

The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 µm x 500 µm region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

A complete study of electric field vectors and efficiencies of diffraction orders for a phase pattern addressed to a pixelated spatial light modulator (SLM) is discussed here. General mathematical expressions of electric field vectors from SLM are explored here analytically for an arbitrary pattern on SLM with a given input electric field. Using the general expression, we calculate orientations of the electric fields of diffraction for sinusoidal and binary patterns of varying duty cycles. The patterns result in diffracted beams of various orders with different efficiencies calculated analytically and matching well with the experimental results. The binary pattern with 50% duty cycle delivers significant distribution of energies where all the even orders are absent, and the diffraction efficiency of the first-order beam can be close to 40% using an appropriately chosen modulation depth. Using the general expression, it is possible to obtain fields and efficiencies of diffraction for any arbitrary pattern on SLM. The discussion might help researchers using SLM in standard optics experiments.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines coherent imaging techniques with waveguide microscopy to realize a super-condenser featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si 3 N 4 waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 μm 2 in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We test the method in simulations and in experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm. While the generated Fourier ptychography reconstructions are found to be prone to image artefacts, an alternative coherent imaging method, rotating coherent scattering microscopy (ROCS), is found to be more robust against artefacts but with less achievable resolution.

In the present Letter, a synthesized pseudothermal light source having high temporal coherence (TC) and low spatial coherence (SC) properties is used. The longitudinal coherence (LC) properties of the spatially extended monochromatic light source are systematically studied. The pseudothermal light source is generated from two different monochromatic laser sources: He-Ne (at 632 nm) and DPSS (at 532 nm). It was found that the LC length of such a light source becomes independent of the parent laser's TC length for a large source size. For the chosen lasers, the LC length becomes constant to about 30 μm for a laser source size of ≥3.3  mm. Thus, by appropriately choosing the source size, any monochromatic laser light source depending on the biological window can be utilized to obtain high axial resolution in an optical coherence tomography (OCT) system irrespective of its TC length. The axial resolution of 650 nm was obtained using a 1.2 numerical aperture objective lens at a 632 nm wavelength. These findings pave the path for widespread penetration of pseudothermal light into existing OCT systems with enhanced performance. A pseudothermal light source with high TC and low SC properties could be an attractive alternative light source for achieving high axial resolution without needing dispersion compensation as compared to a broadband light source.