Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells.

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.

Laser-Generated Scholte Waves in Floating Microparticles.

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.

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells.

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.

On-chip TIRF nanoscopy by applying Haar wavelet kernel analysis on intensity fluctuations induced by chip illumination.

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.

Soft thresholding schemes for multiple signal classification algorithm.

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.

Sampling moiré method: a tool for sensing quadratic phase distortion and its correction for accurate quantitative phase microscopy.

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.

Sub-nanometer height sensitivity by phase shifting interference microscopy under environmental fluctuations.

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.

High space-bandwidth in quantitative phase imaging using partially spatially coherent digital holographic microscopy and a deep neural network.

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.

Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli.

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.

Characterization of color cross-talk of CCD detectors and its influence in multispectral quantitative phase imaging.

Multi-spectral quantitative phase imaging (QPI) is an emerging imaging modality for wavelength dependent studies of several biological and industrial specimens. Simultaneous multi-spectral QPI is generally performed with color CCD cameras. Here, we present a new approach for accurately measuring the color crosstalk of 2D area detectors, without needing prior information about camera specifications. Color crosstalk is systematically studied and compared using compact interference microscopy on two different cameras commonly used in QPI, single chip CCD (1-CCD) and three chip CCD (3-CCD). The influence of color crosstalk on the fringe width and the visibility of the monochromatic constituents corresponding to three color channels of white light interferogram are studied both through simulations and experiments. It is observed that presence of color crosstalk changes the fringe width and visibility over the imaging field of view. This leads to an unwanted non-uniform background error in the multi-spectral phase imaging of the specimens. The color crosstalk of the detector is observed to be the limiting factor for phase measurement accuracy of simultaneous multi-spectral QPI systems.

Effect on the longitudinal coherence properties of a pseudothermal light source as a function of source size and temporal coherence.

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.

Study of electric fields of diffraction from spatial light modulator: discussion.

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.

Multi-modal chip-based fluorescence and quantitative phase microscopy for studying inflammation in macrophages.

Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.

Accurate estimation of the illumination pattern's orientation and wavelength in sinusoidal structured illumination microscopy.

Structured illumination microscopy is able to improve the spatial resolution of wide-field fluorescence imaging by applying sinusoidal stripe pattern illumination to the sample. The corresponding computational image reconstruction requires precise knowledge of the pattern's parameters, which are its phase (ϕ) and wave vector (p). Here, a computationally inexpensive method for estimation of p from the raw data is proposed and illustrated with simulations. The method estimates p through a selective discrete Fourier transform at tunable subpixel precision. This results in an accurate p estimation for all the illumination patterns and subsequently improves the superresolution image recovery by a factor of 10 around sharp edges as compared to an integer pixel approach. The technique as presented here is of major interest to the large variety of custom-build systems that are used. The feasibility of the presented method is proven in comparison with published data.

Single-Input and Multiple-Output Surface Acoustic Wave Sensing for Damage Quantification in Piezoelectric Sensors.

The main aim of the paper is damage detection at the microscale in the anisotropic piezoelectric sensors using surface acoustic waves (SAWs). A novel technique based on the single input and multiple output of Rayleigh waves is proposed to detect the microscale cracks/flaws in the sensor. A convex-shaped interdigital transducer is fabricated for excitation of divergent SAWs in the sensor. An angularly shaped interdigital transducer (IDT) is fabricated at 0 degrees and ±20 degrees for sensing the convex shape evolution of SAWs. A precalibrated damage was introduced in the piezoelectric sensor material using a micro-indenter in the direction perpendicular to the pointing direction of the SAW. Damage detection algorithms based on empirical mode decomposition (EMD) and principal component analysis (PCA) are implemented to quantify the evolution of damage in piezoelectric sensor material. The evolution of the damage was quantified using a proposed condition indicator (CI) based on normalized Euclidean norm of the change in principal angles, corresponding to pristine and damaged states. The CI indicator provides a robust and accurate metric for detection and quantification of damage.

Silicon nitride waveguide platform for fluorescence microscopy of living cells.

Waveguide chip-based microscopy reduces the complexity of total internal reflection fluorescence (TIRF) microscopy, and adds features like large field of view illumination, decoupling of illumination and collection path and easy multimodal imaging. However, for the technique to become widespread there is a need of low-loss and affordable waveguides made of high-refractive index material. Here, we develop and report a low-loss silicon nitride (Si3N4) waveguide platform for multi-color TIRF microscopy. Single mode conditions at visible wavelengths (488-660 nm) were achieved using shallow rib geometry. To generate uniform excitation over appropriate dimensions waveguide bends were used to filter-out higher modes followed by adiabatic tapering. Si3N4 material is finally shown to be biocompatible for growing and imaging living cells.

Rib waveguides for trapping and transport of particles.

Rib waveguides are investigated as an alternative to strip waveguides for planar trapping and transport of microparticles. Microparticles are successfully propelled along the surface of rib waveguides and trapped in the gap between opposing rib waveguides. The trapping capabilities of waveguide end facets formed by a single and opposing waveguide geometries are investigated. The slab beneath a rib waveguide continues to guide light after the end facet of a rib waveguide. Thus particles can be trapped in wider gaps formed by opposing rib waveguides than with strip waveguides. Rib waveguides were found more efficient in trapping a collection of particles in the gap and particles could be moved to different locations in the gap by changing the relative power in the two opposing rib waveguides. Numerical simulations are used to show that the trapping efficiency on the surface of rib and strip waveguides is comparable. The simulations also confirm the advantage of opposing rib waveguides for trapping particles in wide gaps. The low sidewalls of rib waveguides give low propagation losses and make it easy to integrate rib waveguides with other functions in a lab-on-a-chip where particle trapping and transport is required.

Optical transport, lifting and trapping of micro-particles by planar waveguides.

Optical waveguides can be used to trap and transport micro-particles. The particles are held close to the waveguide surface by the evanescent field and propelled forward. We propose a new technique to lift and trap particles above the surface of the waveguides. This is made possible by a gap between two opposing, planar waveguides. The field emitted from each of the waveguide ends diverge fast, away from the substrate and into the cover-medium. By combining two fields propagating at an angle upwards and coming from opposite sides of a gap, particles can be stably lifted and trapped at the crossing of the two fields. Thus, particles are transported by waveguides leading to a gap, where they are lifted away from the substrate and trapped. The experiments are supported by numerical simulations of the forces on the micro-particles. Fluorescence imaging is used to track the particles in 3D with a precision of 50 nm.

Squeezing red blood cells on an optical waveguide to monitor cell deformability during blood storage.

Red blood cells squeeze through micro-capillaries as part of blood circulation in the body. The deformability of red blood cells is thus critical for blood circulation. In this work, we report a method to optically squeeze red blood cells using the evanescent field present on top of a planar waveguide chip. The optical forces from a narrow waveguide are used to squeeze red blood cells to a size comparable to the waveguide width. Optical forces and pressure distributions on the cells are numerically computed to explain the squeezing process. The proposed technique is used to quantify the loss of blood deformability that occurs during blood storage lesion. Squeezing red blood cells using waveguides is a sensitive technique and works simultaneously on several cells, making the method suitable for monitoring stored blood.

Plasmonic nano-bowls for monitoring intra-membrane changes in liposomes, and DNA-based nanocarriers in suspension.

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.