Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling.

Mammalian bodies have more than a billion cells per cubic centimeter, which makes whole-body cell (WBC) profiling of an organism one of the ultimate challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in whole organs and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying mechanisms. Tissue clearing is usually conducted by the following steps: (a) fixation, (b) permeabilization, (c) decolorizing, and (d) refractive index (RI) matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acid probes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various applications, such as identifying cellular circuits across multiple organs and measuring their dynamics in stochastic and proliferative cellular processes, for example, autoimmune and malignant neoplastic diseases.

Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions.

Characterization of the size and material properties of particles in liquid suspensions is in very high demand, for example, in the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, existing methods are limited in their ability to decipher the constituents of realistic samples. Here we introduce iNTA as a new method that combines interferometric detection of scattering with nanoparticle tracking analysis to reach unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multicomponent and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

Transition metal dichalcogenide nanospheres for high-refractive-index nanophotonics and biomedical theranostics.

Recent developments in the area of resonant dielectric nanostructures have created attractive opportunities for concentrating and manipulating light at the nanoscale and the establishment of the new exciting field of all-dielectric nanophotonics. Transition metal dichalcogenides (TMDCs) with nanopatterned surfaces are especially promising for these tasks. Still, the fabrication of these structures requires sophisticated lithographic processes, drastically complicating application prospects. To bridge this gap and broaden the application scope of TMDC nanomaterials, we report here femtosecond laser-ablative fabrication of water-dispersed spherical TMDC (MoS2 and WS2) nanoparticles (NPs) of variable size (5 to 250 nm). Such NPs demonstrate exciting optical and electronic properties inherited from TMDC crystals, due to preserved crystalline structure, which offers a unique combination of pronounced excitonic response and high refractive index value, making possible a strong concentration of electromagnetic field in the NPs. Furthermore, such NPs offer additional tunability due to hybridization between the Mie and excitonic resonances. Such properties bring to life a number of nontrivial effects, including enhanced photoabsorption and photothermal conversion. As an illustration, we demonstrate that the NPs exhibit a very strong photothermal response, much exceeding that of conventional dielectric nanoresonators based on Si. Being in a mobile colloidal state and exhibiting superior optical properties compared to other dielectric resonant structures, the synthesized TMDC NPs offer opportunities for the development of next-generation nanophotonic and nanotheranostic platforms, including photothermal therapy and multimodal bioimaging.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

High Refractive Index Imaging Buffer for Dual-Color 3D SMLM Imaging of Thick Samples.

The current limitations of single-molecule localization microscopy (SMLM) in deep tissue imaging, primarily due to depth-dependent aberrations caused by refractive index (RI) mismatch, present a significant challenge in achieving high-resolution images at greater depths. To extend the imaging depth, we optimized the imaging buffer of SMLM with the RI matched to that of the objective immersion medium and systematically evaluated five different RI-matched buffers, focusing on their impact on the blinking behavior of red-absorbing dyes and the quality of reconstructed super-resolution images. Particularly, we found that clear unobstructed brain imaging cocktails-based imaging buffer could match the RI of oil and was able to clear the tissue samples. With the help of the RI-matched imaging buffers, we showed high-quality dual-color 3D SMLM images with imaging depths ranging from a few micrometers to tens of micrometers in both cultured cells and sectioned tissue samples. This advancement offers a practical and accessible method for high-resolution imaging at greater depths without the need for specialized optical equipment or expertise.

Modeling Absorption Dynamics of Differently Shaped Gold Glioblastoma and Colon Cells Based on Refractive Index Distribution in Holotomographic Imaging.

Herein, it is demonstrated that the toxic effect of gold nanoparticles (Au NPs) on three different cancer cell lines (U-118 and LN-299 glioblastoma and HCT-116 colon) depends on their absorption dynamics by cells, related to the shapes of the NPs. This hypothesis is confirmed by showing that i) based on refractive index (RI) values, typical for cell components and gold nanoparticles, it is possible to show the absorption dynamics and accumulation locations of the latter ones inside and outside of the cells. Moreover, ii) the saturation of the accumulated Au NPs volume in the cells depends on the nanoparticle shape and is reached in the shortest time for star-shaped Au NPs (AuS NPs) and in the longest time for spherical Au NPs (AuSph NPs) and on the cancer cells, where the longest and the shortest saturation are noticed for HCT-116 and LN-229 cells, respectively. A physical model of Au NPs absorption dynamics is proposed, where the diameter and shape of the Au NPs are used as parameters. The obtained theoretical data are consistent with experimental data in 85-98%.

Tissue characterization using axicon probe-assisted common-path optical coherence tomography.

In this work, a common-path optical coherence tomography (OCT) system is demonstrated for characterizing the tissue in terms of some optical properties. A negative axicon structure chemically etched inside the fiber tip is employed as optical probe in the OCT. This probe generates a quality Bessel beam owning a large depth-of-field, ∼700 µm and small central spot size, ∼3 µm. The OCT system is probing the sample without using any microscopic lens. For experimental validation, the OCT imaging of chicken tissue has been obtained along with estimation of its refractive index and optical attenuation coefficient. Afterwards, the cancerous tissue is differentiated from the normal tissue based on the OCT imaging, refractive index, and optical attenuation coefficient. The respective tissue samples are collected from the human liver and pancreas. This probe could be a useful tool for endoscopic or minimal-invasive inspection of malignancy inside the tissue either at early-stage or during surgery.

A Facile One-Step Conversion of Biobased Magnolol (Honokiol) toward High Refractive Materials.

A series of polymers with both high refractive index and high Abbe number have been successfully synthesized through the photoclick thiol-ene reaction between the monomers derived from biobased magnolol (or honokiol) and commercial mercaptans and thiophenols. The polymer films not only exhibit a high refractive index and a high Abbe number but also display a transmittance of up to 90% in a range of wavelengths from 550 to 2000 nm and nearly 0% in the UV region. Moreover, these polymers also display low haze values in the visible-light region as well as exhibit good thermostability. These data indicate that they have potential applications for the fabrication of optical lenses and anti-UV coatings. In particular, this series of polymers are readily used for industrialization due to its excellent optical properties but low expense, simplicity, and efficiency of synthesis.

Refractive-index and density-matched emulsions with programmable DNA interactions.

Emulsion droplets on the colloidal length scale are a model system of frictionless compliant spheres. Direct imaging studies of the microscopic structure and dynamics of emulsions offer valuable insights into fundamental processes, such as gelation, jamming, and self-assembly. A microscope, however, can only resolve the individual droplets in a densely packed emulsion if the droplets are closely index-matched to their fluid medium. Mitigating perturbations due to gravity additionally requires the droplets to be density-matched to the medium. Creating droplets that are simultaneously index-matched and density-matched has been a long-standing challenge for the soft-matter community. The present study introduces a method for synthesizing monodisperse micrometer-sized siloxane droplets whose density and refractive index can be precisely and independently tuned by adjusting the volume fraction of three silane precursors. A systematic optimization protocol yields fluorescently labeled ternary droplets whose densities and refractive indexes match, to the fourth decimal place, those of aqueous solutions of glycerol or dimethylsiloxane. Because all of the materials in this system are biocompatible, we functionalize the droplets with DNA strands to endow them with programmed inter-droplet interactions. Confocal microscopy then reveals both the three-dimensional structure and the network of droplet-droplet contacts in a class of self-assembled droplet gels, free from gravitational effects. This experimental toolbox creates opportunities for studying the microscopic mechanisms that govern viscoelastic properties and self-assembly in soft materials.

Non-invasive Visualization and Characterization of Bile Canaliculus Formation Using Refractive Index Tomography.

The vital role of bile canaliculus (BC) in liver function is closely related to its morphology. Electron microscopy has contributed to understanding BC morphology; however, its invasiveness limits its use in living specimens. Here, we report non-invasive characterization of BC formation using refractive index (RI) tomography. First, we investigated and characterized the RI distribution of BCs in two-dimensional (2D) cultured HepG2 cells. BCs were identified based on their distinct morphology and functionality, as confirmed using a fluorescence-labeled bile acid analog. The RI distribution of BCs exhibited three common features: (1) luminal spaces with a low RI between adjacent hepatocytes; (2) luminal spaces surrounded by a membranous structure with a high RI; and (3) multiple microvillus structures with a high RI within the lumen. Second, we demonstrated the characterization of BC structures in a three-dimensional (3D) culture model, which is more relevant to the in vivo environment but more difficult to evaluate than 2D cultures. Various BC structures were identified inside HepG2 spheroids with the three features of RI distribution. Third, we conducted comparative analyses and found that the BC lumina of spheroids had higher circularity and lower RI standard deviation than 2D cultures. We also addressed comparison of BC and intracellular lumen-like structures within a HepG2 spheroid, and found that the BC lumina had higher RI and longer perimeter than intracellular lumen-like structures. Our demonstration of the non-destructive, label-free visualization and quantitative characterization of living BC structures will be a basis for various hepatological and pharmaceutical applications.

Intracapsular accommodation mechanism in terms of lens curvature gradient.

The intracapsular accommodation mechanism (IAM) may be understood as an increase in the lens equivalent refractive index as the eye accommodates. Our goal was to evaluate the existence of an IAM by analysing observed changes in the inner curvature gradient of the lens. To this end, we fitted a gradient index and curvature lens model to published experimental data on external and nucleus geometry changes during accommodation. For each case analysed, we computed the refractive power and equivalent index for each accommodative state using a ray transfer matrix. All data sets showed an increase in the effective refractive index, indicating a positive IAM, which was stronger for older lenses. These results suggest a strong dependence of the lens equivalent refractive index on the inner curvature gradient.

An Approach to Estimate the Refractive Index and the Electronic Polarizability of the Ternary Ag2O-B2O3-TeO2 Glasses.

Each of the static properties such as refractive index (n0), cation ( ∑ α i $$ \sum {\alpha}_i $$ ), and anion ( α O 2 - $$ {\alpha}_O^{2-} $$ ) oxide polarizabilities for the ternary 30Ag2O⋅xB2O3⋅(70 - x)TeO2 (30AgBTe) glasses has been predicted theoretically from those of the binary 30Ag2O-70B2O3 and 30Ag2O-70TeO2 glasses. This can be done based on two assumptions: that each of these static properties (n0, ∑ α i $$ \sum {\upalpha}_i $$ , and α O 2 - $$ {\upalpha}_{\mathrm{O}}^{2-} $$ ) can be considered as an additive property and that ternary 30AgBTe glasses can be treated as a mixture of two binary 30Ag2O-70B2O3 and 30Ag2O-70TeO2 glasses. In addition, n0 values for the ternary 30AgBTe glasses can be predicted in terms of α O 2 - $$ {\upalpha}_{\mathrm{O}}^{2-} $$ and ∑ α i $$ \sum {\upalpha}_i $$ values for the ternary 30AgBTe glasses, and these later properties can be predicted from that of two binaries like as n0 at first stage. The n0 values obtained by using two methods are exactly the same for the corresponding compositions in the studied glasses, confirming the validity of the two assumptions and the procedure described in the present work. This conclusion is valid for the ternary glasses with a fixed content of either basic former/or modifier oxides for all compositions such as xPbO⋅(40 - x)Sb2O3⋅60B2O3 and 30Ag2O⋅xB2O3⋅(70 - x)TeO2 glasses, respectively.

The influence of solvent refractive index on the photoluminescence decay of thick-shell gradient-alloyed colloidal quantum dots investigated in a wide range of delay times.

Colloidal semiconductor quantum dots have many potential optical applications, including quantum dot light-emitting diodes, single-photon sources, or biological luminescent markers. The optical properties of colloidal quantum dots can be affected by their dielectric environment. This study investigated the photoluminescence (PL) decay of thick-shell gradient-alloyed colloidal semiconductor quantum dots as a function of solvent refractive index. These measurements were conducted in a wide range of delay times to account for both the initial spontaneous decay of excitons and the delayed emission of excitons that has the form of a power law. It is shown that whereas the initial spontaneous PL decay is very sensitive to the refractive index of the solvent, the power-law delayed emission of excitons is not. Our results seem to exclude the possibility of carrier self-trapping in the considered solvents and suggest the existence of trap states inside the quantum dots. Finally, our data show that the average exciton lifetime significantly decreases as a function of the solvent refractive index. The change in exciton lifetime is qualitatively modeled and discussed.

A Label-Free Optical Biosensor Based on an Array of Microring Resonators for the Detection of Human Serum Albumin.

We introduced a label-free sensing system based on an array of microring resonators (MRRs) which was successfully employed for human serum albumin (HSA) detection. The sensing-ring surface was functionalized to immobilize anti-HSA, facilitating HSA binding. Our refractive index sensing system demonstrates high sensitivity at 168 nm/RIU and a low limit of detection (LOD) of 63.54 ng/mL, closely comparable to current HSA detection methods. These findings confirm the potential of MRRs as biocompatible sensors for HSA detection. This system holds great promise as an innovative platform for the detection of HSA, carrying significant importance in medical diagnostics.

A Novel Biosensor for the Detection of Glucose Concentration Using the Dual-Peak Long Period Grating in the Near- to Mid-Infrared.

In this article, we demonstrate an improved efficient fibre sensor with a high sensitivity to measure glucose concentrations in the physiological range of human beings, operating in a broad spectral bandwidth from the near- to mid-infrared. The sensor consists of a dual-peak long period grating (DPLPG) with a period of 150 µm inscribed in an optical fibre with a diameter of 80 µm. The investigation of sensing for refractive index results in a sensitivity of ~-885.7 nm/refractive index unit (RIU) and ~2008.6 nm/RIU in the range of 1.30-1.44. The glucose measurement is achieved by the immobilisation of a layer of enzyme of glucose oxidase (GOD) onto the fibre surface for the selective enhancement of sensitivity for glucose. The sensor can measure glucose concentrations with a maximum sensitivity of -36.25 nm/(mg/mL) in the range of 0.1-3.0 mg/mL. To the best of our knowledge, this is the highest sensitivity ever achieved for a measurement of glucose with a long period grating-based sensor, indicating its potential for many applications including pharmaceutical, biomedical and food industries.

Fibre Refractometry for Minimally Invasive Sugar Content Measurements within Produce.

A minimally invasive needle refractometer is presented for sugar content measurements within produce. A passive sampling cap structure was developed that improves the reliability of the device by avoiding interfering back reflections from the flesh of the produce. It is explained that factory calibration may not be needed for this type of refractometer, potentially reducing production costs. Also demonstrated is an iterative method to correct for temperature variations without the need for an integrated model for how the refractive index changes with temperature for different levels of sugar concentration. The sensor showed a typical standard deviation of 0.4 °Bx for a 10-s-long measurement and was validated against a prism refractometer, showing an average offset of (0.0±0.1) °Bx. In addition, the potential for using the device to investigate sugar distributions within a single fruit sample is demonstrated.

High-Sensitivity Janus Sensor Enabled by Multilayered Metastructure Based on the Photonic Spin Hall Effect and Its Potential Applications in Bio-Sensing.

The refractive index (RI) of biological tissues is a fundamental material parameter that characterizes how light interacts with tissues, making accurate measurement of RI crucial for biomedical diagnostics and environmental monitoring. A Janus sensor (JBS) is designed in this paper, and the photonic spin Hall effect (PSHE) is used to detect subtle changes in RI in biological tissues. The asymmetric arrangement of the dielectric layers breaks spatial parity symmetry, resulting in significantly different PSHE displacements during the forward and backward propagation of electromagnetic waves, thereby realizing the Janus effect. The designed JBS can detect the RI range of 1.3~1.55 RIU when electromagnetic waves are incident along the +z-axis, with a sensitivity of 96.29°/refractive index unit (RIU). In the reverse direction, blood glucose concentrations are identified by the JBS, achieving a sensitivity of 18.30°/RIU. Detecting different RI range from forward and backward scales not only overcomes the limitation that single-scale sensors can only detect a single RI range, but also provides new insights and applications for optical biological detection through high-sensitivity, label-free and non-contact detection.

Label-Free Biosensor Based on Particle Plasmon Resonance Coupled with Diffraction Grating Waveguide.

Particle plasmon resonance (PPR), or localized surface plasmon resonance (LSPR), utilizes intrinsic resonance in metal nanoparticles for sensor fabrication. While diffraction grating waveguides monitor bioaffinity adsorption with out-of-plane illumination, integrating them with PPR for biomolecular detection schemes remains underexplored. This study introduces a label-free biosensing platform integrating PPR with a diffraction grating waveguide. Gold nanoparticles are immobilized on a glass slide in contact with a sample, while a UV-assisted embossed diffraction grating is positioned opposite. The setup utilizes diffraction in reflection to detect changes in the environment's refractive index, indicating biomolecular binding at the gold nanoparticle surface. The positional shift of the diffracted beam, measured with varying refractive indices of sucrose solutions, shows a sensitivity of 0.97 mm/RIU at 8 cm from a position-sensitive detector, highlighting enhanced sensitivity due to PPR-diffraction coupling near the gold nanoparticle surface. Furthermore, the sensor achieved a resolution of 3.1 × 10-4 refractive index unit and a detection limit of 4.4 pM for detection of anti-DNP. The sensitivity of the diffracted spot was confirmed using finite element method (FEM) simulations in COMSOL Multiphysics. This study presents a significant advancement in biosensing technology, offering practical solutions for sensitive, rapid, and label-free biomolecule detection.

Label-Free Three-Dimensional Morphological Characterization of Cell Death Using Holographic Tomography.

This study presents a novel label-free approach for characterizing cell death states, eliminating the need for complex molecular labeling that may yield artificial or ambiguous results due to technical limitations in microscope resolution. The proposed holographic tomography technique offers a label-free avenue for capturing precise three-dimensional (3D) refractive index morphologies of cells and directly analyzing cellular parameters like area, height, volume, and nucleus/cytoplasm ratio within the 3D cellular model. We showcase holographic tomography results illustrating various cell death types and elucidate distinctive refractive index correlations with specific cell morphologies complemented by biochemical assays to verify cell death states. These findings hold promise for advancing in situ single cell state identification and diagnosis applications.

Nanosensors Based on Bimetallic Plasmonic Layer and Black Phosphorus: Application to Urine Glucose Detection.

This paper presents a new biosensor design based on the Kretschmann configuration, for the detection of analytes at different refractive indices. Our studied design consists of a TiO2/SiO2 bi-layer sandwiched between a BK7 prism and a bimetallic layer of Ag/Au plasmonic materials, covered by a layer of black phosphorus placed below the analyte-containing detection medium. The different layers of our structure and analyte detection were optimized using the angular interrogation method. High performance was achieved, with a sensitivity of 240 deg/RIU and a quality factor of 34.7 RIU-1. This biosensor can detect analytes with a wide refractive index range between 1.330 and 1.347, such as glucose detection in urine samples using a refractive index variation of 10-3. This capability offers a wide range of applications for biomedical and biochemical detection and selectivity.