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.

Far-field optical nanoscopy techniques are frequently used to visualize subcellular structures in biological specimens by surpassing the diffraction limit. Optical nanoscopy encompasses a plethora of techniques, including stimulated emission depletion microscopy (1), structured illumination microscopy (SIM) (2), different variants of single-molecule localization microscopy (SMLM), such as photo-activated localization microscopy (3) and direct stochastic optical reconstruction microscopy (dSTORM) (4), and intensity fluctuation–based techniques such as superresolution optical fluctuation imaging (5). These techniques can help detect subcellular structures (<200 nm) of biological specimens such as lipids, proteins, membrane structures, microtubules, and nucleic acids by specific fluorescence tagging (6). Each technique has respective advantages and disadvantages; for example, SIM has gained popularity for live-cell imaging due to its fast image acquisition time but at limited spatial resolution (7). dSTORM, on the other hand, is slower but offers high resolution for characterization of viral proteins (8) and imaging actin filaments in mammalian cells (9, 10), for example. To reduce the complexity of the typical SMLM setup using a total internal reflection fluorescence (TIRF) configuration, a photonic chip-based optical nanoscopy system was recently proposed (11–13). In the chip-based system, a photonic integrated circuit is used to replace the usual free space optics for excitation. The collection, however, is done through free space optics. The main advantage of this configuration is the decoupling of excitation and collection pathways as well as miniaturization of the excitation light path of the system. In chip-based nanoscopy, the TIRF illumination is generated through the evanescent field of waveguides rather than using conventional high magnification and high numerical aperture (N.A.) TIRF lens. The evanescent field in waveguides can be generated over extraordinarily large areas, as it is only defined by the waveguide geometry. The waveguide geometry makes it possible to use any imaging objective lens to image arbitrarily large areas as compared to the traditional TIRF-based dSTORM (12), which is limited by the field of view (FOV) of the TIRF lens.Quantitative phase microscopy (QPM) is a label-free optical microscopy technique, which facilitates sensitive measurements of the refractive index and thickness of both biological specimens (14). Various QPM methods have been proposed so far for extracting optical phase and dynamics of biological cells (15–17). These techniques offer high phase sensitivity (spatial and temporal), transverse resolution, and high imaging speed (15). The spatial and temporal phase sensitivity of the QPM system is highly dependent on the illumination source and the type of interferometric geometry, respectively (17–19). For example, common path QPM techniques offer better temporal phase sensitivity, which can be used to measure membrane fluctuation of the cells (20). In addition, spatial phase sensitivity of the system can be improved by using low-coherence light sources (halogen lamps and light-emitting diodes [LED]) but requires phase-shifting techniques to utilize the whole FOV of the camera (21). A recent advancement in the QPM technique with superior resolution using structured illumination (22, 23) and three-dimensional (3D) information of the samples has been shown by measuring the phase across multiple angles of illumination. This technique facilitates tomography of various biological specimens such as red blood cells, HT29 cells, and bovine embryos (17, 24). Since the lateral resolution of the QPM technique depends on the N.A. of the objective lens, imaging beyond the diffraction limit (<200 nm) is still challenging and limits the study of subcellular structures. Therefore, it is useful to develop multimodality routes in which different microscopy methods can be utilized to provide complementary information about biological specimens such as liver sinusoidal endothelial cells (LSEC).Fig. 1 depicts LSEC that contain large numbers of fenestrations. These transcellular nanopores vary in diameter from 50 to 300 nm, which is just below the diffraction limit of optical microscopy (25–27). Fenestrations are typically clustered in groups of 5 to 100 within areas called sieve plates (28). The porous morphology of LSEC acts as an ultrafilter between blood and the underlying hepatocytes, facilitating the bidirectional exchange of substrates between the interior of the liver and blood. For example, smaller viruses and drugs can pass this barrier, while blood cells are retained within the sinusoidal vessel lumen (25, 29). The typical thickness of sieve plates is around 100 to 150 nm (30), so fenestrations are consequently nanoscale sized in all three dimensions. As shown in Fig. 1, the fenestrations in sieve plates form openings through the entire LSEC cell body, and therefore TIRF illumination is ideally suited for imaging these structures. Determining the diameter and number of fenestrations, as well as the height of sieve plate regions, is important, as it can be affected by several drugs and conditions (31, 32). The loss of LSEC porous morphology, a process called defenestration, compromises the filtration properties of the liver, which may lead to atherosclerosis (33). Moreover, aging results in “pseudocapillarization,” whereby LSEC simultaneously lose fenestrations and become thicker (34) (Fig. 1). This is believed to be a main factor contributing to the age-related need to increase doses of drugs targeting hepatocytes (e.g., statins) that have to pass through the fenestrations (35). The number of fenestrations in vitro can be increased using actin disrupting agents such as cytochalasin B (27). This treatment decreases the height of LSEC outside of the nuclear area, which contributes to the formation of new fenestrations (36).Open in a separate windowFig. 1.Top view (A) and cross-sectional view (B) of LSEC. LSEC have unique morphology, in which nanoscopic fenestrations are grouped in thin sieve plates. The diameter of fenestrations and thickness of sieve plates are below the diffraction limit of conventional optical microscopes. The number and size of fenestrations, but also LSEC thickness, can be affected by aging and in liver diseases. In vitro, the number of fenestrations can be increased using actin disrupting agents, such as cytochalasin B (27).Here, we have developed a multimodal chip-based optical nanoscopy and highly sensitive QPM system to visualize the 3D morphological changes in LSEC. The proposed system decouples the light illumination path from the collection path and thus enables a straightforward integration of dSTORM and QPM. The nanoscale phase sensitivity of the QPM technique is utilized to extract the optical thickness of sieve plates. Moreover, chip-based dSTORM supports superresolution imaging down to 50 nm over an extraordinarily large FOV up to millimeter scale (12). Therefore, integration of dSTORM and QPM allows superresolution imaging in the lateral dimension (with dSTORM) and nanometric sensitivity in the axial direction (with QPM). In this work, we demonstrate the capabilities of the system by imaging LSEC with both diffraction-limited TIRF microscopy and dSTORM. The fenestrations and sieve plates are observable with dSTORM, and the average optical thickness of the sieve plate region is obtained using diffraction-limited QPM. Furthermore, we investigated the change in the interior morphology of sieve plates by treating the cell with cytochalasin B (10 μg/mL). The deficiency of lateral resolution of QPM was compensated by dSTORM, which enabled us to localize the sieve plate regions containing subdiffraction-sized fenestration. Therefore, in the cell membrane regions distal from the nucleus, the 3D morphology of LSEC can be reconstructed reliably using our multimodal approach. The integrated system offers a combination of simultaneous functional and quantitative imaging of the cells with large FOV, providing a compact imaging platform with a potential for high-throughput morphological and nanometric imaging for specific biological applications.     

Improved morbidity with the use of nasal continuous positive airway pressure in I-cell disease

Patients with I-cell disease (mucolipidosis II) present with progressive morbidity failure to thrive, cardiomegaly, and recurrent respiratory tract infections leading to progressive deterioration and early death. We evaluated use of nasal continuous positive airway pressure (NCPAP) for 6 months in a 2-year-old girl with I-cell disease, obstructive sleep apnea (OSA), and craniofacial anomalies. We observed a marked decrease in hospitalizations for respiratory problems and a marked improvement in arterial blood gases with the use of NCPAP. In patients with I-cell disease, anatomical defects with superimposed upper respiratory tract infections cause worsening of OSA, and OSA contributes significantly to morbidity. In such patients NCPAP can lessen morbidity and can improve the quality of life. Pediatr. Pulmonol. 1998; 25:128–129. © 1998 Wiley-Liss, Inc.     

Polydopamine functionalized VEGF gene-activated 3D printed scaffolds for bone regeneration

Bone is a highly vascularized organ and the formation of new blood vessels is essential to regenerate large critical bone defects. In this study, polylactic acid (PLA) scaffolds of 20–80% infill were three-dimensionally (3D) printed using a fused deposition modeling based 3D printer. The PLA scaffolds were coated with polydopamine (PDA) and then were surface-functionalized with polyethyleneimine (PEI) and VEGF-encoding plasmid DNA (pVEGF) nanoplexes (PLA-PDA-PEI-pVEGF). The PLA-PDA-PEI-pVEGF scaffolds with 40% infill demonstrated higher encapsulation efficiency and sustained release of pVEGF than scaffolds with 20, 60 and 80% infill and were therefore used for in vitro and in vivo studies. The PLA-PDA-PEI-pVEGF increased the translation and secretion of VEGF and BMP-2. The PLA-PDA-PEI-pVEGF also yielded a 2- and 4.5-fold change in VEGF and osteocalcin gene expression in vitro, respectively. A tube formation assay using human umbilical vascular endothelial cells (HUVECs) showed a significant increase in tube length when exposed to the PLA-PDA-PEI-pVEGF scaffold, in comparison to PLA and PLA-PDA scaffolds. The PLA-PDA-PEI-pVEGF scaffold in an in vivo rat calvarial critical bone defect model demonstrated 1.6-fold higher new bone formation compared to the PLA-PDA scaffold. H&E and Masson''s trichrome staining of bone sections also revealed that the PLA-PDA-PEI-pVEGF scaffold facilitated the formation of more blood vessels in the newly formed bone compared to the PLA and PLA-PDA scaffold groups. Thus, PLA-PDA-PEI-pVEGF might be a potential 3D printed gene activated scaffold for bone regeneration in clinical situations.

Bone is a highly vascularized organ and the formation of new blood vessels is essential to regenerate large critical bone defects.     

Salt intake and salt‐reduction strategies in South Asia: From evidence to action

The World Health Organization recommends salt reduction as a cost‐effective intervention to prevent noncommunicable diseases. Salt‐reduction interventions are best tailored to the local context, taking into consideration the varying baseline salt‐intake levels, population''s knowledge, attitude, and behaviors. Fundamental to reduction programs is the source of dietary salt‐intake. In South Asian countries, there is a paucity of such baseline evidence around factors that contribute to community salt intake. Upon reviewing the electronic literature databases and government websites through March 31, 2021, we summarized dietary salt intake levels and aimed to identify major sources of sodium in the diet. Information on the current salt reduction strategies in eight South Asian countries were summarized, namely Afghanistan, Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan, and Sri Lanka. One hundred twelve publications (out of identified 640) met our inclusion‐exclusion criteria for full text review. Twenty‐one studies were included in the review. Quality of the included studies was assessed using the US National Heart, Lung, and Blood Institute assessment tool. The primary result revealed that mean salt intake of South Asian countries was approximately twice (10 g/day) compared to WHO recommended intake (< 5 g/day). The significant proportion of salt intake is derived from salt additions during cooking and/or discretionary use at table. In most South Asian countries, there is limited data on population sodium intake based on 24‐h urinary methods and sources of dietary salt in diet. While salt reduction initiatives have been proposed in these countries, they are yet to be fully implemented and evaluated. Proven salt reduction strategies in high‐income countries could possibly be replicated in South Asian countries; however, further community‐health promotion studies are necessary to test the effectiveness and scalability of those strategies in the local context.     

Morphological analysis of the blue cone pathway in the retina of a New World monkey,the marmoset Callithrix jacchus

We have studied the components of the short wavelength-sensitive (SWS or “blue”) cone pathway in the retina of a New World primate, the marmoset Callithrix jacchus. Of particular interest was the small bistratified ganglion cell, which has been identified in macaque monkey to be the morphological substrate of the blue-ON cell (Dacey and Lee [1994] Nature 367:731–735). Small bistratified cells were intracellularly filled with Neurobiotin in an in vitro retinal wholemount preparation. Their morphology, size, and level of dendritic stratification were similar to their homologues in macaque and human retina. A number of different antibodies were applied to vertical cryostat sections, some of which were cut through the processes of injected small bistratified or parasol ganglion cells. We used antibodies against cholecystokinin (CCK) to label blue cone bipolar cells, and antibodies against the human SWS cone photopigment to label SWS cones. Double-labelled preparations showed that blue cone bipolar cell dendrites contact SWS cone pedicles, and the inner dendrites of the small bistratified cell are costratified with the axon terminals of blue cone bipolar cells. A monoclonal antibody against calbindin was used to label a subpopulation of bipolar cells that stratifies in the outer half of the inner plexiform layer. The axon terminals of these bipolar cells occasionally cross the outer dendrites of small bistratified cells and show extensive costratification with the dendrites of OFF parasol cells. We conclude that an SWS cone pathway with similar connectivity is a preserved feature of the primate visual system. J. Comp. Neurol. 379:211–225, 1997. © 1997 Wiley-Liss, Inc.     

Acetylcholinesterase inhibitory potential of a carbazole alkaloid,mahanimbine, from Murraya koenigii

In the search for acetylcholinesterase (AChE) inhibitors from Indian medicinal plants, via bioassay‐guided isolation, a carbazole alkaloid, mahanimbine [3, 5‐dimethyl‐3‐(4‐ methylpent‐3‐enyl)‐11H‐pyrano [5, 6‐a] carbazole], was isolated from the petroleum ether extract of the leaves of Murraya koenigii. Inhibition of AChE was evaluated based on Ellman's method using 96‐well microplate readers. Mahanimbine inhibited AChE activity in a dose‐dependent manner with an IC₅₀ value of 0.03 ± 0.09 mg/mL, while galantamine was used as a standard. The AChE inhibitory activity of this carbazole alkaloid has not been reported so far, and this study is the first to reveal this activity in carbazole alkaloid mahanimbine, isolated from Murraya koenigii. Copyright © 2009 John Wiley & Sons, Ltd.