Promoting Photon-to-Chemical Conversion through a Dielectric Antenna-Hybrid Bilayered Reactor Configuration.

The application of scattered light via an antenna-reactor configuration is promising for converting thermocatalysts into photocatalysts. However, the efficiency of dielectric antennas in photon-to-chemical conversion remains suboptimal. Herein, we present an effective approach to promote light utilization efficiency by designing dielectric antenna-hybrid bilayered reactors. Experimental studies and finite-difference time-domain simulations demonstrate that the engineered SiO2-carbon/metal dielectric antenna-hybrid bilayered reactors exhibit a synergy of absorption superposition and electric field confinement between carbon and metals, leading to the improved absorption of scattered light, upgraded charge carriers density, and ultimately promoted photoactivity in hydrogenating chlorobenzene with an average benzene formation rate of 18 258 µmol g-1 h-1, outperforming the reported results. Notably, the carbon interlayer proves to be more effective than the commonly explored dielectric TiO2 interlayer in boosting the benzene formation rate by over 3 times. This research paves the way for promoting near-field scattered photon-to-chemical conversion through a dielectric antenna-hybrid reactor configuration.

Protein Corona-Directed Cellular Recognition and Uptake of Polyethylene Nanoplastics by Macrophages.

The widespread use of plastic products in daily life has raised concerns about the health hazards associated with nanoplastics (NPs). When exposed, NPs are likely to infiltrate the bloodstream, interact with plasma proteins, and trigger macrophage recognition and clearance. In this study, we focused on establishing a correlation between the unique protein coronal signatures of high-density (HDPE) and low-density (LDPE) polyethylene (PE) NPs with their ultimate impact on macrophage recognition and cytotoxicity. We observed that low-density and high-density lipoprotein receptors (LDLR and SR-B1), facilitated by apolipoproteins, played an essential role in PE-NP recognition. Consequently, PE-NPs activated the caspase-3/GSDME pathway and ultimately led to pyroptosis. Advanced imaging techniques, including label-free scattered light confocal imaging and cryo-soft X-ray transmission microscopy with 3D-tomographic reconstruction (nano-CT), provided powerful insights into visualizing NPs-cell interactions. These findings underscore the potential risks of NPs to macrophages and introduce analytical methods for studying the behavior of NPs in biological systems.

A Rapid and High-Throughput Assay for Light Scattering of SARS-CoV-2 Virion-Sized Particulates via Microfluidic Spray Device Reveals the Protection Performance of Face Masks against Virus Infection.

To prevent interhuman transmission of viruses, new mask types─claiming improved filtration─require careful performance characterization. Here, a microfluidic spray device that can effectively simulate droplets emitted during coughing or sneezing was developed to spray droplets containing gold nanoparticles (AuNPs) that mimic SARS-CoV-2 to overcome the shortcomings associated with using biosamples. The light scattered by the AuNPs passing through the mask is successfully analyzed by using an automated scattering light mapping system within a duration of 2 min, thereby enabling high-throughput analysis of the filtering efficiency of various types of commercial masks. The differences in efficiency in terms of same mask type from different manufacturers, double masking, and prolonged usage, which are challenging to analyze with conventional testing systems, can also be assessed. AuNP-mediated mask performance evaluation enables the rapid determination of mask efficiency according to particle size and can contribute to the rapid response to counter new emerging infectious biohazards.

Silver Nanoparticles Induce Apoptosis in HepG2 Cells through Particle-Specific Effects on Mitochondria.

Silver nanoparticles (AgNPs) have been widely used in biomedical and consumer products. It remains challenging to distinguish the toxicity of AgNPs derived from the particle form or the released silver ions (Ag+). In this study, the toxic effects of two citrate-coated AgNPs (20 and 100 nm) and Ag+ were investigated in hepatoblastoma cells (HepG2 cells). The suppression tests showed that AgNPs and Ag+ induced cell apoptosis via different pathways, which led us to speculate on the AgNP-induced mitochondrial damage. Then, the mitochondrial damages induced by AgNPs and Ag+ were compared under the same intracellular Ag+ concentration, showing that the mitochondrial damage might be mainly attributed to Ag nanoparticles but not to Ag+. The interaction between AgNPs and mitochondria was analyzed using a scattered light imaging method combined with light intensity profiles and transmission electron microscopy. The colocalization of AgNPs and mitochondria was observed in both NP20- and NP100-treated HepG2 cells, indicating a potential direct interaction between AgNPs and mitochondria. These results together showed that AgNPs induced apoptosis in HepG2 cells through the particle-specific effects on mitochondria.

Noninvasive tool for optical online monitoring of individual biomass concentrations in a defined coculture.

Cocultures bear great potential in the conversion of complex substrates and process intensification, as well as, in the formation of unique components only available due to inter-species interactions. Dynamic data of coculture composition is necessary for understanding and optimizing coculture systems. However, most standard online determined parameters measure the sum of all species in the reactor system. The kinetic behavior of the individual species remains unknown. Up to now, different offline methods are available to determine the culture composition, as well as the online measurement of fluorescence of genetically modified organisms. To avoid any genetic modification, a noninvasive online monitoring tool based on the scattered light spectrum was developed for microtiter plate cultivations. To demonstrate the potential, a coculture consisting of the bacterium Lactococcus lactis and the yeast Kluyveromyces marxianus was cultivated. Via partial least squares regression of scattered light spectra, the online determination of the individual biomass concentrations without further sampling and analyses is possible. The results were successfully validated by a Coulter counter-analysis, taking advantage of the different cell sizes of both organisms. The findings prove the applicability of the new method to follow in detail the dynamics of a coculture.

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses.

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.

Novel technique for high throughput measurement of active monooxygenase concentration.

Absorbance measurements via transmitting light spectroscopy in microtiter plates are established for high throughput screening of biological systems. These measurements allow for the determination of important process parameters within a short time. However, absorbance determination via transmitted light measurements is not always feasible. As for carbon monoxide difference absorbance spectroscopy, used for concentration measurements of active P450 monooxygenases (P450s), security standards, and consistent gassing have to be addressed. In this study, a non-invasive online measuring principle for absorbance via scattered light is proposed. Based on optical fiber measurements, a decrease in scattered light signals at 450 nm wavelength of reflecting polymer particles is observed, and P450 concentrations are calculated. In this way, high throughput determination of P450 concentrations in a secure, gas-tight environment is realized. The designed method was successfully applied to concentration measurements and carbon monoxide (CO) saturation kinetics ranging from 0.3 to 5.0 µM P450 BM3 achieving a measurement accuracy of ±0.05 µM P450. Biotechnol. Bioeng. 2017;114: 929-933. © 2016 Wiley Periodicals, Inc.

Hyperspectral backscatter imaging: a label-free approach to cytogenetics.

Current techniques for chromosome analysis need to be improved for rapid, economical identification of complex chromosomal defects by sensitive and selective visualisation. In this paper, we present a straightforward method for characterising unstained human metaphase chromosomes. Backscatter imaging in a dark-field setup combined with visible and short near-infrared spectroscopy is used to monitor morphological differences in the distribution of the chromosomal fine structure in human metaphase chromosomes. The reasons for the scattering centres in the fine structure are explained. Changes in the scattering centres during preparation of the metaphases are discussed. FDTD simulations are presented to substantiate the experimental findings. We show that local scattering features consisting of underlying spectral modulations of higher frequencies associated with a high variety of densely packed chromatin can be represented by their scatter profiles even on a sub-microscopic level. The result is independent of the chromosome preparation and structure size. This analytical method constitutes a rapid, cost-effective and label-free cytogenetic technique which can be used in a standard light microscope. Graphical abstract Hyperspectral backscatter imaging for label-free characterization.

Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light.

The online monitoring of recombinant protein aggregate inclusion bodies during microbial cultivation is an immense challenge. Measurement of scattered and reflected light offers a versatile and non-invasive measurement technique. Therefore, we investigated two methods to detect the formation of inclusion bodies and monitor their production: (1) online 180° scattered light measurement (λ = 625 nm) using a sensor platform during cultivation in shake flask and (2) online measurement of the light reflective interference using a porous Si-based optical biosensor (SiPA). It could be shown that 180° scattered light measurement allows monitoring of alterations in the optical properties of Escherichia coli BL21 cells, associated with the formation of inclusion bodies during cultivation. A reproducible linear correlation between the inclusion body concentration of the non-fluorescent protein human leukemia inhibitory factor (hLIF) carrying a thioredoxin tag and the shift ("Δamp") in scattered light signal intensity was observed. This was also observed for the glutathione-S-transferase-tagged green fluorescent protein (GFP-GST). Continuous online monitoring of reflective interference spectra reveals a significant increase in the bacterium refractive index during hLIF production in comparison to a non-induced reference that coincide with the formation of inclusion bodies. These online monitoring techniques could be applied for fast and cost-effective screening of different protein expression systems.

Longitudinal imaging of microvascular remodelling in proliferative diabetic retinopathy using adaptive optics scanning light ophthalmoscopy.

PURPOSE: To characterise longitudinal changes in the retinal microvasculature of type 2 diabetes mellitus (T2DM) as exemplified in a patient with proliferative diabetic retinopathy (PDR) using an adaptive optics scanning light ophthalmoscope (AOSLO). METHODS: A 35-year-old T2DM patient with PDR treated with scatter pan-retinal photocoagulation at the inferior retina 1 day prior to initial AOSLO imaging along with a 24-year-old healthy control were imaged in this study. AOSLO vascular structural and perfusion maps were acquired at four visits over a 20-week period. Capillary diameter and microaneurysm area changes were measured on the AOSLO structural maps. Imaging repeatability was established using longitudinal imaging of microvasculature in the healthy control. RESULTS: Capillary occlusion and recanalisation, capillary dilatation, resolution of local retinal haemorrhage, capillary hairpin formation, capillary bend formation, microaneurysm formation, progression and regression were documented over time in a region 2° superior to the fovea in the PDR patient. An identical microvascular network with same capillary diameter was observed in the control subject over time. CONCLUSIONS: High-resolution serial AOSLO imaging enables in vivo observation of vasculopathic changes seen in diabetes mellitus. The implications of this methodology are significant, providing the opportunity for studying the dynamics of the pathological process, as well as the possibility of identifying highly sensitive and non-invasive biomarkers of end organ damage and response to treatment.

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells.

BACKGROUND: Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs. METHODS: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues. RESULTS: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells. CONCLUSIONS: The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.

Effect of surface-modified intraocular lenses on long-term postoperative inhibition of posterior capsule opacification.

We compared the posterior capsule opacification incidences at 5 years postoperatively and the neodymium-yttrium-aluminum-garnet capsulotomy rates at 10 years postoperatively for two types of intraocular lenses with different optical properties and shapes. This randomized, controlled, prospective, single-blinded study with intra-individual comparisons was conducted between July 21, 2009, and August 31, 2011, at the Dokkyo Medical University Hospital, Tochigi, Japan. Thirty patients (60 eyes) underwent bilateral cataract surgery and received a XY1 intraocular lens in one eye and a FY-60AD intraocular lens in the other. Both intraocular lenses are acrylic and manufactured by HOYA. The XY1 lens is a single-piece, tinted intraocular lens featuring an ultraviolet/ozone treatment on the posterior surface of the lens optic, aimed at enhancing posterior capsule adhesion to prevent posterior capsule opacification. Conversely, the FY-60AD is a tinted intraocular lens with modified polymethylmethacrylate C-loops and no ultraviolet/ozone treatment of the optic. Scheimpflug images were taken using EAS-1000 (NIDEK Co., Ltd., Aichi, Japan), and the scattered light intensity (computer compatible tape) on the posterior surface of the intraocular lens was calculated and evaluated as the posterior capsule opacification. The scattered light values of the XY1 and FY-60AD groups were 6.50 ± 5.69 and 11.64 ± 5.30 computer compatible tape, respectively, at 5 years postoperatively. The cumulative survival incidence after neodymium-yttrium-aluminum-garnet laser capsulotomy was 74.8 % in the XY1 group and 13.8 % in the FY-60AD group at 10 years postoperatively. The surface-modified intraocular lens XY1 reduced the incidence of posterior capsule opacification even 10 years after surgery. Surface modification to increase the adhesion between the intraocular lens and the capsule effectively prevents posterior capsule opacification.

Excretory products of the cestode, Schistocephalus solidus, modulate in vitro responses of leukocytes from its specific host, the three-spined stickleback (Gasterosteus aculeatus).

Helminth parasites have evolved remarkable strategies to manipulate the immune system of their hosts. During infections of three-spined stickleback (Gasterosteus aculeatus) with the cestode Schistocephalus solidus prominent immunological changes occur, presumably due to manipulative activity of the parasite. We hypothesise that excretory/secretory products of the parasite are involved in the manipulation of the stickleback's immune system and that this may depend on the individual parasite and its origin. We therefore produced S. solidus conditioned cell culture media (SSCM) with parasites from different origins (Norway, Spain and Germany) and exposed head kidney leukocytes (HKL) from un-infected sticklebacks in cell cultures to SSCM. After in vitro culture, HKL were subjected to differential cell counts (granulocytes/lymphocytes) by means of flow cytometry. Leukocyte sub-populations were analysed for cell viability and changes in cell morphology. The respiratory burst activity was measured with a luminescence assay. Exposure of HKL to SSCM induced an up-regulation of respiratory burst activity after already 1 h, which was still elevated at 24 h, but which was in some cases significantly down-regulated after 96 h. Respiratory burst was positively correlated with the number of live granulocytes in the culture, suggesting that the respiratory burst activity was changed by SSCM effects on granulocyte viability. After 1 h and 24 h of HKL culture, no lymphocyte responses to SSCM were detectable, but after 96 h lymphocyte viability was significantly decreased with SSCM from Spanish S. solidus. In these cultures, residual lymphocytes increased in size, suggesting that cell death and activation might have occurred in parallel. The highest respiratory burst activity was induced by SSCM from Spanish parasites, in particular when they were grown in sympatric sticklebacks. The in vitro HKL responses to SSCM depended on the individual parasite and its population of origin, suggesting that in vivo, S. solidus excretory products are regulated individually, possibly to balance the interplay of each individual host-parasite pair.

Engineering the Metal/Dielectric Interface to Unlock the Potential of Scattered Light for Boosted Photoredox Catalysis.

The recycling of scattered light by metals has been emerging as a promising light-manipulation-capture strategy, but how to bring its potential into better play remains to be explored. Herein, we present that constructing dual metal/high-refractive-index dielectric interfaces within the SiO2 core@TiO2 shell-Pd satellite@TiO2 shell effectively strengthens both the scattering efficiency of the dielectric SiO2 support and electric field confinement. Consequently, the absorption of Pd toward near-field scattered light and the interfacial charge carrier separation are both enhanced. The synergy of these effects leads to boosted photoactivity toward the aerobic oxidation of cyclohexanol to cyclohexanone and the anaerobic reduction of proton for hydrogen evolution under visible-light irradiation as compared to the counterparts with a single metal/dielectric interface and dual metal/dielectric interfaces consisting of low-refractive-index dielectric component. Notably, the similar enhancements in both optical absorption and photoactivity can be achieved through the present dual metal/high-refractive-index dielectric interfaces engineering strategy for other metals, such as Pt nanoparticles. This work presents an instructive avenue to upgrade the optical response of metals and thus the photocatalytic performance by engineering metal/dielectric interfaces.

Early Blood Clot Detection Using Forward Scattering Light Measurements Is Not Superior to Delta Pressure Measurements.

The occurrence of thrombus formation within an extracorporeal membrane oxygenator is a common complication during extracorporeal membrane oxygenation therapy and can rapidly result in a life-threatening situation due to arterial thromboembolism, causing stroke, pulmonary embolism, and limb ischemia in the patient. The standard clinical practice is to monitor the pressure at the inlet and outlet of oxygenators, indicating fulminant, obstructive clot formation indicated by an increasing pressure difference (ΔP). However, smaller blood clots at early stages are not detectable. Therefore, there is an unmet need for sensors that can detect blood clots at an early stage to minimize the associated thromboembolic risks for patients. This study aimed to evaluate if forward scattered light (FSL) measurements can be used for early blood clot detection and if it is superior to the current clinical gold standard (pressure measurements). A miniaturized in vitro test circuit, including a custom-made test chamber, was used. Heparinized human whole blood was circulated through the test circuit until clot formation occurred. Four LEDs and four photodiodes were placed along the sidewall of the test chamber in different positions for FSL measurements. The pressure monitor was connected to the inlet and the outlet to detect changes in ΔP across the test chamber. Despite several modifications in the LED positions on the test chamber, the FSL measurements could not reliably detect a blood clot within the in vitro test circuit, although the pressure measurements used as the current clinical gold standard detected fulminant clot formation in 11 independent experiments.

Near-Infrared Transillumination for Macroscopic Functional Imaging of Animal Bodies.

The classical transillumination technique has been revitalized through recent advancements in optical technology, enhancing its applicability in the realm of biomedical research. With a new perspective on near-axis scattered light, we have harnessed near-infrared (NIR) light to visualize intricate internal light-absorbing structures within animal bodies. By leveraging the principle of differentiation, we have extended the applicability of the Beer-Lambert law even in cases of scattering-dominant media, such as animal body tissues. This approach facilitates the visualization of dynamic physiological changes occurring within animal bodies, thereby enabling noninvasive, real-time imaging of macroscopic functionality in vivo. An important challenge inherent to transillumination imaging lies in the image blur caused by pronounced light scattering within body tissues. By extracting near-axis scattered components from the predominant diffusely scattered light, we have achieved cross-sectional imaging of animal bodies. Furthermore, we have introduced software-based techniques encompassing deconvolution using the point spread function and the application of deep learning principles to counteract the scattering effect. Finally, transillumination imaging has been elevated from two-dimensional to three-dimensional imaging. The effectiveness and applicability of these proposed techniques have been validated through comprehensive simulations and experiments involving human and animal subjects. As demonstrated through these studies, transillumination imaging coupled with emerging technologies offers a promising avenue for future biomedical applications.

Two-photon fluorescence diagnostics of femtosecond laser tweezers.

We show how two-photon fluorescence signal can be used as an effective detection scheme for trapping particles of any size in comparison to methods using back-scattered light. Development of such a diagnostic scheme allows us a direct observation of trapping a single nanoparticle, which shows new directions to spectroscopy at the single-molecule level in solution.

Characterization of Intracellular Structure Changes of Microcystis under Sonication Treatment by Polarized Light Scattering.

Cyanobacterial bloom is one of the most urgent global environmental issues, which eventually could threaten human health and safety. Sonication treatment (ST) is a potential effective method to control cyanobacteria blooms in the field. Currently, the bottleneck of extensive application of ST is the difficulty to estimate the ST effect on the cyanobacterial cells and then determine suitable ST times in the field. In this study, cyanobacterial Microcystis samples sonicated at different times were first measured by a spectrophotometer to calculate the removal efficiency of Microcystis cells. Additionally, they were observed by TEM to reveal the intracellular structure changes of the cells. Then the samples were measured by an experimental setup based on polarized light scattering to measure the polarization parameters. Experimental results indicated that the polarization parameters can effectively characterize the intracellular structural changes of Microcystis cells with different ST times, which is quite consistent with the results for removal efficiency and TEM images. Further, the optimal ST time can be inferred by the polarization parameters. These results demonstrate that polarized light scattering can be a potentially powerful tool to explore suitable times for sonication treatment of cyanobacteria blooms.

Photoselective Protective Netting Improves "Honeycrisp" Fruit Quality.

High temperatures, wind, and excessive sunlight can negatively impact yield and fruit quality in semi-arid apple production regions. Netting was originally designed for hail protection, but it can modify the light spectrum and affect fruit quality. Here, pearl, blue, and red photoselective netting (≈20% shading factor) was installed in 2015 over a commercial "Cameron Select® Honeycrisp" orchard. Our research objectives were to (1) describe the light quantity and quality under the colored nets compared to an uncovered control and (2) investigate the effect of Photoselective nets on "Honeycrisp" apple quality for two growing seasons. Light transmittance and scattering for each treatment were measured with a spectroradiometer, and samples for fruit quality analyses were collected at harvest. PAR (photosynthetic active radiation), UV, blue, red, and far-red light were lower underneath all netting treatments compared to an uncovered control. The scattered light was higher under the pearl net compared to other colors, while red and far-red light were lower under the blue net. For two consecutive years, trees grown under the photoselective nets intercepted more incoming light than the uncovered trees with no differences among the three colors. In both years, trees under red and blue nets had more sunburn-free (clean) apples than pearl and control. Red color development for fruit was lower when nets were used. Interestingly, bitter pit incidence was lower underneath red nets for both years. Other than red color development, "Honeycrisp" fruit quality was not appreciably affected by the use of netting. These results highlight the beneficial effect of nets in improving light quality in orchards and mitigating physiological disorders such as bitter pit in "Honeycrisp" apple.

Dissociation of neonatal and adult mice brain for simultaneous analysis of microglia, astrocytes and infiltrating lymphocytes by flow cytometry.

The technical difficulty to isolate microglia, astrocytes and infiltrating immune cells from mouse brain is nowadays a limiting factor in the study of neuroinflammation. Brain isolation requirements are cell-type and animal-age dependent, but current brain dissociation procedures are poorly standardized. This lack of comprehensive studies hampers the selection of optimized methodologies. Thus, we present here a comparative analysis of dissociation methods and Percoll-based separation to identify the most efficient procedure for the combined isolation of healthy microglia, astrocytes and infiltrated leukocytes; distinguishing neonatal and adult mouse brain. Gentle mechanical dissociation and DNase I incubation was supplemented with papain or collagenase II. Dispase II digestion was also used alone or in combination. In addition, cell separation efficiency of 30 % and 30-70 % Percoll gradients was compared. In these experiments, cell yield and integrity of freshly dissociated cells was measured by flow cytometry. We found that papain digestion in combination with dispase II followed by 30 % Percoll separation is the most balanced method to obtain a mixture of microglia, astrocytes and infiltrated immune cells; while addition of dispase II was not an advantage for neonatal brain. These dissociation conditions allowed flow cytometry detection of a slight glial activation triggered by sublethal LPS injection. In conclusion, the enzymes and Percoll density gradients tested here affected differently resting microglia, activated microglia/macrophages, astrocytes and infiltrated lymphocytes. Also, newborn and adult brain showed contrasting reactions to digestion. Our study highlights the strength of flow cytometry for the simultaneous analysis of neuroimmune cell populations once extraction is optimized.