Lipophilic Dye Research Articles

Photoactivatable Carbo- and Silicon-Rhodamines and Their Application in MINFLUX Nanoscopy.

New photoactivatable fluorescent dyes (rhodamine, carbo- and silicon-rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2-nitrobenzyloxycarbonyl unit with an alpha-carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live-cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live-cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single-digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.

Photoactivatable Carbo‐ and Silicon‐Rhodamines and Their Application in MINFLUX Nanoscopy

AbstractNew photoactivatable fluorescent dyes (rhodamine, carbo‐ and silicon‐rhodamines [SiR]) with emission ranging from green to far red have been prepared, and their photophysical properties studied. The photocleavable 2‐nitrobenzyloxycarbonyl unit with an alpha‐carboxyl group as a branching point and additional functionality was attached to a polycyclic and lipophilic fluorescent dye. The photoactivatable probes having the HaloTagTM amine (O2) ligand bound with a dye core were obtained and applied for live‐cell staining in stable cell lines incorporating Vimentin (VIM) or Nuclear Pore Complex Protein NUP96 fused with the HaloTag. The probes were applied in 2D (VIM, NUP96) and 3D (VIM) MINFLUX nanoscopy, as well as in superresolution fluorescence microscopy with single fluorophore activation (VIM, live‐cell labeling). Images of VIM and NUPs labeled with different dyes were acquired and their apparent dimensions and shapes assessed on a lower single‐digit nanometer scale. Applicability and performance of the photoactivatable dye derivatives were evaluated in terms of photoactivation rate, labeling and detection efficiency, number of detected photons per molecule and other parameters related to MINFLUX nanoscopy.

Cardiac Vagal Nerve Activity Increases During Exercise to Enhance Coronary Blood Flow.

The phrase complete vagal withdrawal is often used when discussing autonomic control of the heart during exercise. However, more recent studies have challenged this assumption. We hypothesized that cardiac vagal activity increases during exercise and maintains cardiac function via transmitters other than acetylcholine. Chronic direct recordings of cardiac vagal nerve activity, cardiac output, coronary artery blood flow, and heart rate were recorded in conscious adult sheep during whole-body treadmill exercise. Cardiac innervation of the left cardiac vagal branch was confirmed with lipophilic tracer dyes (DiO). Sheep were exercised with pharmacological blockers of acetylcholine (atropine, 250 mg), VIP (vasoactive intestinal peptide; [4Cl-D-Phe6,Leu17]VIP 25 µg), or saline control, randomized on different days. In a subset of sheep, the left cardiac vagal branch was denervated. Neural innervation from the cardiac vagal branch is seen at major cardiac ganglionic plexi, and within the fat pads associated with the coronary arteries. Directly recorded cardiac vagal nerve activity increased during exercise. Left cardiac vagal branch denervation attenuated the maximum changes in coronary artery blood flow (maximum exercise, control: 63.5±5.9 mL/min, n=8; cardiac vagal denervated: 32.7±5.6 mL/min, n=6, P=2.5×10-7), cardiac output, and heart rate during exercise. Atropine did not affect any cardiac parameters during exercise, but VIP antagonism significantly reduced coronary artery blood flow during exercise to a similar level to vagal denervation. Our study demonstrates that cardiac vagal nerve activity actually increases and is crucial for maintaining cardiac function during exercise. Furthermore, our findings show the dynamic modulation of coronary artery blood flow during exercise is mediated by VIP.

Altered exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of cultured striatal neurons in a knock-in mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive dominantly inherited neurodegenerative disease caused by the expansion of a cytosine-adenine-guanine (CAG) trinucleotide repeat in the huntingtin gene, which encodes the mutant huntingtin protein containing an expanded polyglutamine tract. One of neuropathologic hallmarks of HD is selective degeneration in the striatum. Mechanisms underlying selective neurodegeneration in the striatum of HD remain elusive. Neurodegeneration is suggested to be preceded by abnormal synaptic transmission at the early stage of HD. However, how mutant huntingtin protein affects synaptic vesicle exocytosis at single presynaptic terminals of HD striatal neurons is poorly understood. Here, we measured synaptic vesicle exocytosis at single presynaptic terminals of cultured striatal neurons (mainly inhibitory neurons) in a knock-in mouse model of HD (zQ175) during electrical field stimulation using real-time imaging of FM 1-43 (a lipophilic dye). We found a significant decrease in bouton density and exocytosis of synaptic vesicles at single presynaptic terminals in cultured striatal neurons. Real-time imaging of VGAT-CypHer5E (a pH sensitive dye conjugated to an antibody against vesicular GABA transporter (VGAT)) for inhibitory synaptic vesicles revealed a reduction in bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of HD striatal neurons. Thus, our results suggest that the mutant huntingtin protein decreases bouton density and exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of striatal neurons, causing impaired inhibitory synaptic transmission, eventually leading to the neurodegeneration in the striatum of HD.

Two-dimensional, high-resolution imaging of pH dynamics in the phyllosphere of submerged macrophyte using a new Nano-optode

The phyllosphere pH helps shape the plant microbiome and strongly influences aboveground interactions in plant canopies. Yet little is known about the distribution of pH at a microscale within the macrophyte phyllosphere and the factors promoting them because achieving high-resolution quantitative imaging of phyllosphere pH is a great challenge. Here, new ratiometric pH nano-optodes were prepared by firstly encapsulating the self-synthesized lipophilic dyes (8-acetoxypyrene-N1, N3, N6-trioctadecyl-1, 3, 6-tri-trisulfonamide) to poly(1-vinylpyrrolidone-co-styrene) nanoparticles, and then immobilizing the resulting nanoparticles in polyurethane hydrogel on transparent foils. The nano-optodes presented reversible and fast response (t95 < 80 s) to the pH range from 7.0 to 11.0, with merits of good spatial resolution, photobleaching/leaching resistance and negligible cross-sensitives toward temperature, O2 and ionic strength (< 100 mM). The nano-optodes together with a self-designed phyllosphere chamber were further applied to directly measure the pH distributions at a microscale around single leaves of V. spiralis grown in natural sediment. The pronounced pH microheterogeneity and leaf basification within the V. spiralis phyllosphere were quantitatively visualized. We also provided direct empirical evidence that the dynamic of the phyllosphere pH at high resolution was significantly controlled by the shifting light intensity and temperature. Implementation of the nano-optodes holds great potential for various laboratory applications, which will provide an in-depth insight into phyllosphere activities on the microscale.

Influence of intrinsic plastics characteristics on Nile Red staining and fluorescence

Nile Red is a lipophilic staining dye often used in the detection of microplastics in environmental samples. This work evaluated the influence of characteristics of plastic particles collected from sandy beaches (n = 60) on fluorescent emissions after Nile Red staining. Most particles presented medium to high fluorescence (81.6%). Polymer type (polyethylene, polypropylene, polystyrene), weathering (i.e., carbonyl index, methyl index), and crystallinity did not affect fluorescence intensity (grey value) or color (RGB values). Conversely, particle color significantly influenced fluorescence, with lower fluorescence seen for some blue, green, and red particles, and higher in some white, yellow, and orange particles, suggesting an influence of pigments used in plastics on Nile Red staining. Wide variations in fluorescence were observed for the same particle color, possibly resulting from the use of different pigments. Therefore, future studies should tackle the issue of the interference of plastic pigments in the detection and characterization of plastic particles.

Quantitative assessment of lipophilic membrane dye-based labelling of extracellular vesicles by nano-flow cytometry.

Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40nm in diameter by a laboratory-built nano-flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%-80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%-70% of PFP-EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di-8-ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di-8-ANEPPS and 70%-100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self-aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di-8-ANEPPS and DiI, and the effect of LMD self-aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very-low-density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma-EV labelling. Besides, it was discovered that DSPE-PEG2000 -biotin can only label ∼50% of plasma-EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.

Spatial distribution of atmospheric microplastics in bulk-deposition of urban and rural environments – A one-year follow-up study in northern Germany

Atmospheric microplastic deposition rates play a crucial role for calculating the input of microplastics in the environment and to further understand pollution patterns. In this study, the spatial and temporal distribution of atmospheric microplastic particles in urban and rural areas of Northern Germany was investigated. Therefore, eleven structurally diverse locations in Hamburg and Mecklenburg-Western Pomerania were equipped with bulk-deposition samplers in triplicates and sampled monthly between August 2019 and July 2020. The resulting 306 samples were treated with hydrogen peroxide (30 %) and sodium hypochlorite (6–14 %) to digest biological organic matter. The filters were subsequently stained with the lipophilic dye Nile Red and underwent visual microplastic identification via fluorescence microscopy. Fragments and fibers were quantified down to a cut-off size of 10 μm. The polymer composition of microplastic particles was investigated along a subset of particles via μ-Raman spectroscopy.The microplastic deposition rate for Northern Germany (89 ± 61 MP/m2/day) is in the same order of magnitude as those reported by previous studies. Significant differences in microplastic deposition rates were found between urban and rural sampling sites. Population density was identified as an important factor for greater amounts of microplastics and higher shares of fibers in urban samples. Special attention was given to the canopy cover at two forested sampling sites and an influence of the comb-out effect on atmospheric microplastic deposition was detected.

Comparison of tumor growth assessment using GFP fluorescence and DiI labeling in a zebrafish xenograft model

ABSTRACT DiI is a lipophilic fluorescent dye frequently used to label and trace cells in cell cultures and xenograft models. However, DiI can also transfer from labeled to unlabeled cells, including host organism cells, and label dead cells obscuring interpretation of the results. These limitations of DiI labeling in xenograft models have not been thoroughly investigated. Here we labeled green fluorescent protein (GFP)-expressing MDA-MB-231 cells with DiI to directly compare tumor growth assessment in zebrafish xenografts using the DiI labeling and GFP fluorescence. Our results indicate that the DiI based assessment significantly overestimated tumor growth in zebrafish xenograft models compared to the GFP fluorescence based assessment. The imaging of DiI labeled GFP-expressing MDA-MB-231 cell cultures indicated that the DiI labeling of the membrane is uneven. Analysis of the DiI labeled GFP-expressing MDA-MB-231 cell cultures with flow cytometry indicated that the DiI labeling varied over time while the GFP fluorescence remained unchanged, suggesting that the GFP fluorescence is a more reliable signal for monitoring tumor progression than the DiI labeling. Taken together, our results demonstrate limitations of using DiI labeling for xenograft models and emphasize the need for validating the results based on DiI labeling with other orthogonal methods, such as the ones utilizing genetically encoded fluorophores.

Preparation and application of ZIF‐67/gelatin porous composite membrane via high internal phase emulsion

AbstractThe high internal phase emulsion (HIPE) template method is widely used to prepare porous functional materials because of its designable emulsion components and adjustable porosity. Herein, amphiphilic gelatin (GE) as the sole emulsion stabilizer, and no additional emulsifiers were added to prepare a stable GE‐HIPE membrane and its zeolitic imidazolate framework‐67 (ZIF‐67) hybrid composite membrane. The ZIF‐67/GE‐HIPE composite membrane has good lipophilicity by constructing dual micro‐nano structures by HIPE template and in situ growth of ZIF‐67. When ZIF‐67 was grown in situ, the water contact angle (CA) increased from 58.1 ± 2° for the GE membrane to 121 ± 1.9° for ZIF‐67/GE‐HIPE composite membranes. Besides, the minimum CA of organic solvent is 4.5 ± 0.7° (n‐hexane). The ZIF‐67/GE‐HIPE composite membranes exhibit good O/W emulsion separation properties and adsorption removal ability of organic pollutants. After 10 times of recycling emulsion separation, the emulsion separation capacity of the membrane was still greater than 99.4%. The lipophilic dye removal rate of the ZIF‐67/GE‐HIPE composite membrane was 2.37 times higher than that of GE‐HIPE, and the adsorption process conforms to the pseudo‐second‐order model. Thus, this work provides a strategy for the preparation of GE‐based hydrophobic modified membrane and also presents a typical treatment of adsorption‐separation of lipophilic substances.

Homozygous CDH2 variant may be associated with hypopituitarism without neurological disorders.

Congenital hypopituitarism is a genetically heterogeneous condition. Whole exome sequencing (WES) is a promising approach for molecular diagnosis of patients with this condition. The aim of this study is to conduct WES in a patient with congenital hypopituitarism born to consanguineous parents, CDH2 screening in a cohort of patients with congenital hypopituitarism, and functional testing of a novel CDH2 variant. Genomic DNA from a proband and her consanguineous parents was analyzed by WES. Copy number variants were evaluated. The genetic variants were filtered for population frequency (ExAC, 1000 genomes, gnomAD, and ABraOM), in silico prediction of pathogenicity, and gene expression in the pituitary and/or hypothalamus. Genomic DNA from 145 patients was screened for CDH2 by Sanger sequencing. One female patient with deficiencies in growth hormone, thyroid-stimulating hormone, adrenocorticotropic hormone, luteinizing hormone, and follicle-stimulating hormone and ectopic posterior pituitary gland contained a rare homozygous c.865G>A (p.Val289Ile) variant in CDH2. To determine whether the p.Val289Ile variant in CDH2 affects cell adhesion properties, we stably transfected L1 fibroblast lines, labeled the cells with lipophilic dyes, and quantified aggregation. Large aggregates formed in cells expressing wildtype CDH2, but aggregation was impaired in cells transfected with variant CDH2 or non-transfected. A homozygous CDH2 allelic variant was found in one hypopituitarism patient, and the variant impaired cell aggregation function in vitro. No disease-causing variants were found in 145 other patients screened for CDH2 variants. Thus, CDH2 is a candidate gene for hypopituitarism that needs to be tested in different populations. A female patient with hypopituitarism was born from consanguineous parents and had a homozygous, likely pathogenic, CDH2 variant that impairs cell aggregation in vitro. No other likely pathogenic variants in CDH2 were identified in 145 hypopituitarism patients.

Perilipin 3 promotes the formation of membrane domains enriched in diacylglycerol and lipid droplet biogenesis proteins.

Lipid droplets (LDs) serve as intracellular stores of energy-rich neutral lipids. LDs form at discrete sites in the endoplasmic reticulum (ER) and they remain closely associated with the ER during lipogenic growth and lipolytic consumption. Their hydrophobic neutral lipid core is covered by a monolayer of phospholipids, which harbors a specific set of proteins. This LD surface is coated and stabilized by perilipins, a family of soluble proteins that specifically target LDs from the cytosol. We have previously used chimeric fusion proteins between perilipins and integral ER membrane proteins to test whether proteins that are anchored to the ER bilayer could be dragged onto the LD monolayer. Expression of these chimeric proteins induces repositioning of the ER membrane around LDs. Here, we test the properties of membrane-anchored perilipins in cells that lack LDs. Unexpectedly, membrane-anchored perilipins induce expansion and vesiculation of the perinuclear membrane resulting in the formation of crescent-shaped membrane domains that harbor LD-like properties. These domains are stained by LD-specific lipophilic dyes, harbor LD marker proteins, and they transform into nascent LDs upon induction of neutral lipid synthesis. These ER domains are enriched in diacylglycerol (DAG) and in ER proteins that are important for early steps of LD biogenesis, including seipin and Pex30. Formation of the domains in vivo depends on DAG levels, and we show that perilipin 3 (PLIN3) binds to liposomes containing DAG in vitro. Taken together, these observations indicate that perilipin not only serve to stabilize the surface of mature LDs but that they are likely to exert a more active role in early steps of LD biogenesis at ER subdomains enriched in DAG, seipin, and neutral lipid biosynthetic enzymes.

A Putative Zn(II)2Cys6-Type Transcription Factor FpUme18 Is Required for Development, Conidiation, Cell Wall Integrity, Endocytosis and Full Virulence in Fusarium pseudograminearum.

Fusarium pseudograminearum is one of the major fungal pathogens that cause Fusarium crown rot (FCR) worldwide and can lead to a substantially reduced grain yield and quality. Transcription factors play an important role in regulating growth and pathogenicity in plant pathogens. In this study, we identified a putative Zn(II)2Cys6 fungal-type domain-containing transcription factor and named it FpUme18. The expression of FpUME18 was induced during the infection of wheat by F. pseudograminearum. The ΔFpume18 deletion mutant showed defects in growth, conidial production, and conidial germination. In the responses to the cell wall, salt and oxidative stresses, the ΔFpume18 mutant inhibited the rate of mycelial growth at a higher rate compared with the wild type. The staining of conidia and mycelia with lipophilic dye FM4-64 revealed a delay in endocytosis when FpUME18 was deleted. FpUME18 also positively regulated the expression of phospholipid-related synthesis genes. The deletion of FpUME18 attenuated the pathogenicity of wheat coleoptiles. FpUME18 also participated in the production of the DON toxin by regulating the expression of TRI genes. Collectively, FpUme18 is required for vegetative growth, conidiation, stress response, endocytosis, and full virulence in F. pseudograminearum.

Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

AbstractExtracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell‐free ‘cellular’ therapeutics. Yet, small EVs (&lt;200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano‐porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size‐based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose‐gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72h, while free EVs were rapidly cleared by the liver. Furthermore, microgel‐loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing the targeted delivery of EVs for future therapeutic applications.

Insights into the Strong Emission Enhancement of Molecular Rotor Thioflavin T in Aqueous Cellulose Nanocrystal Dispersion: White Light Generation in Protein and Micellar Media.

Thioflavin t (THT) is a well-known molecular rotor extensively used to detect amyloid-like structures. But THT shows very weak emission in water. In this article, we have found that THT shows very strong emission in the presence of cellulose nanocrystals (CNCs). Steady-state and time-resolved emission techniques have been used to study the strong emission of THT in aqueous CNC dispersion. The time-resolved study showed that in the presence of CNCs, the lifetime increased by ∼1500 fold compared to pure water (<1 ps). To know the nature of interaction and also the reason for this increase in emission zeta potential, stimuli-dependent and temperature-dependent studies have been carried out. These studies proposed that electrostatic interaction is the main factor for this binding of THT with CNCs. Further, the addition of another anionic lipophilic dye, merocyanine 540 (MC540), with CNCs-THT in both BSA protein (CIE: 0.33, 0.32) and TX-100 micellar (4.5 mM) (CIE: 0.32, 0.30) solutions produced excellent white light emission. Lifetime decay and absorption studies proposed a possible fluorescence resonance energy transfer mechanism in this generation of white light emission.

DiI-CT—A bimodal neural tracer for X-ray and fluorescence imaging

Here, we present an X-ray-visible neural tracer, referred to as DiI-CT, which is based on the well-established lipophilic indocarbocyanine dye DiI, to which we conjugated two iodine atoms. The tracer is visible with microfocus computed tomography (microCT) imaging and shares the excellent fluorescent tracing properties of DiI. We document the discovery potential of DiI-CT by analyzing the vibrissa follicle-sinus complex, a structure where visual access is poor and 3D tissue structure matters and reveal innervation patterns of the intact follicle in unprecedented detail. In the brain, DiI-CT tracing holds promise for verification evaluation of indirect connectivity measures, such as diffusion tensor imaging. We conclude that the bimodal dye DiI-CT opens new avenues for neuroanatomy.

Simultaneous adsorption of hydrophilic and lipophilic dyes on iron-doped hydroxyapatite/carbon dots nanocomposites

Nanocomposites of iron-doped hydroxyapatite and carbon dots were synthesized through green synthesis processes, using Yerba Mate (Ilex paraguariensis) as a precursor. A thorough characterization of the particles was carried out by HRTEM, XRD, FTIR, DLS, XPD, and static contact angle. The characterization indicated that the crystallinity, surface charge, and wettability depend on the synthesis parameters.Carbon dots-doped materials, YMnAp@CD and YMFenAp@CD, showed amphiphilic properties with the capacity to adsorb Crystal violet (CV) and Oil Blue A (OBA) from aqueous or toluene-based solutions. The mentioned materials showed the highest number of adsorption sites, with values of 2.95 ± 0.05 and 3.18 ± 0.04 mmol CV/gads and 1.65 ± 0.02 0.97 ± 0.06 mmol OBA/gads on monocomponent systems, and through a multi-component analysis, the competition for adsorption sites was elucidated.

Identifying lipid particle sub-types in live Caenorhabditis elegans with two-photon fluorescence lifetime imaging.

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.

Identification and Characterization of Alveolar and Recruited Lung Macrophages during Acute Lung Inflammation.

To precisely identify mouse resident alveolar macrophages (AMs) and bone marrow (BM)-derived macrophages, we developed a technique to separately label AMs and BM-derived macrophages with a fluorescent lipophilic dye followed by FACS. We showed that this technique overcomes issues in cell identification related to dynamic shifts in cell surface markers that occurs during lung inflammation. We then used this approach to track macrophage subsets at different time points after intratracheal (i.t.) instillation of Escherichia coli LPS. By isolating BM-derived macrophages and AMs, we demonstrated that BM-derived macrophages were enriched in expression of genes in signal transduction and immune system activation pathways whereas resident AMs were enriched in cellular processes, such as lysosome/phagosome pathways, efferocytosis, and metabolic pathways related to fatty acids and peroxisomes. Taken together, these data indicate that more accurate identification of macrophage origin can result in improved understanding of differential phenotypes and functions between AMs and BM-derived macrophages in the lungs.

Ca2+-induced myelin pathology precedes axonal spheroid formation and is mediated in part by store-operated Ca2+ entry after spinal cord injury.

The formation of axonal spheroid is a common feature following spinal cord injury. To further understand the source of Ca2+ that mediates axonal spheroid formation, we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca2+. We performed two-photon excitation imaging of spinal cords isolated from Thy1YFP+ transgenic mice and applied the lipophilic dye, Nile red, to record dynamic changes in dorsal column axons and their myelin sheaths respectively. We selectively released Ca2+ from internal stores using the Ca2+ ionophore ionomycin in the presence or absence of external Ca2+. We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 mM Ca2+ artificial cerebrospinal fluid. In contrast, removal of external Ca2+ significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment. Using mice that express a neuron-specific Ca2+ indicator in spinal cord axons, we confirmed that ionomycin induced significant increases in intra-axonal Ca2+, but not in the absence of external Ca2+. Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation. Pretreatment with YM58483 (500 nM), a well-established blocker of store-operated Ca2+ entry, significantly decreased myelin injury and axonal spheroid formation. Collectively, these data reveal that ionomycin-induced depletion of internal Ca2+ stores and subsequent external Ca2+ entry through store-operated Ca2+ entry contributes to pathological changes in myelin and axonal spheroid formation, providing new targets to protect central myelinated fibers.