תמיכה של מענקים EB007279 ו HL081273 מארה&quot;ב NIH היא הודתה בהכרת תודה.

<ol>
	<li>Vanderkooi, J. M., Maniara, G., Green, T. J., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+method+for+measurement+of+dioxygen+concentration+based+on+quenching+of+phosphorescence.">An optical method for measurement of dioxygen concentration based on quenching of phosphorescence.</a> J. Biol. Chem. 262, 5476-5482 (1987).</li><li>Rumsey, W. L., Vanderkooi, J. M., Wilson, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+phosphorescence:+A+novel+method+for+measuring+the+distribution+of+oxygen+in+perfused+tissue.">Imaging of phosphorescence: A novel method for measuring the distribution of oxygen in perfused tissue.</a> Science. 241, 1649-1651 (1988).</li><li>Lebedev, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+phosphorescent+probes+for+oxygen+Imaging+in+biological+systems.">Dendritic phosphorescent probes for oxygen Imaging in biological systems.</a> Acs Applied Materials and Interfaces. 1, 1292-1304 (2009).</li><li>Finikova, O. S., Cheprakov, A. V., Beletskaya, I. P., Carroll, P. J., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+versatile+synthesis+of+substituted+tetrabenzoporphyrins.">Novel versatile synthesis of substituted tetrabenzoporphyrins.</a> Journal of Organic Chemistry. 69, 522-535 (2004).</li><li>Lindsey, J. S., Schreiman, I. C., Hsu, H. C., Kearney, P. C., Marguerettaz, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rothemund+and+Adler-Longo+Reactions+revisited:+Synthesis+of+tetraphenylporphyrins+under+equilibrium+conditions.">Rothemund and Adler-Longo Reactions revisited: Synthesis of tetraphenylporphyrins under equilibrium conditions.</a> Journal of Organic Chemistry. 52, 827-836 (1987).</li><li>Lebedev, A. Y., Troxler, T., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+metalloporphyrin-based+dendritic+nanoprobes+for+two-photon+microscopy+of+oxygen.">Design of metalloporphyrin-based dendritic nanoprobes for two-photon microscopy of oxygen.</a> J. Porphyrins and Phthalocyanines. 12, 1261-1269 (2008).</li></ol>

<table border="1" cellspacing="0" cellpadding="0"&gt;<tr<td<strong&gt; קיצור</strong<strong</strong</td&gt;<td<strong&gt; שם מלא</strong<strong</strong</td&gt;</tr&gt;<tr<td&gt; NMP</td&gt;<td&gt; N-methylpyrrolidinone</td&gt;</tr&gt;<tr<td&gt; TFA</td&gt;<td&gt; Trifluoroacetic חומצה</td&gt;</tr&gt;<tr<td&gt; DIPEA</td&gt;<td&gt; Diisopropylethylamine</td&gt;</tr&gt;<tr<td&gt; HBTU</td&gt;<td&gt; 2 - (-1H benzotriazol-1-י.ל.) -1,1,3,3-tetramethyluronium hexafluorophosphate</td&gt;</tr&gt;<tr<td&gt; DMSO</td&gt;<td&gt; Dimethylsulfoxide</td&gt;</tr&gt;<tr<td&gt; CDMT</td&gt;<td&gt; CDMT = 1-כלורו-3 ,5-dimethoxytriazine</td&gt;</tr&gt;<tr<td&gt; המוזיאון</td&gt;<td&gt; המוזיאון = N-methylmorfoline</td&gt;</tr&gt;</table

synthesis and calibration of phosphorescent nanoprobes for oxygen imaging in biological systems

מדידת חמצן על ידי מרווה זרחני [1, 2] מורכב מהשלבים הבאים: 1) בדיקה מועבר לתוך המדיום של עניין (למשל דם או נוזל interstitial); 2) את האובייקט מואר עם אור באורך גל מתאים כדי להלהיב בדיקה למצב שלישיה שלה; 3) זרחני הנפלט נאסף, ואת כמובן הזמן שלו הוא ניתח להניב את חייו זרחני, אשר מומר ריכוז החמצן (או הלחץ החלקי, ת.ד.<sub&gt; 2</sub&gt;). החללית אסור באינטראקציה עם הסביבה הביולוגית ובמקרים מסוימים להיות 4) מופרשים מן המדיום עם סיום המדידה. כל אחד משלבים אלה מטילה דרישות על עיצוב המולקולרי של בדיקות זרחני, המהווים מרכיב רק פולשני של פרוטוקול המדידה. כאן אנו סוקרים את העיצוב של nanosensors זרחני הדנדריטים למדידות חמצן במערכות ביולוגיות. הבדיקות כוללות Pt או Pd porphyrin מבוססי polyarylglycine (AG) dendrimers, שונה שולי עם פוליאתילן גליקול (PEG של) שאריות. עבור עירור שני הפוטונים יעיל, טרמיני של dendrimers עשוי להיות שונה עם שני הפוטונים chromophores האנטנה, אשר ללכוד את האנרגיה עירור וערוץ אותו ליבות שלישיה של בדיקות באמצעות intramolecular סריג (פורסטר תהודה העברת אנרגיה). אנו מתארים את המאפיינים photophysical המפתח של בדיקות כיול להציג פרוטוקולים מפורטים.

Watch this Scientific Journal Video about Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems at JoVE.com

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

אנו מציגים עקרונות של מדידות חמצן על ידי מרווה זרחני ועיצוב סקירה של porphyrin מבוססי nanosensors הדנדריטים הדמיה חמצן במערכות ביולוגיות.

סינתזה כיול nanoprobes זרחני עבור הדמיה חמצן במערכות ביולוגיות

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

synthesis-calibration-phosphorescent-nanoprobes-for-oxygen-imaging

Research

JoVE Journal

Biology

13.8K Views.  University of Pennsylvania. אנו מציגים עקרונות של מדידות חמצן על ידי מרווה זרחני ועיצוב סקירה של porphyrin מבוססי nanosensors הדנדריטים הדמיה חמצן במערכות ביולוגיות.

Video: Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

שימוש Micro-Electro-Mechanical Systems (MEMS) לפתח כלים לאבחון

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

נורמליזציה נולד עבור טומוגרפיה Fluorescence הקרנה אופטית עבור הדמיה בלב שלם

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopic Fluorescence טומוגרפיה עבור In-vivo הדמיה של פיתוח תסיסנית</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

הכנת פיתוח למבוגרים תסיסנית מוח רשתיות עבור הדמיה חיה

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

אלקטרונים ספין תהודה מיקרו הדמיה המינים Live עבור מיפוי חמצן

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

תא ההדמיה ממברנה פגיעה ותהליכי subcellular המעורבים בתיקון

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

מדידה של רמות הרן סינתזה בתאי יונקים

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

מערכות ביטוי חופשי הנייד מבוסס הלה לביטוי של<em&gt; Plasmodium</em&gt; Rhoptry חלבונים

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

זמן לשגות הדמיה ברזולוציה גבוהה וניתוח אוטומטי של Dynamics microtubule In Living האדם טבורי וריד לתאי אנדותל

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.