The authors have no acknowledgements.

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The authors have nothing to disclose.

القدرة على بدقة وبتكاثر قياس حالة الكبد البطانة الجيبية هي خطوة هامة في فهم بيولوجيا هذه الخلايا المتخصصة للغاية. والتقنيات الحديثة مثل منظم إضاءة المجهر 32، قوة المجهر الذري 33 و D-STORM (مباشر الاستوكاستك البصرية Reconstrucion الميكروسكوب) 34 نقل المعلومات ا?...

الكبد الخلايا البطانية الجيبية (LSECs) هي غاية الخلايا البطانية المتباينة التي تبطن جدار جيبي الكبدي. ومثقبة LSECs مع fenestrations التي هي غير diaphragmed، العابر للالمسام 50-250 نانومتر في القطر. ما يصل الى 20٪ من سطح LSECs تغطيها fenestrations، التي عادة ما تكون في مجموعات من عشرات إلى مئات دعا لوحات غربال 1-3 (الشكل 1). Fenestrations سماح نقل البلازما وnanosubstrates بين الدم وخلايا الكبد، وخلق نظام الترشيح الفائق كفاءة عالية. Fenestrations هي البنى الدينامية - سواء من حيث الحجم و / أو رقم يمكن تغييرها استجابة لمختلف الدول الفسيولوجية، والمخدرات، والمرض. على سبيل المثال، fenestrations هي أكبر في صمت مما كانت عليه في الدولة التي تغذيها 4؛ 2-دي-iodoamphetamine يزيد عدد تنوفذ، 5،6 وانخفاض في حجم وعدد fenestrations في حدوث خلية في الشيخوخة والمرض العديد من الدول 13/7 </sup&gt;. قياس دقيق لحجم وعدد fenestrations مهم لفهم كيفية تأثر LSEC التشكل المرض المختلفة، وسامة، والدول الفسيولوجية. تأثيرها على وظائف الكبد. ومن أجل تطوير تنوفذ تحوير التدخلات العلاجية 1. دراسة fenestrations صعبة. قطر fenestrations يكمن تحت قرار من المجهر الضوئي التقليدي، لذلك فقد كان الوحيد سابقا الملاحظة باستخدام المجهر الإلكتروني في كل من أنسجة الكبد أو مثقف LSECs سليمة ممكن. وأكثر ما تم استخدام المجهر الإلكتروني الماسح (SEM) لدراسة حجم تنوفذ والتردد والمسامية (النسبة المئوية للغشاء LSEC التي مخترقين fenestrations) لSEM يسمح لمراقبة مناطق واسعة من سطح البطانية وقياس الآلاف، إن لم يكن عشرات الآلاف من fenestrations. على الرغم من فائدتها، والنتائج التي أبلغ عنها من دراسات SEM المستندة لالمعلمات LSEC مثل حجم تنوفذ، عدد وتواتر والمسامية وتختلف على نطاق واسع في الأدب (الجدول 1). Fenestrations ولوحات غربال هي الهياكل الهشة التي العقد، كسر، تمدد أو تلتحم خلال إعداد العينات، لا بد من معالجة بالتالي الحذر للحفاظ على سلامتهم. ارتفاع ضغط التروية 14؛ الأسمولية غير صحيحة من تثبيتي، ومخازن 15؛ عدم كفاية التثبيت أو التثبيت الزمن؛ وسرعة الجفاف بعد تثبيت وتجفيف كلها مجالات تجهيز لSEM التي قد تنتج القطع الأثرية التي تتداخل مع الحفاظ على التركيب الدقيق (الشكل 2). فقدان fenestrations (&#39;القذف من النافذة&#39;) وتنوفذ انكماش يمكن أن يحدث نتيجة لسوء التثبيت، مما أدى إلى انخفاض قطرها تنوفذ والمسامية الخلية. وقد وصفت وسائل لتحسين الحفاظ على عينات لتحليل SEM سابقا 15-17 وسيتم مناقشتهاهنا مع نصائح إضافية حول كيفية تحسين الحفاظ على العينة. الأهداف الرئيسية لحفظ العينات هي لإزالة الدم من الجيوب بحيث يمكن تصور سطح LSEC وتجنب الضرر LSEC إما من ارتفاع ضغط أو تأخير التثبيت. نضح الكبد كاملة من تثبيتي عبر الوريد البابي هو الأسلوب المفضل لتثبيت الكبد. كما هو موضح بالتفصيل في مكان آخر يجب القيام 16،18 نضح تحت ضغط منخفض (على سبيل المثال 10 سم من H 2 0) لتجنب القطع الأثرية نضح المتعلقة الضغط والأضرار التي لحقت LSEC، والذي تجلى عادة فجوات كبيرة كما ضمن في غشاء الخلية. ومع ذلك، يمكن في كثير من الأحيان الحصول على تثبيت معقول باستخدام إبرة نضح من خزعات الكبد من البشر والحيوانات، كما هو موضح بالتفصيل في مكان آخر (19). تتضمن هذه التقنية عن طريق الحقن مباشرة تثبيتي إلى الأنسجة حتى يتم مسح الدم من العينة والنسيج حازم وثابت. تثبيت العينات للفحص المجهري الإلكترون يجب أن يكون الأداء الإقتصادي الأداءormed في أسرع وقت ممكن بعد وقف تدفق الدم لمنع التغييرات التركيبية التي تحدث نتيجة للكبد عمليات الانحلال الذاتي سريعة للغاية. كما نقدم طريقة لتحليل الصور التي تقلل إدراج القطع الأثرية، وتوحد قياس fenestrations. وقد أدى الاختلاف في اختيار الجيوب لالميكروسكوب، تحليل الصور من القطع الأثرية، وقياس مساحة الخلية لالمسامية وتنوفذ تردد إلى تناقضات كبيرة في نشر النتائج. نهج موحد لتقييم وقياس fenestrations ومتطلبات الحد الأدنى لعرض البيانات لم يتم التطرق بوضوح في الأدب سابقا 4،10،20-31.

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Name of material</td>
			<td style="width:100px;">Company</td>
			<td style="width:93px;">Catalogue Number</td>
			<td style="width:311px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">EM grade Glutaraldehyde</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C001</td>
			<td style="width:311px;">Store stock at -20 C until needed, avoid refreeze</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Paraformaldehyde powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>158127</td>
			<td style="width:311px;">Always prepare Paraformaldehyde fresh</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:155px;">Sodium Cacodylate powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C0250</td>
			<td style="width:311px;">Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:155px;">Calcium Chloride</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C1016</td>
			<td style="width:311px;">Prepare 1 M CaCl2by dissolving powder in dH2O</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:155px;">Osmium tretroxide</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C011</td>
			<td style="width:311px;">Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Ethanol- Absolute</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>459836&#160;</td>
			<td style="width:311px;">100 % Ethanol must be high grade and stored with Molecular Sieve</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Other grades of Ethanol</td>
			<td style="width:100px;">Labtech</td>
			<td style="width:93px;">EL5</td>
			<td style="width:311px;">Prepare graded Ethanols with dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Hexamethyldisilazane</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>52619&#160;</td>
			<td style="width:311px;">Allow to reach room temperature before use</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:155px;">Cannulas</td>
			<td style="width:100px;">Terumo</td>
			<td style="width:93px;">TSROX1832C, TSROX2225C, TSROX2419C</td>
			<td style="width:311px;">18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Conductive Carbon tape</td>
			<td style="width:100px;">ProSciTech</td>
			<td>IA0201</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Carbon Paint</td>
			<td style="width:100px;">ProSciTech</td>
			<td>I003</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Ketamine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be optained under licence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Xylazine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be obtained under licence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Molecular Sieve</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">208647</td>
			<td style="width:311px;">Removes water from the 100 % Ethanol</td>
		</tr>
	</tbody>
</table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

التصور الأولي في التكبير المنخفض عن طريق المسح الضوئي المجهر الإلكتروني يكشف سطح مستو للعينة الكبد وتبلغ مساحتها يتعرض كبيرة بما يكفي لمراقبة العديد من السفن الكبد الكبيرة والجيوب (الشكل 1A). ضمان الصحيح وضع كتلة الكبد على كعب متزايدة من الضروري للحصول على ص...

Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

طريقة موحدة لتحليل خلايا الكبد خاص بالمنحنى البطانية وFenestrations من خلال المجهر الإلكتروني

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

a-standardized-method-for-analysis-liver-sinusoidal-endothelial-cells

Research

JoVE Journal

Biology

14.2K Views.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Video: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

في فيفو تتبع 4 الأبعاد للالجذعية المكونة للدم والسلف الكبار في خلايا الفأر قبي نخاع العظم

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

عزل خلايا الفئران صمام غشائي

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

المجهر الإلكتروني من الأنسجة متآكلة إلى تصور مصفوفة خارج الخلية

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

دراسة منظمة Supramolecular من الضوئى الأغشية داخل الأنسجة ورقة بكسر تجميد من قبل كرو مسح الميكروسكوب الالكتروني

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

مسح بروتوكولات المجهر الإلكتروني الماسح (SEM) لإشكالية النبات، طلائعيات بيضية، وعينات الفطرية

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

إعداد الكائنات الحية بدائية وحقيقية النواة باستخدام التجفيف الكيميائية للتحليل المورفولوجي في المسح الضوئي المجهر الإلكتروني (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

إعداد العينات البيولوجية بالتجميد ذات الضغط العالي، وتعزيز النقيض ساعدت الموجات الدقيقة، والحد الأدنى راتنج التضمين لتصوير حجم

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

طريقة CryoAPEX لتحليل المجهر الإلكتروني لتوطين بروتين الغشاء داخل الخلايا المحفوظة فائقة الهيكلية

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

سير عمل التصوير المقطعي للصفيف للحصول المستهدف على معلومات الحجم باستخدام المجهر الإلكتروني المسح الضوئي

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

المجهر الإلكتروني المتسلسل لمسح الوجه الكتلي (SBF-SEM) لعينات الأنسجة البيولوجية

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...