Мы хотели бы поблагодарить Канадский фонд инноваций, знаний британской Колумбии Фонд развития и Национального наукам и инженерным исследованиям Совета (NSERC) Канады за поддержку проводимых исследований на регионах с низким уровнем кислорода в прибрежных и открытых водах океана. Кроме того, ОУПЖ была поддержана стипендии NSERC, Киллам и ТУЛА основу финансируемого Центром микробного разнообразия и эволюции.

Часть 1: для сбора воды <ol><li> Глубины присваиваются каждому актеров. </li><li> Глубины на отбор проб и общего объема должны быть собраны для текущего литых определяются. </li><li> Он заверил, что Go-Фло и Niskin бутылки открыты, и что их клапаны и краны закрыты. </li><li> CTD загружается на нижней линии и ниже в воду. </li><li> Бутылки для глубокой глубине, отобранных в литых первой загружается на линии после CTD, он заверил, что линия правильно установлены в скобы и винты затянуты. </li><li> Посланник прилагается к каждой бутылке (за исключением самых глубоких один) перед спуском. </li><li> Линия опускается расстояние между первым глубину и затем; еще одну бутылку, затем загружены. Следует отметить, что для глубин, где 20L собираются для фильтрации, в общей сложности 2 12L бутылки загружаются последовательно около 0,75 м друг от друга. </li><li> После того как все бутылки на линии, линии опускают на необходимую глубину и 2 минуты разрешается проход. </li><li> Гонец направляется вниз и через несколько минут разрешено пройти, чтобы все бутылки близко. </li><li> Линия наматывается внутри и бутылки выгружаются по мере их появления на стойках. </li><li> Для глубин, которые требуют 20L пробы для ДНК и РНК, две бутылки Niskin собираются. Одна бутылка используется для образцов химических параметров (см. ниже), в то время как другие сливают в бутыль. РНК собирается сразу со второго Niskin. Остальная часть первой также слить в бутыль, чтобы дать ~ 20L конечного объема. Для низкого разрешения образцов только 1л воды, предназначенные для фильтрации в стерильные бутылки Nalgene. </li></ol> Часть 2: сбор пробы газа <ol><li> Если кислорода образцы нужны, они собираются в первую очередь. </li><li> Кусок тонкой трубки крепится к Niskin / Go-Flo кран, а другой конец трубки находится место в стеклянные бутылки сыворотки. </li><li> Сыворотке бутылки промыть 3 раза пробы воды, и наполнил до краев, что нет воздушных пузырей присутствуют. </li><li> 50 мкл хлоридом ртути (HgCl 2) будут добавлены в сыворотке бутылку так, чтобы не вводить воздушных пузырьков в пробе. </li><li> Бутылка запечатана с резиновой пробкой и обжимается с алюминиевой крышкой, а затем хранится на льду в кулере. </li></ol> Часть 3: Питательный сбор образцов <ol><li> 60 мл шприца промывают пробы воды, (0,2 мкм) прилагается в линию и немного воды выталкивается через Acrodisc. </li><li> Полиэтиленовые бутылки промыть ~ 10 мл фильтрованной воды 3 раза (взболтайте бутылку, когда промывка). </li><li> Фильтрованный пробы воды добавляется в бутылки, около ¾ полном объеме. </li><li> Образец хранится на лед в холодильнике. </li></ol> Часть 4: коллекции образцов Аммиак <ol><li> Пример трубы промываются пробы воды. </li><li> Пример трубы заполняются полностью с образцом воды. </li><li> 5 мл пробы воды берутся с помощью пипетки 10 мл, остальное вода в трубке отбрасывается и 5 мл в пипетки наконечник заменяются в пробирку. </li><li> 7.5ml аммония рабочий раствор добавляют в каждую пробирку раз образцы из всех глубинах были собраны. </li></ol> Часть 5: сбор проб для сотовых перечисления <ol><li> Предварительно меченных 50 мл Сокол пробирку, содержащую 5,4 мл 37% формальдегида доверху для конечной концентрации 4%. Уход берется не расплескать. </li></ol> Часть 6: сбор проб для измерения водородного сульфида <ol><li> Предварительно меченных 50 мл Сокол трубки, содержащей 20% ацетат цинка (Zn (O 2 CCH 3) 2) заполняется до самого верха. Уход берется не расплескать. </li></ol> Часть 7: Небольшая коллекция объем биомассы концентрация <ol><li> 1-литровые бутылки доверху. </li></ol> Часть 8: Большая коллекция объем биомассы концентрация <ol><li> 20-литровых бутылях заполнены. </li></ol> Часть 9: Представитель Результаты Окончательные результаты отбора проб морской воды приводит к приблизительно 12 растворенные образцы кислорода, 32-48 образцов газа, 16 питательных проб, 32 пробы аммония, 16 образцов сероводорода, 16 образцов для клеток, 6 20-литровых бутылях 16, 1-литровых бутылок и 4 Sterivex 0.22μm фильтры, содержащие микробной биомассы сохранились с РНК позже.

<ol>
	<li>Cline, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+determination+of+hydrogen+sulfide+in+natural+waters.">Spectrophotometric determination of hydrogen sulfide in natural waters.</a> Limnology &#38; Oceanography. 14, 454-458 (1969).</li><li>Holmes, R. M., Aminot, A., Kerouel, R., Hooker, B. A., Peterson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+precise+method+for+measuring+ammonium+in+marine+and+freshwater+ecosystems.">A simple and precise method for measuring ammonium in marine and freshwater ecosystems.</a> Canadian Journal of fisheries and Aquatic Science. 56, 1801-1808 (1999).</li></ol>

The authors have nothing to disclose.

В целом, существует большая подготовка и планирование необходимых для этого типа выборки, поэтому бюджет за 2 дня до поездки, чтобы получить вещи в порядок и потом в один прекрасный день для очистки все вверх. В общем, не давайте соленой воде вступает в контакт с перистальтического насос...

<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>20L Carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>02-960-15</td>
<td>&nbsp;</td>
<tr>
<td>3/8" Tygon</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-36</td>
<td>&nbsp;</td>
<tr>
<td>Sterivex filters</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SVG010RS</td>
<td>&nbsp;</td>
<tr>
<td>Prefilter Housing Unit</td>
<td>&nbsp;</td>
<td>AMD</td>
<td>501200</td>
<td>&nbsp;</td>
<tr>
<td>Luer Fitting</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>31507-27</td>
<td>&nbsp;</td>
<tr>
<td>1/4" Tygon tubing</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-24</td>
<td>&nbsp;</td>
<tr>
<td>1/4" Tygon tubing</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-24</td>
<td>&nbsp;</td>
<tr>
<td>1/4" Tygon tubing</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>06429-24</td>
<td>&nbsp;</td>
<tr>
<td>50ml Plastic tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass Serum Bottles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Glass Sample Collection</td>
</tr>
<tr>
<td>Septa and aluminum caps for glass serum bottles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hand Crimper</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>20-200ul Pipette and Tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass Tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ammonium Sample Collection</td>
</tr>
<tr>
<td>30ml Plastic Bottles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Nutrient Collection</td>
</tr>
<tr>
<td>Acrodisc Filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5 ml Tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Nitrite Sample Collection</td>
</tr>
<tr>
<td>37% Formaldehyde</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For cell count samples</td>
</tr>
<tr>
<td>20% Zinc Acetate</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For sulfide samples</td>
</tr>
<tr>
<td>Mercuric Chloride</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For gas samples</td>
</tr>
<tr>
<td>milliQ water</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Working Reagent</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Borate buffer, sodium sulfite, orthophaladialdehyde</td>
</tr>
<tr>
<td>Sulfanilamide</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5g into 450ml water and 50ml HCl</td>
</tr>
<tr>
<td>N-(1-naphthy)-ethylenediamine dihydrochloride solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.5g into 500ml water</td>
</tr>
</tbody>
</table>

seawater sampling and collection

Это видео документы методы сбора проб воды прибрежных морских и обработка их для различных приложений, включая вниз по течению концентрация биомассы, нуклеиновые кислоты очистки, сотовые изобилие, питательных веществ и следов анализ газа. Для демонстрации сегодняшней образцы были собраны с палубы HMS Джон Стрикленд, работающих в Саанич входе. А-образные вышки, с многоцелевым лебедки и кабельная система, используется в сочетании с Niskin или Go-Flo бутылки отбора проб воды. Проводимости, температуры и глубины (CTD) датчики используются также для образца основной водной массы. Чтобы свести к минимуму дегазации, следов газа образцы собраны в первую очередь. Затем, питательные вещества, химический состав воды, а также клеток определяются. Наконец, воды собираются для биомассы фильтрации. Настройка и время сбора для одного приведение ~ 1,5 часа при максимальной глубине 215 метров. Таким образом, в общей сложности 6 часов, как правило, необходимых для завершения коллекции серии описаны здесь.

Watch this Scientific Journal Video about Seawater Sampling and Collection at JoVE.com

Seawater Sampling and Collection

Это видео документы методы сбора проб воды прибрежных морских и обработка их для различных приложений, включая вниз по течению концентрация биомассы, нуклеиновые кислоты очистки, сотовые изобилие, питательных веществ и следов анализ газа.

Отбор проб морской воды и сбор

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Research

JoVE Journal

Biology

19.8K Views.  University of British Columbia - UBC. Это видео документы методы сбора проб воды прибрежных морских и обработка их для различных приложений, включая вниз по течению концентрация биомассы, нуклеиновые кислоты очистки, сотовые изобилие, питательных веществ и следов анализ газа.

Video: Seawater Sampling and Collection

Collection and Cryopreservation of Hamster Oocytes and Mouse Embryos

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse embryos could be frozen and stored at -196 &deg;C and, a few years later, Parkening et al. 3 reported the birth of live offspring resulting from in vitro fertilization (IVF) of cryopreserved oocytes. Since then, the use of cryopreservation techniques has rapidly spread to become an essential component in the practice of human and animal assisted reproduction and in the conservation of animal genetic resources. Currently, there are two main methods used to cryopreserve oocytes and embryos: slow freezing and vitrification. A wide variety of approaches have been used to try to improve both techniques and millions of animals and thousands of children have been born from cryopreserved embryos. However, important shortcomings associated to cryopreservation still have to be overcome, since ice-crystal formation, solution effects and osmotic shock seem to cause several cryoinjuries in post-thawed oocytes and embryos. Slow freezing with programmable freezers has the advantage of using low concentrations of cryoprotectants, which are usually associated with chemical toxicity and osmotic shock, but their ability to avoid ice-crystal formation at low concentrations is limited. Slow freezing also induces supercooling effects that must be avoided using manual or automatic seeding 4. In the vitrification process, high concentrations of cryoprotectants inhibit the formation of ice-crystals and lead to the formation of a glasslike vitrified state in which water is solidified, but not expanded. However, due to the toxicity of cyroprotectants at the concentrations used, oocytes/embryos can only be exposed to the cryoprotectant solution for a very short period of time and in a minimum volume solution, before submerging the samples directly in liquid nitrogen 5. In the last decade, vitrification has become more popular because it is a very quick method in which no expensive equipment (programmable freezer) is required. However, slow freezing continues to be the most widely used method for oocyte/embryo cryopreservation. In this video-article we show, step-by-step, how to collect and slowly freeze hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.

Сбор и Криоконсервация ооцитов хомяка и мыши Эмбрионы

Embryos and oocytes were first successfully cryopreserved more than 30 years ago, when Whittingham et al. 1 and Wilmut 2 separately described that mouse ...

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

Большой объем (20 л +) Фильтрация прибрежной морской воды Образцы

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

Small Volume (1-3L) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents small volume (~1 L) filtration of microbial biomass from the water column. The protocol is an extension of the large volume sampling protocol described earlier, with one major difference: here, there is no pre-filtration step, so all size classes of biomass are collected down to the 0.22 &mu;m filter cut-off. Samples collected this way are ideal for nucleic acid analysis. The set-up, filtration, and clean-up steps each take about 20-30 minutes. If using two peristaltic pumps simultaneously, up to 8 samples may be filtered at the same time. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use.

Small Volume 1-3L Filtration of Coastal Seawater Samples

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

Малого объема (1-3л) Фильтрация прибрежной морской воды Образцы

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

Диагностическое вскрытие и выбранной коллекции тканей и образцов у крыс и мышей

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Методы сбора молока для мышей и Ривза олень мунжтак

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

Номера для терминалов методы проб крови у морских свинок

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Забор крови из боковой хвостовой вены крыс

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

Сбор и идентификация пыльцы из пчелиных семей

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

Удаленный сбор данных криоэлектронной томографии и усреднение субтомограмм

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Mushing, Sieving, and Sugar Floatation for Fruit Fly Larvae Collection

Измельчение, просеивание и плавание сахара для сбора личинок плодовой мухи  

To begin, select fallen, overripe, or ripe fruit with signs of damage, over position scars and soft spots. Cut the fruit into large pieces and place them into a four-liter Ziploc storage bag. Add water to the bag until the water covers the chopped fruit by 25 to 50 millimeters.Squeeze the fruit gently by hand until all the pulp has dislodged from the peel and has a smooth consistency. Use large sieves to process around five fruits at once. Stack the sieve with a large mesh sieve atop a small mesh sieve, and place a third sieve on the bottom of the stack to detect early instars.Pour the pulp into the top sieve, and thoroughly wash the pulp through the stack of sieves using water from a hose, faucet or a bottle until the fine pulp has passed through the sieves. Visually scan the top sieves for late instar larvae that might have been retained with the peel or any large pieces of fruit. And carefully inspect the second sieve for late instar larvae.Collect larvae from the sieves with larval forceps and place them into vials with 70%ethanol. Premix the sugar solution by dissolving 453 grams of dark brown sugar in two liters of tap water. Wash the pulp from the finer mesh sieves to the edge of the sieve with tap water, and then move the material to an 11-liter plastic dish pan.Add the brown sugar solution until it covers the pulp by 25 to 50 millimeters, and add two drops of anti-foamer. After five minutes, collect larvae that float to the surface of the solution with larval forceps into vials with 70%ethanol. Label a vial with the collection location, date, type of fruit, and collector for later examination and identification.The mean number of Anastrepha suspensa larvae and the mean number of larvae collected per minute from five guavas and processed by cutting and by mushing, sieving and floating or MSF method are shown here. The result shows that the MSF method yielded greater numbers of larvae and more larvae per minute than hand cutting. This MSF method was validated using manual infestation of mango and papaya to simulate low Bactrocera dorsalis infestation.More larvae were found using the MSF method for both papaya and mango when compared to the fruit cutting method. For both mangoes and papayas, the full MSF method resulted in higher numbers of larval detections and was faster than fruit cutting.