我们感谢 Dr. r. 伯格, 在唐纳德丹佛斯植物科学中心的综合显微镜设施主任, 为他的指导和帮助与共焦显微镜。作者没有利益冲突的声明。

1. 用荧光标记防御
<ol>
	<li>选择一种对抗菌性能影响最小的荧光, 以及对活细胞内防御的吸收和定位。 注意: 选择最佳荧光取决于特定的实验目标。还应考虑荧光的光谱和化学性质、光、大小和电荷。</li>
	<li>标签防御与选定的荧光使用适当的肽标签套件可从商业供应商。另外, 化学合成荧光标记的防御。在这项研究中, 标签 MtDef5 防御与 DyLight550 使用商业标签套件根据制造商的协议和四甲基罗丹明 (TAMRA) 标记 MtDef4 防御是化学合成商业。 注: 建议使用化学合成的荧光标记的多肽 (特别是50氨基酸或更少的短肽), 因为荧光可以附着在肽的 n-或 C 端残留物上, 以确保对其的最小影响抗菌活性。合成具有三以上二硫化键的长肽具有挑战性。在这种情况下, 建议使用适当的商业可用的肽标记试剂盒, 将肽标记为最佳荧光。</li>
	<li>测试体外标记防御的抗菌活性, 并测定对真菌的最小抑菌浓度 (MIC)。 注意: 在本研究中, DyLight550-MtDef5 和 TMR-MtDef4 的 MIC 是针对孢菌和镰刀菌菌确定的。</li>
</ol>
2. 真菌培养和生长培养基
<ol>
	<li>将孢子从N. 菌砧木培养物转移到含有伏戈尔琼脂培养基21的斜管上, 在室温下孵育5天。</li>
	<li>区域性F. 菌PH-1 在包含完整介质 (CM)22的板上的应变为 28 c 5 天。为了生产孢子, 接种 4 (10 毫米直径) 5 天的老文化的插头F. 菌PH-1 为50毫升羧甲基纤维素 (CMC) 培养基 (15 克羧甲基纤维素, 1 克酵母提取物, 0.5 g MgSO4. 7 H2O, 1 g NH4没有3和 1 g 的2PO4) 和文化4-7 天在 28 c 在旋转振动筛 180 rpm。</li>
</ol>
3. 准备的孢子悬挂
<ol>
	<li>F. 菌孢子悬挂
	<ol>
		<li>涡流的F. 菌液体培养和收集孢子通过过滤1.5 毫升真菌培养通过两层过滤材料 (如 Miracloth) 到2毫升离心管。离心孢子悬浮在 13226 x g 速度在一个离心为2分钟。</li>
		<li>丢弃上清液, 加入1毫升的无菌水清洗颗粒。</li>
		<li>离心机孢子悬浮在 13226 x g 速度在一个离心 2 min. 丢弃上清和重在1毫升的2X 可持续森林 (合成真菌培养基)23中的颗粒。</li>
		<li>在光显微镜下用例计数孢子。</li>
		<li>将孢子悬浮液调整为 105生孢子/毫升, 2X 可持续使用。</li>
	</ol>
	</li>
	<li>N. 菌孢子悬浮
	<ol>
		<li>在N. 菌中使用一个接种回路将少量的生长培养物 (5 天老) 转移到含有2毫升液体介质的离心管上。</li>
		<li>涡流的孢子悬浮和过滤通过过滤材料进入一个新的离心管。</li>
		<li>将孢子悬浮液以最大速度离心离心2分钟, 在1毫升液体介质中丢弃上清和重孢子颗粒。</li>
		<li>在光显微镜下用例计数孢子。</li>
		<li>将孢子悬浮液调整到 105的生孢子/毫升, 并使用伏地尔液体介质。</li>
	</ol>
	</li>
</ol>
4. 样品制备和共聚焦显微镜
<ol>
	<li>MtDef4 防御的亚细胞定位
	<ol>
		<li>吸管50µL 每孢子悬浮液 (105生孢子/毫升) 入10毫米微35毫米玻璃底部微菜。为观察分生孢子, 直接进行下一步, 或为 germlings 准备, 吸管50µL 的N. 菌和F. 菌分生孢子进入 35 mm 培养皿, 并让发芽3-6 小时室温。</li>
		<li>添加50µL 荧光标记防御在麦克风每微菜和孵化2.5 小时。此处使用的荧光标记防御的麦克风 (步骤 1.3) 为N 菌和F菌的3µM, TMR-MtDef4 菌和f菌的麦克风分别为1µM 和12µM。</li>
		<li>在培养皿中加入2µL 的膜选择性染料 FM4-64 (最终浓度: 5 µM), 并在共聚焦显微镜下孵育30分钟, 立即装入成像。</li>
		<li>选择白光激光器 (环路)。使用两个激光源 488 nm 和 550 nm 分别激发 TMR-MtDef4 和 FM4-64 染料。检测荧光 TMR-MtDef4 在 580-700 nm 和检测 FM4-64 染料在 690-800 nm。 注: 在暗室中进行共聚焦显微镜。</li>
	</ol>
	</li>
	<li>MtDef5 防御内部化的时间推移成像
	<ol>
		<li>吸管50µL 的N. 菌和F. 菌孢子悬浮液 (105生孢子/毫升) 成 10 mm 微的 35 mm 玻璃底微菜。10毫米微无涂层, 并在底部有一个 No. 1.5 盖玻璃。对于分生孢子的观察, 直接进行下一步。为观察 germlings, 在室温下孵育孢子3-6 小时, 然后再进行显微镜检查。</li>
		<li>打开。选择 550 nm 的激光线, 以激发荧光标记的防御 (步骤 1.3) 和 FM4-64, 并使用1.00% 强度并激活相应的探测器。在这里使用的荧光标记的防御 (步骤 1.3) 在 560-600 nm 检测到, FM4-64dye 在 690-800 nm 检测到。 注: 使用低激光强度的活细胞成像作为高激光强度可能会造成对活菌细胞的损害。</li>
		<li>在显微镜上安装微盘。首先找到使用 10 x 目标的单元格, 然后切换到 100 x/1.44 油目标, 以获得更高的放大倍数。 注意: 将扫描模式设置为 xyzt (z 堆栈和延时模式的组合)、z 位置、缩放、图像捕获频率等。</li></ol></li></ol>在继续下一步之前。
		<li>添加50µL 的荧光标记防御使用这里 (步骤 1.3) 的麦克风3µM 和2µL 膜选择性染料 FM4-64 (最终浓度5µM) 每微菜, 并开始延时成像。将小块湿滤纸放在微盘中, 以防蒸发。三十年代的图像采集频率为3分钟, 总周期为2小时30分钟。 注: 在显微镜下安装微盘后添加防御和荧光可以使感兴趣的区域散焦。因此, 在将防御和荧光添加到微后, 需要重新调整感兴趣区域, 这会导致图像获取的延迟。</li>
		<li>使用共聚焦显微镜进行所有共聚焦成像, 并在暗室的室温下进行显微镜观察。</li>

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作者没有什么可透露的。

在这项研究中, 一个可靠的活细胞成像方法, 使用荧光标记的抗真菌防御被描述为研究的动力学, 这些肽内化的真菌细胞和确定其亚型靶点。这种方法是一个强大的工具, 以可视化的动态的互动之间的防御和真菌细胞的时空。
采用多种方法研究了真菌细胞内植物防御的内部化和细胞定位。在这些方法中, 防御处理的细胞通常是固定的, 然后处理的 immunolocalization, 电子显微镜, 或 X ...

植物进化了一个复杂的先天免疫系统, 以抵御微生物植物病原体1。他们表达了许多基因编码的宿主防御肽与假定的抗菌活性2。事实上, 这些肽中的许多都是在体外3中显示抗菌活性的。防御包括植物王国中最大的寄主防御肽组之一4。这些富含半胱氨酸的阳离子肽在 micromolar 浓度下表现出对真菌和卵病原体的有效生长抑制作用, 并且是抵御这些病原体的第一条防线5,6。由于其强大的抗真菌活性, 防御可以被利用在 agribiotechnological 的应用, 以产生抗病作物。几种植物防御的本构表达提高了温室的抗病性和转基因作物的田间试验6。重要的是要解开这些抗真菌肽的作用机制 (MOA), 以便充分利用其潜力作为有效的作物保护工具。然而, 对这些植物防御的 MOA 了解得很差。目前的证据表明, 他们表现出不同的 MOA5,6,7,8。一些防御行为 extracellularly 真菌和靶向特定细胞壁/细胞膜驻留鞘, 扰乱膜完整性和激活细胞毒性通路9,10,11。最近, 然而, 抗真菌防御, 转移到真菌细胞已被发现12,13,14。这些防御结合到膜驻留生物活性磷脂, 形成聚复合物和 permeabilize 等离子膜15,16,17。从而阐明了植物防御的某些方面。然而, 植物防御的 MOA 可能涉及一系列复杂的事件, 它们尚未被识别并集成到一个综合模型中。特别是, 在我们对这些肽的细胞靶点的理解上仍然存在很大的差距。
近年来, 随着显微镜技术的发展和新的荧光探针的开发, 活体细胞成像技术已被广泛应用于抗菌肽 (安培) 的研究。这些技术补充了广泛使用的方法, 如 immunolocalization, 电子显微镜, 原子力显微镜或 X 射线断层摄影术18, 主要用于分析抗真菌肽对形态学的影响和真菌细胞的生长, 包括细胞壁完整性的研究, 细胞生长/分支模式的改变, 以及血浆膜性和杀戮。然而, 这些研究仅限于在治疗后的某个时间点的影像细胞, 而不是在同一个细胞上进行延时成像, 以监测它们对防御挑战的动态变化。近年来, 使用荧光标记多肽与活细胞成像结合使用共焦显微镜, 使 real-time 可视化动力学的 AMP–microbe 相互作用。天然纯化和化学合成的抗真菌肽可以标记为荧光标签 (如, DyLight, 罗丹明, BODIPY, 或 Alexa 的染料), 并直接观察在他们与细胞的互动时, 延时活细胞成像。这些标记多肽的使用大大增加了我们对其 MOA 的不同方面的理解, 包括进入模式、亚细胞定位、胞内贩运以及活真菌细胞中的抗真菌作用部位18。
最近, 有几项研究表明, 包括植物防御在内的各种抗真菌肽都是由活真菌细胞内化的12,14,19,20。这些防御的 MOA 可能涉及与细胞内目标的相互作用。我们最近报告了植物防御 MtDef4 在两个囊真菌的抗真菌作用,孢菌和镰刀菌菌。MtDef4 被证明使用不同的途径真菌细胞进入和亚细胞定位这些真菌14。本研究采用化学合成的四甲基罗丹明 (TAMRA) 标记 MtDef4 与生命荧光染料 (膜选择染料, FM4-64; 膜 permeant 染料, SYTOX 绿色; 细胞死亡记者染料, 碘碘化物) 和代谢抑制剂。这些分析显示了 MtDef4 内化的动力学, 其胞内转运机制和亚细胞靶向14。
本文提出了一种利用共焦显微镜进行活细胞成像的协议。该协议利用荧光标记多肽与生命荧光染料结合, 研究植物防御真菌相互作用, 特别是真菌细胞的易位途径和胞内防御的细胞靶点。

<table>
	<tbody>
		<tr>
			<td>FM4-64 Dye</td>
			<td>Life Technologies</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight 550 Antibody Labeling Kit</td>
			<td>Thermo Scientific</td>
			<td>84530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Mat TeK</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mira cloth</td>
			<td>EMD Millipore Corp</td>
			<td>475855-1R</td>
			<td></td>
		</tr>
		<tr>
			<td>SP8-X confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>FiJi</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
	</tbody>
</table>

live-cell imaging of fungal cells to investigate modes of entry and subcellular localization of antifungal plant defensins

小的半胱氨酸丰富的防御是在所有植物中存在的最大的寄主防御肽群之一。许多植物防御展示了强有力的在体外抗真菌活性的 broad-spectrum 霉菌病原体, 因此有潜力被用作抗真菌剂在转基因作物。为了充分发挥植物防御对疾病控制的潜力, 阐明其作用机制是至关重要的。随着先进的显微技术的问世, 活细胞成像已经成为了解植物防御抗真菌的动力学的有力工具。在这里, 用两个荧光标记植物防御 (MtDef4 和 MtDef5) 结合生命荧光染料描述了一种基于共聚焦显微镜的活体细胞成像方法。这一技术使 real-time 可视化和分析的动态事件的 MtDef4 和 MtDef5 内化为真菌细胞。重要的是, 这种分析产生了丰富的信息, 包括内化动力学, 进入方式和亚细胞定位这些肽。这些方法与其他细胞生物工具一起, 为这些肽的动态和复杂性提供了重要的洞察力。这些工具也可以用来比较这些肽对不同真菌的 MOA。

从苜蓿蒺藜中进行了活细胞成像, 以追踪和比较两个防御、MtDef4 和 MtDef5 的内化和亚单位定位;在真菌细胞中。TMR-MtDef4 是化学合成的, 而 MtDef5 被标记为 Dylight550 (Dylight550-MtDef5)。用防御与膜选择性染料 FM4-64 联合培养孢子。图 1显示, 与F. 菌相比, TMR-MtDef4 在N. 菌中具有不同的贩运路径。在N. 菌中, FM4-64 不 co-localize 与防御, 而?...

Watch this Scientific Journal Video about Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins at JoVE.com

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

植物防御在植物防御病原体中起着重要的作用。为了有效地使用这些抗真菌肽作为抗真菌剂, 了解其作用方式 (MOA) 是至关重要的。在这里, 描述了一个活细胞成像方法, 以研究这些肽的 MOA 的关键方面。

真菌细胞的活细胞成像研究抗真菌植物防御的进入和亚单位定位

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

live-cell-imaging-fungal-cells-to-investigate-modes-entry-subcellular

Research

JoVE Journal

Biology

6.6K 次观看.  Donald Danforth Plant Science Center. 植物防御在植物防御病原体中起着重要的作用。为了有效地使用这些抗真菌肽作为抗真菌剂, 了解其作用方式 (MOA) 是至关重要的。在这里, 描述了一个活细胞成像方法, 以研究这些肽的 MOA 的关键方面。

视频: Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

科研

生物学

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动:Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

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.

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

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

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

活细胞成像血小板脱水和分泌流量

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...