Name: autofluorescence imaging to evaluate red algae physiology
Uploaded: 2023-02-17T13:25:00
Description: Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Red Algae Physiology at JoVE.com

这项研究是作为TIN2015-68454-R和20961/PI/18项目的一部分进行的，该项目由西班牙经济和竞争力部以及穆尔西亚地区的塞内卡基金会资助。穆尔西亚大学科学与研究区统计支持科的艾琳·埃尔南德斯·马丁内斯和弗朗西斯科·哈维尔·伊瓦涅斯·洛佩斯（Irene Hernández Martínez）是使用施维雅医学艺术的图片绘制的。施维雅医疗艺术获得知识共享署名 3.0 未移植许可 （https://creativecommons.org/licenses/by/3.0/）

藻类物种 Chroothece mobilis 用于本研究。该物种是从西班牙微藻Edaphic SE，MAESE 20.29培养物收集中获得的。该协议的概述如图 1 所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64533/64533fig01.jpg"> 图1：研究概述。 在极端栖息地条件下（例如不同的单色光）孵育 Chroothece mobilis2 </...

<ol>
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一些单细胞或殖民红藻，如Chroothece，在体外生长缓慢，但含有多种自发荧光化合物，可以通过共聚焦显微镜下的光谱分析进行分析，其中可以检测到色素发射峰的差异。光谱共聚焦荧光显微镜使我们能够进行体内研究，以评估光合生物的适应或适应8，10，17，18，19，<sup cl...

红藻，如Chroothece属，可以在极端的栖息地生长，在那里它们经常不得不应对明显的环境变化1。在可以找到该属的半干旱地区，洪水和干旱频繁发生，据报道，在小溪，悬崖，洞穴甚至温泉水中都报告了一些物种2。然而，大多数时候，生物变量，如竞争或放牧，将物种降级到非最佳生长条件。由于这些生物通常难以培养，并且在实验室条件下生长或生长非常缓慢，因此一个主要限制是可用的样本量。因此，遵循非破坏性方法或涉及最少样品操作的方法非常重要3，4。
在这些恶劣环境中生存所需的生理技能可以通过跟踪其光合作用系统的变化来监测。代谢机制、光合作用效率以及对光或培养条件的敏感性可以通过色素荧光发射曲线来揭示，因为它们的能量转移或捕获5，6，7，8的准确变化。
细胞化合物的自发荧光可用作细胞诊断的标志物，或作为细胞状态或代谢的自然指标，通过发射变化响应外部和内部信号9。它还可用于区分不同分类学的光合生物群10。根据光养微生物的系统发育位置，人们可以发现不同的 体内 荧光特征。因此，基于光养荧光的 体内 特性（包括荧光吸收和发射光谱）的分类学鉴定已被多次尝试11，12。由于浮游植物分类群中辅助色素的多样性，激发叶绿素a（Chl a）荧光的波长差异或发射光谱的差异可用于推断分类学13。这些标本的 体内 荧光激发和发射光谱不仅依赖于藻类门，还依赖于光系统适应14。能量转移到Chl a的效率，或Chl a与辅助色素的比例，以及细胞色素含量对生长条件敏感5。
红藻，特别是Chroothece，具有几种辅助荧光色素 - 藻胆蛋白和类胡萝卜素;前者集中在附着在叶绿体类囊体上的藻胆体中。藻胆蛋白（藻蓝蛋白、藻红蛋白和别藻蓝蛋白）可以捕获不同波长的光并将其传输到 Chl a，这使得在非常不同的光和培养条件下进行光合作用成为可能15.例如，Chroothece物种可以在洞穴内生长，或者几乎出现在略带盐分的钙质溪流中2。
单色光影响光合生物的生长和色素组成， 并已被研究用于预防或控制光合生物在洞穴中的生长.Mulec等人表明，富含红色的照明可促进蓝藻，藻类和植物的生长16。以前的研究还报道了绿光影响蓝藻的色素组成17，而其他人则揭示了绿光阻止大多数光合生物的生长，一些蓝藻表现出类囊体减少和平均荧光强度较弱18。
为了了解 Chroothece 作为模式生物克服恶劣条件的能力，培养的细胞已经暴露在不断增加的光强度和单色光（绿色或红色）15下，以了解它如何应对洞穴（红光占主导地位）的昏暗条件。本文提出的协议使用其自身的自发荧光在细胞水平上重现了上述变量对 Chroothece藻胆蛋白的影响。
如今，荧光通常用作研究维管植物，微藻，大型藻和蓝藻的生理反应的工具13，14，16。光谱共聚焦荧光显微镜是体内研究的绝佳工具，可在单细胞水平10，17，18，19，20上评估光合标本的生理学，避免与实验室低生长率相关的问题以及难以获得足够的生物量用于相关的提取和生化方法8.一旦细胞在不同的培养条件下处理2周，就可以在体内测量λ扫描谱。尽管有几篇出版物使用了3，4，10，17共聚焦成像激发的不同波长，但大多数藻胆蛋白和Chl a可以使用561 nm波长激发线进行检测，并且检测到的发射范围为570至760nm波长。这些标准基于先前通过共聚焦成像对商业纯颜料（表1）进行的分析10以及在不同藻类物种中获得的结果20，21，22。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">颜料</td>
			<td rowspan="2">λ最大流径 （纳米）</td>
			<td colspan="8">λ EXC （纳米）</td>
		</tr><tr><td>351</td>
			<td>364</td>
			<td>458</td>
			<td>476</td>
			<td>488</td>
			<td>514</td>
			<td>543</td>
			<td>633</td>
		</tr><tr><td>Chl a</td>
			<td>660.9-678.1</td>
			<td>43.4 ± 1.8</td>
			<td>11.2 ± 0.2</td>
			<td>1.8 ± 0.05</td>
			<td>2.0 ± 0.08</td>
			<td>12.2 ± 0.7</td>
			<td>6.0 ± 0.3</td>
			<td>4.2 ± 0.16</td>
			<td>80.7 ± 1.5</td>
		</tr><tr><td rowspan="2">R-PE</td>
			<td>569.2-583.3</td>
			<td>5.9 ± 0.6</td>
			<td>5.9 ± 0.16</td>
			<td>11.1 ± 0.04</td>
			<td>42.2 ± 0.3</td>
			<td>100.0 ± 0</td>
			<td>90.0 ± 0.3</td>
			<td>99.2 ± 0.08</td>
			<td>-</td>
		</tr><tr><td>652.1-668.6</td>
			<td>-</td>
			<td>-</td>
			<td>1.5 ± 0.01</td>
			<td>3.7 ± 0.04</td>
			<td>26.7 ± 0.5</td>
			<td>8.7 ± 0.16</td>
			<td>11.1 ± 0.16</td>
			<td>11.3 ± 0.2</td>
		</tr><tr><td>C-PC</td>
			<td>636.2-676.4</td>
			<td>2.3 ± 0.04</td>
			<td>1.0 ± 0.01</td>
			<td>0.6 ± 0.004</td>
			<td>0.7 ± 0.008</td>
			<td>2.0 ± 0.08</td>
			<td>2.0 ± 0.04</td>
			<td>3.3 ± 0.16</td>
			<td>33.6 ± 0.9</td>
		</tr><tr><td>APC-XL</td>
			<td>667.3-683.8</td>
			<td>15.1 ± 1.5</td>
			<td>9.6 ± 0.98</td>
			<td>1.0 ± 0.04</td>
			<td>1.2 ± 0.08</td>
			<td>5.9 ± 0.7</td>
			<td>4.1 ± 0.5</td>
			<td>23.2 ± 3.5</td>
			<td>91.4 ± 2.3</td>
		</tr></tbody></table>表 1：用于运行 lambda 扫描分析的纯颜料信息。该表显示了通过共聚焦成像分光光度法对所有激发波长的不同荧光染料/颜料的发射峰和肩部/荧光带最大值，以及颜料/荧光染料的发光百分比。值由公式计算：= MFI*100/255。每个值都是 SE ±平均值（平均值±平均值的标准误差）。纯颜料用于校准共聚焦扫描激光显微镜，如下所示1，2，10。叶绿素a取自油菜，R-藻红蛋白（R-PE）取自卟啉，C-藻蓝蛋白（C-PE）取自螺旋藻属。 所有物种都溶解在过滤的蒸馏水中。从乳腺藻中获得同种异藻蓝蛋白-XL（APC-XL），将其溶解在硫酸铵（60%）和磷酸钾（pH = 7）中，浓度达到38mM。使用8孔盖玻底室使用400 μL每种颜料溶液（浓度为1 mg/mL）进行扫描。
对单个激发波长的研究是一个非常有用的第一近似值。然而，在这种情况下，有必要阐明荧光信号中不同复合物的相对贡献，建议在多个波长下进行荧光比或光谱分析等方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Dish 35 mm, high Glass Bottom</td>
			<td>Ibidi&#160;</td>
			<td>81158</td>
			<td>-</td>
		</tr>
		<tr>
			<td>24 black well plate</td>
			<td>Ibidi</td>
			<td>82406</td>
			<td>&#160;flat and clear bottom for high throughput microscopy</td>
		</tr>
		<tr>
			<td>Algae Incubator</td>
			<td>Panasonic</td>
			<td>MLR-352-PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica Microsystems</td>
			<td>SP8 TCS</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Fisher Scientific</td>
			<td>15380591</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>green filter</td>
			<td>PNTA, LEE filters</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>HC PL APO 63X/1.30 GLYC CORR CS2</td>
			<td>Leica Microsystems</td>
			<td>506353</td>
			<td>Glycerol immersion lens</td>
		</tr>
		<tr>
			<td>Image acquisition software. LAS X</td>
			<td>Leica Microsystems</td>
			<td>SP8 TCS</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Panasonic</td>
			<td>FL40SSENW/37MLR-352-PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum photoradiometer</td>
			<td>DeltaOhm&#160;</td>
			<td>DO 9721</td>
			<td>-</td>
		</tr>
		<tr>
			<td>R software</td>
			<td>R Core Team, 2020</td>
			<td>4.0.2.</td>
			<td>-</td>
		</tr>
		<tr>
			<td>red filter</td>
			<td>PNTA, LEE filters</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>SWES medium</td>
			<td>University of Murcia</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Type G Immersion liquid</td>
			<td>Leica Microsystems</td>
			<td>11513910</td>
			<td>Glycerol&#160;</td>
		</tr>
	</tbody>
</table>

autofluorescence imaging to evaluate red algae physiology

红藻（Rhodophyta）含有藻胆蛋白，并在光线昏暗的栖息地定居，但是一些（例如，一些 Chroothece 物种）也可以在充足的阳光下发育。大多数红藻是红色的，但有些可能呈现蓝色，这取决于蓝色和红色胆蛋白（藻蓝蛋白和藻红蛋白）的比例。不同的藻胆蛋白可以捕获不同波长的光并将其传输到叶绿素a，这使得在非常不同的光条件下进行光合作用成为可能。这些色素对光的栖息地变化做出反应，它们的自发荧光可以帮助研究生物过程。以 Chroothece mobilis 为模式生物，在共聚焦显微镜下使用光谱λ扫描模式，在细胞水平上研究了光合色素对不同单色光的适应性，以猜测物种的最佳生长条件。结果表明，即使将所研究的菌株从洞穴中分离出来，它也适应了昏暗和中等光强度。所提出的方法对于研究在实验室条件下生长不慢或生长非常缓慢的光合生物特别有用，这通常是生活在极端栖息地的人的情况。

叶绿素a通常吸收蓝色和红色波长的可见光，而藻胆蛋白使用绿色，黄色和橙色波长7。这些色素的自发荧光使得在实验和现场条件下研究藻胆蛋白和叶绿素行为的第一种方法成为可能。
通过比较获得的数据并绘制在不同的图表上，可以区分平均荧光强度（MFI）的显着变化（图4）。对λ扫描轮廓（620、648、660和680 nm）中获得的不同发射峰...

Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Red Algae Physiology at JoVE.com

Autofluorescence Imaging to Evaluate Red Algae Physiology

本协议描述了基于光谱分析的红藻中藻胆蛋白变化的分步自发荧光成像和评估。这是一种无标记且无损的方法，用于评估细胞对极端栖息地的适应，当只有稀缺材料可用并且细胞在实验室条件下生长缓慢或根本不生长时。

自发荧光成像评估红藻生理学

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...

这种共聚焦显微镜方案将允许使用其色素的自发荧光在实验室中解释微藻的生态行为和适应，这些环境在实验室中生长缓慢。这种技术不是侵入性的，并且由于不使用化合物，因此可以最大限度地减少伪影。了解自养生物色素的性能和相应的生长将允许设计控制方法，这是旅游洞穴和建筑古迹的最大兴趣。通过将Chroothece mobilis的接种物从琼脂培养物转移到海水培养基或SWES液体培养基中，开始制备Chroothece mobilis的接种物。在低白光强度下，在20摄氏度下用16至8光暗光周期将所有培养物维持两周，并且不摇晃，直到获得所需的细胞密度。使用一毫升指数期培养物，每毫升细胞密度为10至第三个细胞的五倍，接种在24孔板中用于不同的实验。为了重现单色光效果，请使用绿色滤光片，允许绿光从 470 到 570 纳米通过，峰值在 506 纳米波长处，并使用 590 到 720 纳米之间的滤光片调整红光，峰值在 678 纳米。将细胞培养物暴露在这种光线下两周。打开倒置共聚焦激光扫描显微镜的所有组件，包括激光器。将细胞从24孔板的每个实验孔安装在SWES生长培养基中，到35毫米玻璃底培养皿中进行成像。选择63X或1.30数值孔径或NA甘油浸没物镜，并将甘油放在镜头上。接下来，将孔板放在显微镜载物台上，确保标本在图像采集过程中不会移动。通过选择荧光强度最高的平面，将标本置于光路的中心，并聚焦在细胞的中心。完成后，打开图像采集软件，从采集模式的下拉列表中选择XY lambda。选择激光的激发线 561 纳米、8 位动态范围和 1024 x 1024 像素。在 10 纳米带宽内收集荧光发射光谱和 570 至 760 纳米范围内 4 纳米的λ步长。将针孔设置在一个空气单元上并运行λ扫描采集。在不同的视野和红绿灯的不同条件下重复此过程后，保存所有数据。获取λ扫描后，单击软件顶部的量化窗口以评估收集的荧光发射光谱。转到打开的项目窗口，然后选择一个 XY lambda 文件。在成像软件中选择堆叠轮廓分析，并在细胞中心定义一个四微米见方的感兴趣区域，以分析平均荧光强度。导出数据和 CSV，然后在不同条件下对不同的单元格重复该过程。接下来，打开CSV文件，通过在CSV文件中分别选择藻红蛋白藻蓝蛋白、藻蓝蛋白、别藻蓝蛋白和叶绿素A的最大荧光数据，打开CSV文件以选择所有被测区域的不同荧光发射峰。完成后，创建一个新表，其中包含从每个藻胆蛋白和叶绿素峰获得的所有最大荧光值，并将数据绘制在图表上。观察到平均荧光强度的显着差异。当细胞暴露于单色绿光时，藻红蛋白藻蓝蛋白的平均荧光强度显着降低。相比之下，与对照组相比，红光没有观察到差异。绿光对别藻蓝蛋白和叶绿素A具有相反的影响，其中平均荧光强度显着增加，这是由于天线复合物的数量增加或连接性改善而导致天线复合物的适应。红光产生别藻蓝蛋白和叶绿素荧光的不显着增加.为了研究不同生长条件对培养细胞的影响，使用完全相同的显微镜设置进行测量至关重要。可以分析与自发荧光生物培养相关的其他环境参数，以及可见光谱内的荧光信号。该技术是研究具有几种自发荧光色素的生命生物体的一种非常有效的方法。

Research

JoVE Journal

Biology

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

科研

生物学

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

小龙虾开瓶器肌肉NMJ的历史观和生理学示范

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

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

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 ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

生理学实验室演示：肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Intracavernosal 压力记录对啮齿动物勃起功能的评价

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

单细胞共轭藻类无性系培养的建立

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

体外细胞生理学研究中视网膜微动脉的分离

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...