Name: design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications
Uploaded: 2017-02-19T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications at JoVE.com

我们要感谢莫莉拉扎尔和Veronica德尔加多协助在测试协议，基兰·斯威尼有益的讨论和编辑，和泰勒·斯科特，我的安adirekkun，和斯蒂芬妮盖勒的手稿的批判性阅读。梅根妮可麦克林博士拥有事业奖从宝来惠康基金科学接口。

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作者什么都没有透露。

我们设计时考虑的灵活性本设备。所有使用的代码是自由和开放源码。默认的图像分析过程分割单元简单，运行速度很快。定制分析可以通过记录用户输入，同时与斐济图形用户界面进行分析的代表性图像，所述输入转换为BeanShell的脚本，然后设置插件调用脚本来实现。当它被调用时，该脚本将被送到称为"图像"包含文件路径到最近一套扣除背景的图像的字符串数组。图像和数据，因为它们被收集，这样，如果一个实验突然结束，他们不会丢失保存。蓝色发光二极管阵列被选择用于诱导我们的光遗传学系统，但它可以用不同颜色的LED来代替。有额外MOSFET开关和继电器开关PCB上的微控制器，以及通孔提供额外的输入和输出管脚，可以很容易此系统也可以适于更复杂的用途。例如，为了使该装置作为一恒浊操作，使用额外的引脚上的微控制器来驱动LED和从绑在培养容器中的光传感器读出的值，然后修改来测量浊度的软件，并相应地稀释容器。 必须小心，以确保培养物不被污染，以保护敏感显微镜设备，并确保了流体流速是一致的。现有当培养容器故意接种到污染物可以通过接种容器，并验证里面有没有生长之前等待几天来检测。为了避免在显微镜物镜溢出细胞培养，检查微流体装置不会被第一激发细胞培养，通过过来吸水布泄漏。如果介质流入培养容器是不一致的，这通常是因为管周围蠕动泵的辊已经变得太松。如果空气从水族馆泵流是不一致的，确保有在流出管，其中液体可以汇集和不一致抗蚀空气流没有U形弯曲。如果因为媒体已经在它的内部干涸管成为一个实验堵塞后，浸泡堵塞的管中热水浴溶解堵塞。 有内在的局限性该协议计划的实验或分析结果时要考虑的。取决于稀释率和使用的管的长度，有大约10分钟，在此期间，细胞培养物，从培养容器到显微镜泵送的延迟。因此，不能很好地适合于发生在较短的时间尺度学习事件。此外，在实验可在10天左右，仅通过的介质的体积限定连续运行，所述细胞培养物的进化必须视为的实验的增加的持续时间。该LIMI媒体婷营养对代谢和基因表达35，36，37，38，39深远的影响，因此应根据所研究的生理方面来确定。当分析结果中，该图像 - 特别是从外围数据图像点-应当为在该感兴趣区分别由图像分析程序确定的方式错误审查。这是可能被误认为是细胞的气泡或假象，从而扭曲测量荧光。消除这种错误测量的一个简单方法是丢弃所有ROI与自体荧光强度低于荧光强度。 该协议将是在可在连续培养生长的生物体测量荧光强度和/或流出物细胞培养物的细胞形态响应于光有用，设置TLE到时不搅拌一致焦平面，并且可以通过图像分析算法自动识别。这里使用的默认小区识别算法将是最直接适用于类似于出芽酵母大致球形的微生物。一个替代方案40至该协议是一组分批培养的生长的微生物，然后品尝一批每个时间点，并通过流式细胞表征样品。然而，在这里描述的方案的优点是，来自相同培养采取样品，许多样品可采取，并且处理是自动的。该协议还改进了类似的方法，其中光遗传学酵母是连续培养，通过获得多个图像很快，而不是由荧光施力的ROI识别一个显微镜34下成像。该协议的一个巨大优势是，单个细胞的许多测量可以在几天内被无覆盖定期收购UT用户输入。测量微生物培养物，以光照射的响应之后，下一个步骤可以是在响应硅片闭环控制来实现。

光遗传学系统使用光来控制细胞过程，包括基因表达，1，2，3，4，5蛋白质定位，6蛋白活性，6，7，8蛋白结合，8，9，10和蛋白质降解的一个增长的名单。 11，用于在与编程光学刺激受控环境中培养的细胞，并用于测量其响应在生物学相关的时间尺度的方法，有必要利用这些工具的潜力在细胞生物学和生物技术研究。我们的方法利用chemostasis的优势，在充分混合以保持恒定的细胞生长速率，AE额定，并且暴露于编程照度温度控制玻璃培养容器12，13。在倒置显微镜培养流出我们图像单个细胞来测量培养的以编程照明的响应。培养，取样，成像和图像分析完全自动化，使得荧光强度和流出物细胞培养物的细胞形态可以在多天进行测量，而无需用户输入。 这种协议可以在熟悉生长的细胞培养和显微镜检查大多数实验室来实现，并且所使用的装置是廉价且随时提供的组件。透明培养容器被放置的发光二极管（LED）能够发光的1μW/ cm 2的-10毫瓦/厘米2的矩阵的上方。微生物生长在连续培养容器; 1蠕动泵用于在添加媒体稀释率，另一个用于在较小速度到显微镜撤回培养，差值逸出通过溢流出口。一个加热垫维持温度。空气连续地泵入培养容器保持正压以及混合和通气培养。除了空气泵，功率到这些设备是由还接收来自温度计和连接台式计算机输入微控制器调节。流出物细胞培养物被泵送到微流体装置上的倒置显微镜的阶段。非荧光和荧光图像自动获得的。在影像中的细胞是由位于每个小区作为感兴趣区域（ROI）的区域，并测量每个ROI的属性的算法表征。 为了证明该协议的应用，我们测量了蓝光responsi工程酵母细胞的不同光强的响应已经控制荧光蛋白的转录光遗传学系统。 酿酒酵母 ，俗称面包酵母，被选中是因为在这一系统中控制基因表达的多个光遗传学系统已经存在14，15，16。此外，该模型生物体通常在系统生物学17和作为用于生物技术应用18，19，20中的底盘用于研究。我们的代表性的结果表明，该协议可以用于通过改变输入光强度和测量的生产荧光报道的以控制培养多天的转录。

<table><tbody><tr><td>Extensive lab manual</td><td>GitHub</td><td>NA</td><td>An extensive, regularly updated lab manual is available in the &amp;ldquo;Optogenetic Chemostat Files&amp;rdquo; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files). This also includes a description of the microfluidic mold used to generate the representative results.</td></tr><tr><td>Fritzing Design Viewer</td><td>Fritzing</td><td>NA</td><td>The free, open-sourced software to view and edit the .fzz type circuit board designs is available at &quot;http://fritzing.org/download/&quot;</td></tr><tr><td>Arduino Uno R3 (Atmega328 - assembled)</td><td>Adafruit</td><td>50</td><td>Microcontroller. 1 required.</td></tr><tr><td>Arduino Stackable Header Kit</td><td>SparkFun Electronics</td><td>10007</td><td>Female pin headers for connecting PCB to microcontroller. 1 required.</td></tr><tr><td>Adjustable 30W 110V soldering iron - XY-258 110V</td><td>Adafruit</td><td>180</td><td>For making electrical connections to the PCB. 1 required.</td></tr><tr><td>Soldering iron stand</td><td>Adafruit</td><td>150</td><td>For making electrical connections to the PCB. 1 required.</td></tr><tr><td>Mini Solder spool - 60/40 lead rosin-core solder 0.031&quot; diameter - 100g</td><td>Adafruit</td><td>145</td><td>For making electrical connections to the PCB. 1 required.</td></tr><tr><td>0.1 &amp;mu;F capacitor</td><td>SparkFun Electronics</td><td>COM-08375</td><td>Stabilizes voltage in PCB. 1 required.</td></tr><tr><td>10 &amp;mu;F capacitor</td><td>SparkFun Electronics</td><td>COM-00523</td><td>Stabilizes voltage in PCB. 1 required.</td></tr><tr><td>MAX7219CNG LED Matrix/Digit Display Driver - MAX7219</td><td>Maxim</td><td>MAX7219CNG</td><td>LED driver. 1 required.</td></tr><tr><td>8 pin IC Socket</td><td>Mouser Electronics</td><td>575-144308</td><td>16 required. These will be stacked on top of each other to support the culture vessel above the LED matrix.</td></tr><tr><td>24 Pin IC socket</td><td>Mouser Electronics</td><td>535-24-3518-10</td><td>Optional. Use this to reversibly attach the MAXIM 7219CNG driver to the PCB.</td></tr><tr><td>Digital multimeter</td><td>Adafruit</td><td>2034</td><td>For troubleshooting electronics. 1 required.</td></tr><tr><td>Break Away Headers - 40-pin Male (Long Centered, PTH, 0.1&quot;)</td><td>SparkFun Electronics</td><td>PRT-12693</td><td>Male pin headers for connected LED matrix to printed circuit board. Ends can be trimmed with wire cutters. 1 set required.&amp;nbsp;</td></tr><tr><td>Flush diagonal wire cutters</td><td>Adafruit</td><td>152</td><td>For trimming long pin headers and cutting power cables. 1 required.</td></tr><tr><td>Premium Female/Female Jumper Wires - 40 x 12&quot; (300mm)</td><td>Adafruit</td><td>793</td><td>Wire ribbon for connecting breadboard to LED matrix. Can be connected end-to-end with male pin-headers to be longer. 1 required.</td></tr><tr><td>Half-size breadboard</td><td>Adafruit</td><td>64</td><td>The LED matrix will connect to this and the culturing vessel will rest above it.</td></tr><tr><td>Miniature 8x8 Blue LED Matrix</td><td>Adafruit</td><td>956</td><td>Light source. Dominant wavelength is 470nm (blue). 1 required. Alternative miniature LED matrices from the same vendor are available with dominant wavelengths: 624 nm (red), 588 nm (yellow), 525 nm (green), 572 nm (yellow-green), and white.</td></tr><tr><td>Stackable header-3 pin</td><td>SparkFun Electronics</td><td>13875</td><td>8 required.</td></tr><tr><td>Resistor Kit - 1/4W (500 total)</td><td>SparkFun Electronics</td><td>10969</td><td>For electronics. 1 required.</td></tr><tr><td>&amp;nbsp;IRL520N MOSFET</td><td>International Rectifier</td><td>IRL520N</td><td>Voltage regulating switch for controlling DC current. 4 required.</td></tr><tr><td>Hook-Up Wire - Assortment (Solid Core, 22 AWG)</td><td>SparkFun Electronics</td><td>PRT 11367</td><td>Wire for electronics. 1 required.</td></tr><tr><td>5V 2A (2000mA) switching power supply - UL Listed</td><td>Adafruit</td><td>276</td><td>Power supply for the heating pad and Arduino. 2 required.</td></tr><tr><td>12 VDC 1000mA regulated switching power adapter - UL listed</td><td>Adafruit</td><td>798</td><td>For peristaltic pumps. 2 required.</td></tr><tr><td>Electric Heating Pad - 10cm x 5cm</td><td>Adafruit</td><td>1481</td><td>For heating the bioreactor. 1 required.</td></tr><tr><td>Low flow variable flow peristaltic pump</td><td>Fisher Scientific</td><td>13-876-1</td><td>For pumping media. 1 required</td></tr><tr><td>Medium flow variable flow peristaltic pump</td><td>Fisher Scientific</td><td>13-876-2</td><td>For pumping culture. 1 required.</td></tr><tr><td>9 VDC 1000mA regulated switching power adapter - UL listed</td><td>Adafruit</td><td>63</td><td>For microcontroller power supply. Order 1.</td></tr><tr><td>High Temp Waterproof DS18B20 Digital temperature sensor + extras</td><td>Adafruit</td><td>642</td><td>Thermometer for the bioreactor. 1 required.</td></tr><tr><td>Micromanager</td><td>Micromanager</td><td>NA</td><td>The free, open-sourced microscope control software is available at &quot;https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release&quot;</td></tr><tr><td>FIJI</td><td>ImageJ</td><td>NA</td><td>The free, open-sourced image analysis software is available at &quot;http://fiji.sc/&quot;</td></tr><tr><td>Arduino Integrated Development Environment</td><td>Arduino</td><td>NA</td><td>The free, open-sourced IDE is available at &quot;https://www.arduino.cc/en/Main/Software&quot;</td></tr><tr><td>Custom code</td><td>GitHub</td><td>NA</td><td>The custom microcontroller code and &quot;Bioreactor Controller&quot; plugin are available in the &amp;ldquo;Optogenetic Chemostat Files&amp;rdquo; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td></tr><tr><td>USB Cable A to B - 6 Foot</td><td>SparkFun Electronics</td><td>CAB-00512</td><td>Used to download data to microcontroller. 1 required.</td></tr><tr><td>bioreactorTimecourse_example.csv</td><td>GitHub</td><td>NA</td><td>The advantage of loading LED matrix values from a CSV file is that a program can be called by the plugin to update those values based on image analysis results, and those values can be reloaded to the microcontroller, enabling feed-back control. It is available from the &amp;ldquo;Optogenetic Chemostat Files&amp;rdquo; GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).</td></tr><tr><td>Tota-frost gels (diffusion paper)</td><td>B&amp;amp;H</td><td>B&amp;amp;H # LOFSFTL&lt;br/&gt; MFR # T1-72</td><td>For LED matrix. 1 required.</td></tr><tr><td>Kitting Sheet Crosslink 1/4x12x24in</td><td>Grainger, inc</td><td>20JL37</td><td>Black foam for culturing vessel enclosure. 4 required.</td></tr><tr><td>Standard Photodiode Power Sensor, Si, 200 - 1100 nm, 50 mW&amp;nbsp;</td><td>Thorlabs</td><td>S120VC</td><td>For measuring light intensity. 1 required.</td></tr><tr><td>Labelling Tape</td><td>Fisher Scientific</td><td>159015N</td><td>For labelling and securing loose components. 1 required.</td></tr><tr><td>Compact Power and Energy Meter Console, Digital 4&quot; LCD</td><td>Thorlabs</td><td>PM100D</td><td>For measuring light intensity. 1 required.</td></tr><tr><td>100mL GL45 hybridization glass bottle</td><td>Bellco Glass, Inc.</td><td>(7910-40150)</td><td>Bioreactor vessel. 1 required.</td></tr><tr><td>Six port assembly</td><td>Bellco Glass, Inc.</td><td>Custom&amp;nbsp;</td><td>For the bioreactor vessel. Tubing Specs: .125&quot; OD x .055&quot;ID. Port A: 1.0&quot; long above cap slug and to bottom of tube. Ports B,C,E,F: 1.0&quot; long above cap slug, 33 mm long below. Port D: 1.0&quot; long above cap slug, 65 mm&amp;nbsp; long below. 1 required. Includes 45 mm diameter polypropylene open top screw cap and a white silicone gasket to ensure a tight seal between the cap and the vessel.&amp;nbsp;</td></tr><tr><td>Scotch Magic Tape 3105, 3/4 x 300 Inches, Pack of 3</td><td>Amazon</td><td>B0009F3P3U</td><td>Clear scotch tape. This is available from many other vendors. It is used to cover markings on the culturing vessel and to secure the coverglass with the PDMS channel to the aluminum support frame.</td></tr><tr><td>1/16&quot; ID x 3/16&quot; OD x 1/16&quot; Wall Tygon Sanitary Silicone Tubing</td><td>United States Plastic Corp.</td><td>57288</td><td>Tubing. ~25&#039; required.</td></tr><tr><td>Cole-Parmer Twistit white rubber stopper, size 10</td><td>Cole-Parmer</td><td>EW-62992-32</td><td>Media flask stopper and effluent flask stopper. 2 required.</td></tr><tr><td>2L Laboratory Flask</td><td>Pyrex</td><td>4980</td><td>Media flask and effluent flask. 2 required.</td></tr><tr><td>Day pinchcock</td><td>Fisher Scientific</td><td>5867</td><td>For pinching tubes shut. 3 required.</td></tr><tr><td>Replacement tubing assembly 1/16&quot; ID</td><td>Traceable Products</td><td>3372</td><td>The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td></tr><tr><td>Replacement tubing assembly 1/50&quot; ID</td><td>Traceable Products</td><td>3371</td><td>The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.</td></tr><tr><td>Male luer with lock ring x 1/16&quot; hose barb, Nylon, 25/pk</td><td>Cole-Parmer</td><td>EW-45505-00</td><td>Connectors. ~10 luers are required.</td></tr><tr><td>Male luer with lock ring x 1/8&quot; hose barb, Nylon, 25/pk</td><td>Cole-Parmer</td><td>EW-45505-04</td><td>Connectors. 5 required, one for each rubber stopper hole to fill with tubing.</td></tr><tr><td>Female luer x 1/16&quot; hose barb adapter, Nylon, 25/pk</td><td>Cole-Parmer</td><td>EW-45502-00</td><td>Connectors. ~10 luers required.</td></tr><tr><td>Female luer x 3/16&quot; hose barb adapter</td><td>Cole-Parmer</td><td>EW-45502-08</td><td>Connectors. ~10 luers required.</td></tr><tr><td>Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk</td><td>Cole-Parmer</td><td>SC-45502-28</td><td></td></tr><tr><td>Cole-Parmer Luer Accessory, Male Luer Lock Plug, Nylon, 25/Pk</td><td>Cole-Parmer</td><td>EW-45505-56</td><td></td></tr><tr><td>Microbore PTFE Tubing, 0.022&quot;ID x 0.042&quot;OD, 100 ft/roll</td><td>Cole-Parmer</td><td>EW-06417-21</td><td>Tubing. 1 roll required.</td></tr><tr><td>Masterflex platinum-cured silicone tubing, L/S 13, 25 ft</td><td>Cole-Parmer</td><td>EW-96410-13</td><td>Tubing. ~25&#039; required.</td></tr><tr><td>3/16&quot; ID x 1/4&quot; OD x 1/32&quot; Wall Tygon Sanitary Silicone Tubing</td><td>United States Plastic Corp.</td><td>57293</td><td>Tubing. ~1&#039; required.</td></tr><tr><td>Vacuum filter</td><td>Fisher Scientific</td><td>974107</td><td>Nalgene vacuum filter for sterile filtering media.</td></tr><tr><td>Aquel Oxy-Boost 200</td><td>Rena Aquatic Supply</td><td>AP200</td><td>Dual diaphram adjustable flow air pump for aerating and mixing media. 1 required.&amp;nbsp;</td></tr><tr><td>0.2 &amp;mu;m pore syringe filter</td><td>Corning International</td><td>431229</td><td>This ensures that air from the aquarium pump does not contaminate the apparatus. 1 required.</td></tr><tr><td>Slygard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>Slygard 184</td><td>For microfluidic device. 1 required.</td></tr><tr><td>American Safety Razor GEM Scientific Single-Edge Razor Blades</td><td>Fisher Scientific</td><td>17989000</td><td>For cutting tubes and PDMS. 1 blade required.</td></tr><tr><td>Harris Uni-Core hole puncher 1.2mm ID</td><td>Sigma-Aldrich</td><td>WHAWB100028 ALDRICH</td><td>For punching inlet/outlet in microfluidic device. 1 required.</td></tr><tr><td>Microscope cover glass 22x60-1.5</td><td>Fisher Scientific</td><td>12-544-G</td><td>For microfluidic device. 1 required.</td></tr><tr><td>Rectangular aluminum frame with a square window</td><td>Custom</td><td>Custom</td><td>To support the microfluidic channel. Outer dimensions: 3 inches x 1.25 inches.&lt;br/&gt; Inner dimmensions (cut out portion): 7/8 inches x 7/8 inches&lt;br/&gt; Thickness: ~1/32 inches</td></tr></tbody></table>

design and implementation of an automated illuminating, culturing, and sampling system for microbial optogenetic applications

光遗传学系统利用改变构响应于特定波长的光，以改变细胞过程遗传编码的蛋白质。有需要用于培养和测量对于包含编程光遗传学系统的照明和刺激系统。我们提出了构建和使用连续培养装置照亮的微生物细胞与编程的剂量的光，并自动采集和分析流出物中的细胞的图像的协议。该装置作为一个恒化器的操作允许的生长速率和细胞的环境中进行严格控制。连续细胞培养物的流出物定期采样，将细胞由多通道显微镜成像。培养，取样，成像和图像分析完全自动化，使得在荧光强度和从培养流出物取样细胞的细胞形态的动态响应多天测量无需用户输入。我们通过动态诱导蛋白生产中与激活转录的光遗传学系统设计的酿酒酵母的菌株证明这种培养装置的效用。

此装置用于通过基于所述CRY2 / CIB1蛋白对30可诱导光遗传学转录系统刺激酿酒酵母的表达响应于蓝光黄色荧光蛋白（YFP）的培养。细胞在磷酸盐限制培养基chemostatically生长为0.2±0.008的平均稀释率。磷酸盐限制在酿酒酵母恒化实验通常用于控制生长速率和磷酸盐限制的效果很好的特点。 31，32，从连续稀释的培养容器33的流出物进行采样，以在微流体装置上的倒置显微镜。 如图8中的图像进行自动分析。单个细胞中扣除背景的相衬图像和它们的YFP concentra被确定从扣除背景的荧光图像测量和灰是从他们的荧光估计。 169677细胞的荧光，从来自28位置中的微流体装置在实验的70小时获得的流出物的33,600图像进行分析。我们使用装有荧光照明系统和CMOS照相机倒置显微镜。 YFP的图像与二十零分之五百nm激发滤光器，三十零分之五百三十五nm发射滤波器，和一个T515lp分色获得。图像用一个40X相衬物镜，在科勒照明。图片记录了16位色彩深度87像素每平方微米。将培养物暴露于不同的蓝色光的强度为6小时的间隔，随后为6小时的时间间隔完全黑暗。培养已暴露前的第一测量，这也就是为什么它的荧光强度在第一黑暗时期减少点亮。&lt;/ P&gt; 图9示出响应于照亮培养容器由于YFP生产经光遗传学系统的激活的荧光。它表明荧光的单小区测量的以上人口平均单小区测量揭示荧光强度的人口分布的效用。注意在42小时26分钟的异常。审查相应的图像后，很明显，有细胞的团块在大多数用于产生背景的合成图像的图像的，导致在类似细胞中扣除背景的图像中的伪影。此外，从培养容器中的泡沫被泵送到微流体通道和其边缘的部分被误认为如通过图像分析算法的细胞。由于既没有气泡也没有从减去背景的器物符合实际的细胞，其测量荧光强度比细胞的自发荧光低。该图演示了数据的质量，可以在3天中自动获取。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54894/54894fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54894/54894fig8.jpg" /> 图8: 图像分析算法的视觉描绘。这些图像裁切和便于观看的扩大。 （A）的六张图像获取以产生细胞培养物的扣除背景的相位对比图像。主图像被获取之后，另外五个图像获取与流出物取样泵每个采集之间简要地接通，以确保细胞位移。背景的复合图像从五个分量图像生成。每个像素的值在背景图像在5成分的图像，同一像素的中间值。 （B）中的扣除背景的图像然后被转换为二进制。则二进制被扩张和二进制的连续部分中的孔填充。黄色轮廓对应于感兴趣的选定区域（ROI），根据尺寸和圆度的标准。 （C）的那些ROI被映射到的荧光图像，其中每个细胞的荧光作为ROI中最亮的像素的值进行测定。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54894/54894fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/54894/54894fig9.jpg" /> 图9: 随着时间的推移荧光强度的人口分布 。在这些子图测得氟的对数escence显示，继流式细胞仪的标准做法。在A和B的线表示光吸收或文化，这是通过通过细胞培养在培养容器无菌传输媒体和光透射光之间的强度差衍射测定的强度。它绘制在第二纵轴。 （A）盒须情节人口随时间的测量荧光的对数（ 第 5百分位，25 百分位数，中位数，75 百分位数，95 百分位数）。 （B）中相同的数据，其中，颜色对应于细胞与在对应的容器的范围内的测得的荧光的归一化频率的二维直方图。颜色分别在42小时包括26分钟调整为无异常的数据范围。离群的最低荧光纸槽归一化频率为0.7。 （ 三 ）人口的第一次记录曝光前的测量荧光的对数的一维直方图和最大曝光后。它表明YFP的在该菌株的光诱导表达是双峰的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54894/54894fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications at JoVE.com

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

我们设计用于光遗传学系统一起使用，照亮微生物的培养，并定期图像细胞出水倒置显微镜连续培养装置。培养，取样，成像和图像分析完全自动化，使得光照动态响应可以在几天进行测量。

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

design-implementation-an-automated-illuminating-culturing-sampling

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.6K 次观看.  University of Wisconsin-Madison. 我们设计用于光遗传学系统一起使用，照亮微生物的培养，并定期图像细胞出水倒置显微镜连续培养装置。培养，取样，成像和图像分析完全自动化，使得光照动态响应可以在几天进行测量。 

视频: Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

科研

遗传学

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

神经科学

研究斑马鱼光遗传学信号转导操作的发育

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

作者聚焦：操纵斑马鱼胚胎中的信号转导以解码细胞命运决定 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

斑马鱼胚胎中的光遗传学信号激活

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

发育生物学

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

为光遗传学实验构建简单通用的照明系统

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...

生物工程

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

用于微生物化学和蛋白质生产的光控发酵

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

人类神经肌肉接头光遗传学模型的工程和表征

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

使用 Lustro 自动化技术进行酵母光遗传学反应分析

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems.
Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of&#160;their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro&#39;s components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

作者聚焦:使用 Lustro 推进光遗传学研究 

This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in yeast.

使用自动化平台 Lustro 在酵母中进行高通量光遗传学实验

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to ...

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

摘要

探索更多视频

此视频中的章节

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

在啮齿动物中枢神经系统的体内光遗传学刺激

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

斑马鱼胚胎中的光遗传学信号激活

为光遗传学实验构建简单通用的照明系统

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

用于微生物化学和蛋白质生产的光控发酵

人类神经肌肉接头光遗传学模型的工程和表征

使用自动化平台 Lustro 在酵母中进行高通量光遗传学实验