An open source toolkit for repurposing Illumina sequencing systems as versatile fluidics and imaging platforms.
An open source toolkit for repurposing Illumina sequencing systems as versatile fluidics and imaging platforms.
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DOI：
10.1038/s41598-022-08740-w
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发表时间：
2022-03-24
影响因子：
4.6
通讯作者：
Maniatis S
中科院分区：
文献类型：
--
作者：
Pandit K;Petrescu J;Cuevas M;Stephenson W;Smibert P;Phatnani H;Maniatis S
荧光显微技术是生命科学研究的重要手段。现有技术的组学方法将联合收割机荧光显微术与复杂的方案相结合，以使样品中数百万像素中的每一个中的数十至数千个特征可视化。这些组学方法需要在数天或数周的过程中进行的迭代化学和成像循环期间精确控制温度、试剂应用和图像采集参数。这种方法的自动执行使得能够生成鲁棒的和可再现的数据。然而，很少有商业解决方案存在的温度控制，流体耦合荧光成像，定制仪器的实施需要专门的工程专业知识。在这里，我们提出PySeq2500，一个开源的Python代码库和流动池设计，它将Illumina HiSeq 2500仪器（包括集成流体的落射荧光显微镜）转换为可编程应用程序的开放平台，而无需专门的工程或软件开发专业知识。可定制的PySeq2500协议使实验设计能够在扩展实验中同时进行4通道图像采集、温度控制、试剂交换、稳定定位和样品完整性。为了证明复杂的多天工作流程的可访问自动化，我们使用PySeq2500系统来无人值守地执行迭代间接免疫荧光成像（4i）。我们的自动化4i方法在染色、成像和抗体洗脱的多个循环中使用现成的抗体，以在小鼠和死后人类脊髓切片中构建细胞类型和病理特征的高度多重图谱。鉴于HiSeq 2500平台的广泛可用性以及重新使用这些系统所需的修改的简单性，PySeq 2500使非专业人员能够在广泛可用的台式系统中开发和实施最先进的流体耦合成像方法。
Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument, comprising an epifluorescence microscope with integrated fluidics, into an open platform for programmable applications without need for specialized engineering or software development expertise. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. Given the widespread availability of HiSeq 2500 platforms and the simplicity of the modifications required to repurpose these systems, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.
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DOI：
10.1126/science.aay3446
发表时间：
2021-02-26
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Payne AC;Chiang ZD;Reginato PL;Mangiameli SM;Murray EM;Yao CC;Markoulaki S;Earl AS;Labade AS;Jaenisch R;Church GM;Boyden ES;Buenrostro JD;Chen F
通讯作者：
Chen F
影响因子：
48
作者：
Giesen, Charlotte;Wang, Hao A. O.;Bodenmiller, Bernd
通讯作者：
Bodenmiller, Bernd
影响因子：
--
作者：
Geser, Felix;Brandmeir, Nicholas J.;Trojanowski, John Q.
通讯作者：
Trojanowski, John Q.
影响因子：
64.5
作者：
Cho CS;Xi J;Si Y;Park SR;Hsu JE;Kim M;Jun G;Kang HM;Lee JH
通讯作者：
Lee JH
使用迭代免疫荧光成像的衰老齿状回的神经源性生殖位的表征。
影响因子：
7.7
作者：
Cole JD;Sarabia Del Castillo J;Gut G;Gonzalez-Bohorquez D;Pelkmans L;Jessberger S
通讯作者：
Jessberger S