Super-Resolution Microscopes

DNA-PAINT Imaging

单分子定位显微镜在纳米级地形中的点积累

Explore DNA-PAINT Solutions

The Only Out-of-the-Box Solution for Multiplexed, Multicolor DNA-PAINT Imaging

Combined with Bruker's software-controlled, automated, fully-integratedMicrofluidics Unit, 这Vutara VXLsuper-resolution microscope is唯一的商业系统that offers out-of-the-box multiplexed DNA-PAINT capabilities. Performed on these tools, DNA-PAINT imaging allows for sub-10 nm localization precision in single-molecule localization microscopywith unlimited multiplexing potential

Moreover, the Vutara microscope with microfluidics system provides the added benefits of:

  • No-hassle setup with expert service and support to expedite time to data collection
  • 与使用翻新系统收集的数据更可靠和重复的结果
  • Automation and multi-condition/multi-user programming for easily scalable experiment design

了解更多有关DNA涂料溶液的信息

或与我们联系,以了解有关使用Vutara VXL显微镜进行DNA斑点实验的更多信息。

什么是DNA粉末?

What is DNA-PAINT?

DNA-PAINT is a technique to enable single-molecule localization through the binding of short (<10 nucleotides) oligonucleotides labeled with a fluorophore to a complementary oligonucleotide bound to a target molecule, typically an antibody. It allows for extensive imaging modalities, from whole-cell extensive Z-stacks to high-resolution multi-target images.

Understanding the Advantages of DNA-PAINT Imaging

The binding of the short oligos is transient in nature, and thus creates a blinking effect akin to dSTORM or PALM, yet DNA-PAINT has numerous advantages over other blinking techniques. The advantages of using DNA-PAINT imaging for nucleic acid nanostructures include:
  • Higher photon yield:眨眼通常比常规DSTORM更长。与DSTROM和PALM等方法相比,这会导致较高的光子产生的荧光团产量,因此可以具有更高的定位精度(<10 nm)。
  • Practically unbleachable imaging:The sample is bathed in an excess of fluorophore, allowing for extremely long-lasting imaging.
  • 无限的多路复用潜力:由于目标特异性是由核苷酸序列设置的,因此有可能(使用正确的工具)标记具有不同寡核序列的多个目标。使用Vutara的集成微流体单元,可以从样品中洗涤给定靶的成像链,并可以添加不同的成像链标记不同的生物学靶标。

How Does DNA-PAINT Work?

How Does DNA-PAINT Work?

DNA-PAINT通过短暂的“成像寡核苷酸”的瞬时结合,该结合含有荧光团到互补的寡核苷酸(称为“停靠链”)的目标,例如感兴趣的目标,例如抗体,纳米体,Aptamer,Aptamer或suicide酶体。

Using Super-Resolution Microscopy with DNA-PAINT

Super-resolution imaging with DNA-PAINT involves several steps, including:

  1. The sample is labeled with the “docking strand” through conventional techniques and prepared for imaging.
  2. For imaging, the sample is bathed in imaging buffer (typically PBS but can include oxygen scavengers) and a low (typically 0.1-1 nM) concentration of imaging oligo complementary to the docking strand. The imaging oligo is typically 9-10 nucleotides in length and contains a fluorophore. We recommend Cy3B for DNA-PAINT due to its fluorogenicity and thus lower background.
  3. 进入成像缓冲液后,可以成像样品。成像链与对接链的瞬态结合阻止了荧光团的扩散,从而使其在相机上成像。

由于样品被沐浴在大量不断交换成像链中,因此目标本质上是不可漂白的,因此可以将大量框架和扩展的Z堆栈进行分解。

上面的卡通显示了DNA斑点的工作原理。靶蛋白(微管蛋白)用带有对接链寡核的抗体标记。然后将样品沐浴在成像链寡醇中。荧光标记的成像链与对接链的瞬态结合导致样品看起来眨眼,然后可以将其定位在Vutara SRX软件中。重复此过程,直到形成超分辨率图像为止。

为什么使用DNA-PAINT ?

为什么要将DNA-Paint用于3D细胞超分辨率成像?

3 d细胞骨架微管蛋白网络的成像BS-C-1 cells. Cells were labeled with alpha-tubulin primary antibodies and anti-rabbit oligo conjugated secondary antibodies. Left: 3D tubulin network colored by depth. Right: close up view of the tubulin polymers showing the hollow labeling lumen. Secondary DNA-PAINT antibodies were purchased from Massive-Photonics.com

高精度本地化

DNA-PAINT allows sub-10 nm localization precision, making it one of the most precise microscope techniques available.

在这里,将带有水浸入水1.2 Na物镜的Vutara显微镜用于DNA-Paint实验。该图像显示了整个BS-C-C-1细胞的小管蛋白网络,该网络用与DNA-Paint二抗结合的微管蛋白抗体标记。插图显示了微管蛋白网络的放大部分。微管的管腔清晰可见。

Multicolor Unbleachable Imaging

通过DNA-Paint使多重多色超分辨率成像成为可能。

DNA-PAINT has the potential for large-scale multicolor Z-stacks due to the fact the sample is bathed in a practically limitless supply of fluorophore. This enables large-scale z-stack imaging composed of millions of localizations.

Here, a two-color DNA-PAINT experiment was performed on the Vutara single-molecule localization microscope. Tubulin is labeled in cyan and clathrin in magenta. Furthermore, due to the unbleachable nature of DNA-PAINT large Z-stacks are possible.

BS-C-1细胞中微管蛋白网络和网状蛋白笼的3D细胞骨架成像。细胞用α-微管蛋白和抗拉取重链原代抗体和抗兔和抗小鼠寡核苷的二抗二抗标记。顶部:用α-微管蛋白(青色)和网状蛋白笼(Magenta)标记的BS-C-1细胞。底部:与上述图像相同的电影的电影,显示了数据的3D性质。次级DNA粉末抗体购自大量 - photonics.com
Left: Workflow for DNA-PAINT. Each target (actin, tom20, tubulin and clathrin) is labeled with primary and secondary antibodies. The secondary antibodies have orthogonal docking strands. Each target is imaged sequentially by flowing in the complementary imaging oligo to the desired target followed by a wash step before flowing in the next imaging strand for the next target. Fluidics and imaging are handled automatically with the Vutara microscope and SRX software. Right: The images from each target are combined automatically in SRX software. Secondary DNA-PAINT antibodies were purchased from Massive-Photonics.com

无限的多路复用ing Potential

DNA-PAINT has enormous potential for multiplexed imaging.

在这里,使用VUTARA VXL和集成的流体单元进行了多目标DNA-Paint实验。在不同的探针上使用正交对接链,可能有可能无限的目标数量。

还图所示是在Vutara单分子定位显微镜和集成的流体单元上进行的四个目标DNA漆实验。F-Actin-Magenta,Tom20-Cyan,微管蛋白 - 黄色和网状蛋白绿色。

示例图像

Images automatically collected using the Vutara VXL with fluidics

示例图像: DNA-PAINT for Improved Super-Resolution Imaging

BS-C-1用抗微管蛋白,肌动蛋白,抗TOM20和抗克拉蛋白标记。购买正交2ºDNA涂料是从中购买的巨大的 - photonics.com

左上:肌动蛋白 - 马根塔,微管蛋白黄色,线粒体 - 橙色。


Top right: mitochondria-cyan, F-actin-magenta, tubulin-yellow, clathrin heavy chain-green.
左下:f-肌动蛋白 - 克拉辛笼绿色。
右下角:线粒体 - 小管,微管蛋白 - 马根塔。