3D Multiplexed Super-Resolution Imaging with DNA-PAINT and the Vutara VXL | Bruker - 雷竞技 lpl夏季赛六支俱乐部赛前变化,雷竞技贴吧

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,Vutara VXLsuper-resolution microscope isthe only commercial systemthat 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:

无争议的设置与专家服务和支持expedite time to data collection

Results that are more reliable and repeatable than data collected with retrofitted systems

Automation and multi-condition/multi-user programming for easily scalable experiment design

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What Is DNA-PAINT?

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:The blinks are typically longer-lasting than in conventional dSTORM. This results in higher photon yields from the fluorophore and therefore allows for a much higher localization precision (<10 nm) when compared to such methods as dSTORM and PALM.

Practically unbleachable imaging:The sample is bathed in an excess of fluorophore, allowing for extremely long-lasting imaging.

Unlimited multiplexing potential:Since target specificity is set by nucleotide sequence, it is possible — with the right tools — to label multiple targets with different oligo sequences. With the Vutara’s integrated microfluidics unit, the imaging strand for a given target can be washed from the sample and different imaging strands labeling different biological targets can be added.

For further reading on Super-Resolution Microscopy with DNA-PAINT, see:

How Does DNA-PAINT Work?

DNA-PAINT works through the transient binding of a short “imaging oligonucleotide” containing a fluorophore to a complementary oligonucleotide — called the “docking strand” — on the target of interest, such as an antibody, nanobody, aptamer or suicide enzyme ligand.

Using Super-Resolution Microscopy with DNA-PAINT

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

The sample is labeled with the “docking strand” through conventional techniques and prepared for imaging.
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.
一次in the imaging buffer, the sample can be imaged. The transient binding of the imaging strand to the docking strand stops the diffusion of the fluorophore allowing it to be imaged on the camera.

Since the sample is bathed in a large excess of constantly exchanging imaging strand, the target is essentially unbleachable, making it possible to batch-process a large number of frames and extended Z-stacks.

The cartoon above shows how DNA-PAINT works. The target protein (tubulin) is labeled with an antibody labeled with the docking strand oligo. The sample is then bathed in imaging strand oligos. The transient binding of the fluorescently labeled imaging strand to the docking strand causes the sample to appear to blink, which can then be localized in the Vutara SRX software. This process is repeated until the super-resolution image is formed.

Why Use DNA-PAINT?

Why Use DNA-PAINT for 3D Cellular Super-Resolution Imaging?

3D cytoskeletal imaging of the tubulin network in 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

High-Precision Localization

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

在这里,水浸1 Vutara显微镜.2 NA objective was utilized for a DNA-PAINT experiment. The image shows a whole BS-C-1 cell’s tubulin network labeled with tubulin antibodies conjugated to a DNA-PAINT secondary antibody. The inset shows a zoomed-in section of the tubulin network. The lumen of the microtubule is clearly visible.

Multicolor Unbleachable Imaging

Multiplexed multicolor super-resolution imaging is made possible with DNA-PAINT.

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.

3D cytoskeletal imaging of the tubulin network and clathrin cages in BS-C-1 cells. Cells were labeled with alpha-tubulin and anti-clathrin heavy chain primary antibodies and anti-rabbit and anti-mouse oligo conjugated secondary antibodies. Top: BS-C-1 cells labeled with alpha-tubulin (cyan) and clathrin cages (magenta). Bottom: movie of the same image as above showing the 3D nature of the data. Secondary DNA-PAINT antibodies were purchased from Massive-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

Unlimited Multiplexing Potential

DNA-PAINT has enormous potential for multiplexed imaging.

Here, a multi-target DNA-PAINT experiment was performed using the Vutara VXL and integrated fluidics unit. Using orthogonal docking strands on different probes, a potentially unlimited number of targets are possible.

Also pictured is a four-target DNA-PAINT experiment performed on the Vutara single-molecule localization microscope and integrated fluidics unit. F-actin-magenta, tom20-cyan, tubulin-yellow and clathrin-green.