Improving Imaging Techniques with Dual-Labeled DNA Strands

Fluorophore-labeled DNA strands are important tools used in advanced microscopy techniques. These tiny strands help scientists see very small things, like structures in cells, more clearly. When they are combined with special imaging methods, they allow for better images of biological samples.

#Key Techniques

Some of the advanced microscopy techniques that utilize these DNA strands include:

• Single-Molecule Localization Microscopy (SMLM): This method helps identify where individual molecules are located in a sample.
• DNA Points Accumulation in Nanoscale Topography (DNA-PAINT): This technique uses short DNA strands to create detailed images of structures.
• Super-resolution Optical Fluctuation Imaging (SOFI): This method provides high-resolution images by analyzing the fluctuations of light.
• Stimulated Emission Depletion (STED) Microscopy: This technique offers super-resolution images by using a special light pattern to turn off background signals.

A major benefit of using these DNA strands is their ability to label multiple targets at once. By washing away and re-adding different labeled strands, scientists can take repeated images that help build a more complete picture of the sample.

#Optimize Imaging with DNA Labels

One specific advantage of DNA-PAINT is that it allows scientists to measure how much of a specific molecule is present. This can be done through a process called kinetic analysis. However, using weak-affinity DNA labels can lead to challenges. These labels need to remain in the imaging buffer during experiments, which can increase background noise and slow down the acquisition of images.

To tackle this issue, scientists have introduced FRET-quenched fluorophores. These are special types of fluorophores that, when linked to longer DNA strands, can reduce background noise. Recently, scientists have shown that using DNA strands at both ends with identical fluorophores can result in self-quenching. This means that when the strands are not bound to their target, they exhibit lower fluorescence, leading to clearer images.

#Characterizing New Probes

Scientists designed several new fluorophore-labeled DNA strands, known as "imager strands." They used a common DNA sequence and attached fluorophores at the ends or at just one end. The fluorophores selected for this study were Cy3B, silicon-rhodamine (SiR), and tetramethylrhodamine (TMR).

They characterized these strands by examining their behavior in solution when they are single and when they bind to a complementary strand. By looking at their absorption and fluorescence properties, they could see how effective these new strands were.

#Observations

When the imager strands carry two fluorophores, their fluorescent signals change when they are free in solution versus when they are bound to a target. Specifically, the fluorescence signal increases significantly upon binding, indicating that the dual-labeled strands are effective for imaging.

They also observed changes in the speed at which the strands bind and unbind from their targets, which helps determine how long they stay attached during imaging.

#Using in STED Microscopy

The new dual-labeled imager strands were tested in STED microscopy, where it was hoped they would lower the background fluorescence seen in unbound probes. Previous results indicated that certain combinations of dual-labeled strands effectively reduced background noise while increasing the brightness of bound probes.

The images obtained showed that while some strands exhibited a decrease in fluorescence intensity when bound, all dual-labeled strands displayed lower background signals compared to their single-labeled counterparts. This led to a better signal-to-noise ratio for each dual-labeled strand used in imaging.

In addition, the increase in signal-to-noise ratio was coupled with improved image resolution. This improvement means that scientists could distinguish smaller details in their samples.

#Two-color Imaging

Utilizing dual-labeled strands also enabled scientists to conduct simultaneous two-color imaging. This means they could visualize two different structures in a single experiment, which provides even richer information about the biology being studied.

#Advancing DNA-PAINT Microscopy

The scientists also applied these dual-labeled imager strands to DNA-PAINT microscopy. They focused on Cy3B and SiR, known for their stability and spectral separation. By comparing the performance of single-labeled and dual-labeled strands in DNA-origami structures, they were able to assess how well these strands performed together.

The findings indicated that dual-labeled strands offered improved fluorescence properties, leading to better imaging outcomes. The increase in photon yield was significant, and dual-labeled strands provided enhanced resolution.

#Visualizing Biological Structures

Using the dual-labeled imager strands, scientists visualized important structures like the nuclear pore complex protein Nup96. They compared the photon output of single and dual-labeled strands, confirming that dual-labeled strands delivered more photons and provided better image detail.

#Conclusion

This research highlights how self-quenching dimers attached to short DNA strands can significantly improve imaging in advanced microscopy techniques. By utilizing dual-labeled DNA probes, scientists can gain higher signal-to-background ratios and improved image resolution.

The implications of this work extend beyond just one area of microscopy. The principles governing these dual-labeled strands could be adapted for other techniques, leading to further advancements in biological imaging. Ultimately, this research opens the door for more precise observations of biological processes and structures at the molecular level.