NanoPlex: A New Way to See Cells

Fluorescence Microscopy is a widely used tool in cell biology. It helps scientists see specific parts of cells or tissues by using special colors of light. Scientists can often look at multiple targets at once by using different colors. This is beneficial when studying complex biological samples.

#The Basics of Fluorescence Microscopy

In fluorescence microscopy, scientists use special dyes that glow under certain lights. The most common way to see different colors is to use four channels: blue, green, red, and deep-red. To see multiple targets in a sample, scientists often use a method called indirect immunofluorescence (IF). This method relies on two types of antibodies.

1. Primary Antibodies (1.Abs): These bind to specific targets in the sample.
2. Secondary Antibodies (2.Abs): These are linked to a fluorescent dye and bind to the primary antibodies.

To look at three different targets in a sample, for example, scientists need three primary antibodies from three different species. This way, they can attach the secondary antibodies specifically to each primary antibody.

#Challenges in Multiplexing

While this method has been helpful, it has limitations. Sometimes, scientists need to observe a larger number of targets at once, especially for detailed studies like looking at proteins in single cells.

In the past, some scientists tried to visualize six targets by removing antibodies using chemical methods. This was done through processes like chemical denaturation or photobleaching and allowed for more than 10 targets to be visualized. However, these methods could damage the structure of the cells.

More recent approaches have explored using special types of DNA to tag signals, allowing for large area imaging. These allow for bigger studies but can also come with challenges, such as reduced precision in locating molecules. Therefore, scientists need new methods that maintain the integrity of the samples while allowing for imaging of multiple targets.

#Advancements in Super-Resolution Microscopy

Super-resolution microscopy is a newer technique that offers better detail. It allows scientists to see more targets simultaneously than traditional methods. One of the best-known techniques is called Exchange-PAINT. In this method, antibodies or other binding tools are linked to single-stranded DNA (ssDNA). Each ssDNA serves as a unique tag, allowing scientists to identify targets accurately.

Recent developments have allowed researchers to visualize up to 30 different targets in a sample using a more advanced technique called SUM-PAINT. However, these techniques can be complicated and require specialized equipment and expertise.

#A New Approach: NanoPlex

To help simplify the process of imaging multiple targets, a new method called NanoPlex has been developed. This method utilizes engineered secondary nanobodies (2.Nbs) that can attach to primary antibodies in a straightforward way. The technology can be used with regular light microscopes as well as with advanced super-resolution techniques like dSTORM or STED.

The key to NanoPlex is that it doesn’t rely on harsh treatments that could damage the cells. Instead, it uses gentle methods to remove fluorescent signals.

#Photolabile Molecule: OptoPlex

The first method within NanoPlex, named OptoPlex, employs a special light-sensitive molecule. When this molecule is illuminated with a specific wavelength of light, it releases its fluorescent group. This allows scientists to quickly and selectively erase signals in regions of interest without harming the sample.

This is particularly useful because it allows for detailed study without extensive damage to the cellular structure. After erasing a signal, scientists can add more antibodies and repeat the imaging process.

#Enzymatic Cleavage: EnzyPlex

The second method is called EnzyPlex. This approach uses a specific protease enzyme that cleaves a linker connected to the fluorophore. Scientists have found that when they use this enzyme, they can effectively remove fluorescent signals from the nanobodies without much damage to the specimen.

In tests, EnzyPlex achieved high signal removal efficiency, allowing cells to be imaged multiple times in short periods. This method is also easier to implement since the enzyme can work under many different conditions.

#Redox Chemistry: ChemiPlex

Lastly, there is the ChemiPlex method, which is the simplest among the three. It uses a chemical reaction to break a bond between the nanobody and the fluorescent tag. A reducing agent is applied, leading to quick and even removal of signals across multiple targets.

ChemiPlex has shown to be efficient and uniform in its signal removal capabilities, allowing for high-quality imaging even after several cycles.

#Achieving Versatile Imaging with NanoPlex

With its three approaches, NanoPlex allows scientists to image many targets in a single sample without the need for harsh treatments. This makes it a versatile method applicable in various fields, ranging from basic biology to advanced medical research.

#Experimenting with Neurons

To demonstrate the capabilities of NanoPlex, scientists performed experiments on primary hippocampal neurons. They managed to visualize 21 different proteins by applying the ChemiPlex method in a straightforward manner. This included looking at proteins involved in synapses, filament structures, and other cellular parts.

By studying these neurons, researchers could analyze the behavior of synaptic proteins and the interactions between them. They could even examine how certain treatments affected the localization and interaction of these proteins.

#Correlating Protein Interactions

In another experiment, scientists focused on understanding the relationships between nine synaptic proteins. By examining their co-localization in excitatory and inhibitory synapses, they could assess how the proteins interacted with one another.

They found certain proteins, like alpha-Synuclein and Synapsin-1, had strong correlations, indicating their roles in synaptic functions. After testing with 1,6-hexanediol, known to disrupt specific interactions, they observed changes in how these proteins correlated. This suggested a deeper level of understanding about how synaptic proteins function and interact within cells.

#Conclusion

In summary, NanoPlex offers a valuable solution for scientists looking to visualize multiple targets in cells. Its gentle signal removal methods lead to improved imaging quality with minimal damage. As the need for advanced imaging techniques grows, methods like NanoPlex could pave the way for groundbreaking discoveries in cell biology.

By making multiplex imaging accessible to many labs, this new strategy has the potential to expand our understanding of complex biological systems. The ability to visualize numerous targets simultaneously will be pivotal in various fields, from research to clinical diagnostics.