What's inside the cage?
Researchers at Harvard University, working under the leadership of Professor Peng Yin, have developed a new microscopy method that allows you to obtain images that simultaneously represent dozens of different biomolecules that make up a single cell. Such images will help to understand the complex intracellular mechanisms and can be used in the development of new methods for diagnosing diseases and predicting and course, as well as monitoring the effectiveness of therapy at the cellular and molecular level.
Dozens and even hundreds of different proteins and RNA molecules are often involved in performing complex cellular functions. Modern methods, as a rule, allow a maximum of three or four types of molecules to be visualized simultaneously. However, for a complete understanding of cellular functions, it is extremely important to visualize all or at least most of the participants in the process, since considering only a few components at a time, it is impossible to assess the full picture of what is happening.
To accomplish this task, Yin's group had to overcome the laws of physics that had hampered the work of microscopists for a century. The problem is that if the distance between two objects is less than 200 nanometers (the so-called diffraction limit corresponding to about 1/5 of the thickness of a human hair), they cannot be distinguished from each other using a traditional light microscope. Instead of two objects, a person looking through the eyepiece sees only one blurry spot.
Since the mid-1990s, new methods have been available to scientists to overcome this barrier by combining specialized optical systems and special fluorescent proteins or dyes that label cellular components.
One of these methods, called DNA-PAINT, was developed by Dr. Ralf Jungmann (Ralf Jungmann) from Professor Yin's laboratory in the process of completing a dissertation project. DNA-PAINT allows you to get very clear images of three molecules at the same time by labeling them with various dyes.
To get more detailed images of the processes taking place in the cell, the researchers modified DNA-PAINT, resulting in a new visualization method called Exchange-PAINT.
The new method is based on the ability of DNA strands to bind specifically to complementary DNA strands. The researchers label the target molecule with a short DNA chain, after which they add a complementary chain to the solution, labeled with a fluorescent dye that begins to glow only when two chains are connected. The two chains are connected for a short period of time, the duration of which is precisely regulated by researchers. The resulting burst of glow allows you to get an ultra-sharp image.
Repetition of this process provides visualization of the second, third and subsequent target molecules. When the resulting images are superimposed, a composite picture is obtained in which each target molecule is colored with its own color. This allows you to create images whose components are colored with additional artificial colors, so that the visualization capabilities go far beyond the limits limited by the number of existing fluorescent dyes. And the resulting images in the smallest detail reflect the studied molecular process.
To test Exchange-PAINT, the researchers synthesized 10 unique DNA sequences – the so-called DNA origami – depicting numbers from 0 to 9. When using the new imaging method, these numbers were distinguishable at a distance of less than 10 nanometers, which corresponds to 1/12 of the diffraction limit. They also managed to get clear images of all 10 DNA origami in one picture.
10-color ultrafine images (artificially composed into one composition) of artificial DNA nanostructures in the form of digits from 0 to 9, obtained using the Exchange-PAINT method using only one dye and a single-beam laser. Photo: Johannes B. Woehrstein/Wyss InstituteMoreover, the authors used the Exchange-PAINT method to obtain detailed ultra–sharp images of fixed human cells, on which important cellular components – microtubules, mitochondria, Golgi apparatus and peroxisomes - were colored in different colors.
In this ultra-sharp image obtained using the Exchange-PAINT method, microtubules (green), mitochondria (purple), Golgi apparatus (red) and peroxisomes (yellow) of one human cell are simultaneously visible. Photo: Maier Avendano/Wyss InstituteThe developers believe that further improvement of the Exchange-PAINT method will allow it to be used for simultaneous visualization of dozens of cellular components.
This will provide biologists with a powerful new tool for a comprehensive study of the processes occurring in the cell.
Article by Ralf Jungmann et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT is published in the journal Nature Methods.
Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on materials from Harvard University:
Capturing ultrasharp images of multiple cell components at once.
10.02.2014