Fluorescence-lifetime encoding for scalable single-protein-resolution imaging
This paper introduces RE-FLIM, a fluorescence-lifetime encoding technique that overcomes the quadratic scaling limitations of previous methods to achieve single-protein-resolution imaging, enabling the visualization of individual HIV-1 Env trimer protomers in intact cell membranes.
Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer
To see the machinery of life, scientists often turn to microscopes that can peer far beyond the limits of ordinary light. For decades, a technique called DNA-PAINT has allowed researchers to map the positions of individual proteins with a precision of about 15 nanometers. This is incredibly small, yet many protein complexes, the functional units that drive cellular processes, are organized at scales even smaller than that, often just a few nanometers apart. When these tiny components sit too close together, standard super-resolution methods blur them into a single, indistinct spot, hiding their true structure. To solve this, scientists developed a method called RESI, which acts like a slow, careful sorting process. It labels different parts of a protein with unique DNA tags and then images them one tag at a time. By waiting for each tag to blink on and off in sequence, the method can pinpoint the center of each part with extreme accuracy. However, this sequential approach has a major bottleneck: as the number of parts in a protein increases, the time required to image them all grows explosively, making it impractical for large, complex structures.
A team of researchers led by Sabrina Simoncelli at University College London has now introduced a faster, more efficient way to see these tiny details, a method they call RE-FLIM. Instead of waiting to image each protein part one by one, this new technique uses the "lifetime" of light to tell different parts apart simultaneously. Every fluorescent dye molecule glows for a slightly different amount of time after being hit by a laser before it fades away. By measuring this precise duration, the microscope can distinguish between different types of dye molecules even when they are glowing at the same time. The researchers combined this lifetime measurement with the high-precision averaging of the older RESI method. This allowed them to resolve multiple protein parts in a single shot, cutting the number of imaging rounds needed by three times compared to the previous best method.
The team first tested their idea on a flat, rectangular structure made of DNA, known as a DNA origami, which served as a perfect, rigid ruler. They attached pairs of docking strands to this structure that were separated by just 3.2 nanometers, a distance too small for standard DNA-PAINT to resolve. By using two different fluorescent dyes that glowed for different lengths of time, they were able to see both strands clearly in a single image. The new method pinpointed the location of each strand with a precision of about 1 nanometer, an eightfold improvement over the standard technique. They repeated this success with a more complex triangular arrangement of three strands, spaced roughly 5 nanometers apart. In this case, they used three different dyes simultaneously. The resulting images clearly showed the three distinct corners of the triangle, confirming that the method could handle multiple targets at once without blurring them together.
Encouraged by these results on the artificial DNA structures, the researchers applied RE-FLIM to a real biological target: the HIV-1 envelope protein, a spike that sits on the surface of the virus and helps it infect cells. This protein exists as a trimer, meaning it is made of three identical subunits arranged in a tight circle. In its closed, inactive state, these subunits are separated by about 15.3 nanometers. Previous attempts to image this structure with standard methods resulted in a blurry cluster where the three subunits could not be distinguished. Using their new technique, the team labeled the protein with six different DNA tags and imaged them in just two rounds, with three different dye types glowing at once in each round. The resulting images revealed the individual subunits of the HIV-1 spike with a precision of about 3 nanometers.
The data showed that the three parts of the spike were indeed arranged in a tight triangle, with the distance between them measuring roughly 16 to 17 nanometers, which matches the known structure of the virus. This was the first time researchers have been able to clearly see the individual building blocks of the HIV-1 envelope protein while it sits in a living cell membrane. By proving that fluorescence lifetime can be used to read multiple DNA barcodes at the same time, this work offers a scalable path to understanding the intricate architecture of complex proteins. It suggests that scientists can now map the precise arrangement of many different parts within a single protein complex without spending days waiting for sequential images, opening the door to studying the molecular machinery of life with unprecedented clarity and speed.
Drowning in papers in your field?
Get daily digests of the most novel papers matching your research keywords — with technical summaries, in your language.