New molecules for high-resolution cell imaging

Like our own body, cells also have their own skeleton called "cytoskeletons", which are made up of proteins rather than bones. These reticular structures maintain the shape of the cells, provide mechanical support, and participate in key processes in the cell life cycle. The cytoskeleton is the subject of intense research in science and medicine, and these studies often require direct observation within the cell. Ideally, highly fluorescent molecules that can bind to highly specific cytoskeletal proteins without toxicity to the cells will be involved.

In a study published in Nature Methods on May 25th, 2014, EPEL scientists at the University of Lausanne in Switzerland used the properties of a new fluorescent molecule (also developed by EPEL) to generate two powerful probes. The needles image the cytoskeleton with unprecedented resolution. These probes paved the way for easier and higher-quality cell imaging, and also provide many scientific and medical advantages.

The cytoskeleton is a large intracellular structure that provides mechanical support for cells, maintaining their three-dimensional shape and internal structure, allowing them to move and divide. It consists of three major substructures within the cell. These substructures consist of long filamentous proteins (tubulin and actin).

Current techniques for observing the cytoskeleton are difficult to enter into living cells and may be toxic, often with limited resolution and time, as the signal will fade over time. One technique that is commonly used is fluorescence microscopy, in which fluorescent molecules ("probes") attach to cellular structures and then "light up" in contrast to a black background.

The Kai Johnsson research team of EPFL has developed new fluorescent probes that can easily enter living cells, are non-toxic and have long-lasting signals, and most importantly, provide unprecedented image resolution. In 2013, researchers developed a fluorescent molecule called silicon-rhodamine (SiR) that turns on only when it binds to the charged surface of a protein that looks like a protein found on the cytoskeleton. When the SiR is turned on, it emits light of far-infrared wavelengths.

The challenge is how to make SiR specifically bind to cytoskeletal proteins - actin and tubulin. To do this, scientists fused SiR molecules with compounds that bind tubulin or actin. The resulting mixed molecules include a SiR molecule (providing a fluorescent signal) and a natural compound (which can bind to a target protein). One such compound is docetaxel (an anticancer drug that binds tubulin), and another jasplakinolide (actin that specifically binds to the cytoskeleton). These two compounds, used here in very low, non-toxic concentrations, can easily pass through the cell membrane and enter the cell itself.

This probe, called SiR-tubulin and SiR-actin, was used to visualize cytoskeletal dynamics in human skin cells. Because the probe's optical signal is emitted in far-red light, it is easy to separate it from background noise. When a technique called a super-resolution microscope is used, unprecedented high-resolution images are produced.

Another advantage is the utility of the probe. Kai Johnsson said: "You just add them directly to the cells that you are cultivating, and they will be accepted by the cells." The probe also does not require any washing or cell preparation before use, nor does it require any subsequent washing steps. This helps them to maintain their environmental and natural biological stability.

Scientists believe that they can extend this work to other types of proteins and tissues. Johnsson said: "Biologists have been imaging cytoskeletal structures. So far, there is no one probe that allows you to use high-quality live microtubules and microfilament images without using a certain genetic modification. In this work, we have provided two high-performance, high-contrast fluorescent probes that can emit non-phototoxic parts of the spectrum, even for whole blood samples."