Researchers have developed fluorescent molecules that permit imaging how DNA is packaged inside living cells at unprecedented resolution, and in preserved cells, close to the width of the double helix itself.
The team tested the fluorescent probes on slices of bowel tissue taken from three cancer patients. These are ordinary wax-preserved samples, which is how almost all hospitals store patient biopsies.
In the tumours, the DNA was noticeably looser and more spread out than in the healthy tissue sitting right beside it.
Other studies have found that DNA unpacks steadily as cancer takes hold, and the researchers suggest that how loosely a cell's DNA is folded could eventually serve as a measure of how far a tumour has progressed or how aggressive it is.
Doctors currently examine these types of biopsies by eye, using a staining method more than a century old. The advance, described today in the journal Molecular Cell, raises the possibility that one day they could also look at how DNA occupies three-dimensional space inside cells as an additional clue for diagnosing and treating cancer.
"With the same dye we can do two very different things. In a living cell we can watch DNA moving, which tells us how chromatin, the natural state of DNA in cells, behaves. In a preserved cell we can zoom in until we are almost at the scale of the DNA molecule itself. Combining both approaches helps us see one of the main layers of control in human biology in unprecedented resolution," explains ICREA Research Professor Pia Cosma, senior author of the study.
Every cell in the human body holds two metres of DNA squeezed into a very small space. How tightly it is folded up decides which genes are switched on and which stay off.
Almost all images of DNA folding come from cells that are already dead. Powerful microscope techniques needed to track individual components of DNA in cells typically require treating samples with harsh chemicals and strong laser lights that living cells cannot survive.
A team at the Centre for Genomic Regulation (CRG) in Barcelona, the City University of Hong Kong and the Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences) Southern Medical University, found a new way to overcome this challenge by designing new fluorescent probes.
Called HoTs, the dyes can navigate inside living cells on their own and stick to DNA. The probes are designed to blink intermittently, meaning they flicker on and off like fairy lights. The researchers tested them in living human skin cells and in living HeLa cancer cells grown in the laboratory.
If every dye lit up at once, microscopes would take a blurry picture. By blinking one at a time, an advanced microscope works out exactly where each probe is. After taking thousands of snapshots, a computer programme builds up a picture ten times sharper than conventional microscopes.
"The key challenge was to design fluorophores with the right blinking behaviour for super-resolution imaging," says Professor Hongyan Sun at City University of Hong Kong, co-senior author of the study.
An ordinary microscope blurs anything closer together than about 200 nanometres. The new probes allowed the researchers to use an advanced microscopy technology called STORM to visualise DNA at a resolution of 20 nanometres within living cells.
The team could zoom in closer still using cells killed and preserved with chemicals so that their structures are locked in place. Using a technique called MINFLUX on preserved laboratory cells, they pinpointed single dye molecules to within three billionths of a metre, or 3 nanometres, close to the width of the DNA strand itself.
"The diameter of the DNA double helix is approximately 2 nanometres, so 3 nanometre localisation precision brings us remarkably close to the physical scale of the DNA molecule itself," said Aiping Wang, first author of the study.
The probes also worked in non-human tissues. The team used them on slices of zebrafish eye, an animal that can regrow damaged retina. It is thought that retinal regeneration in these animals involves cells loosening their DNA to become flexible again.
In a separate experiment, the researchers showed that an AI could learn to read these images. They used AINU — short for "AI of the Nucleus" — a program the same team unveiled in 2024 that scans super-resolution pictures of a cell's DNA and picks out patterns far too subtle for the human eye. Trained on the new living-cell images, AINU correctly told skin cells apart from stem cells between 96 and 98 per cent of the time.
The two cell types carry identical DNA but fold it differently, and the AI could see the difference. When AINU was first developed it could only study cells that had been killed and preserved; pairing it with the new dyes means it can now work on living cells. It is the same principle the team hopes could one day be turned to telling cancerous tissue from healthy, or to picking out the most promising stem cells to speed up advances in regenerative medicine.
The fluorescent molecules have their limits. They coat the whole of the DNA rather than picking out one particular gene. The sharpest pictures required preserved cells, not living ones, though the team says it is working on that now in follow-up studies and MINFLUX has the potential to image the probes in living cells.
