Behind the scenes: how Leonardo secrets could finally come to light

Coming inside after the bright sunlight of Florence's Piazza della Signoria, away from the spot where the puritanical Savonarola burned to death in 1498 as a finale to his own Bonfire of the Vanities, it was hard to see anything at first. The wide brim of a hardhat did not help either. So, once I had climbed the scaffolding along one side of the grand hall of the Palazzo Vecchio, I had to use the pale light of my mobile phone screen to look into the tiny holes that had been made in the large wall painting.

It was an unconventional tool, but then for many years this mural, the site of one of the deep mysteries of Italian art, has been subjected to a succession of experimental techniques, from fibre optic cameras to x-rays and ultrasound. So far, the authorities have drawn the line at a neutron gun, for fear of collateral radiation.

The theory is that behind the innocuous battle scene by Giorgio Vasari lies a great, lost masterpiece, painted half a century earlier – Leonardo da Vinci's abandoned painting, The Battle of Anghiari.

At an international press conference last week, Professor Maurizio Seracini, the controversial "art diagnostician" who has spent 30 years attempting to uncover what lies behind Vasari's work, announced that he had fresh evidence. New black pigments of paint, plucked from small holes made in existing cracks, matched those in the brown used on Leonardo's Mona Lisa. It was a far from conclusive find, but Seracini, speaking in Florence this weekend, admitted he was elated. "I don't call it a proof, but I was incredibly excited," he said. "If you don't feel that excitement, knowing that you are trying to unveil the secrets of such great men, then don't do this job."

Seracini's high-profile scientific hoopla is not appreciated in every corner of the city, and reaction here to his latest announcement reveals an irritation with the increasing interest in Florence's art detectives; those experts who inhabit the glamorous, intersection between science and art, now known as diagnostics.

With plenty of enduring historical puzzles to solve, as well as a series of fictional riddles courtesy of thriller writers from Dan Brown to Sarah Dunant, the work of the skilled teams that interrogate the stories behind the art of the Renaissance has never looked so alluring. That this trade also allows practitioners to live among the chief exhibits just adds to the appeal.

"Many people want to do this work, but, actually, maintenance is at least as important as these big, exciting investigations. If we do prove the Leonardo is there, what do we next? How do we proceed?" said Andrea Todorow, a heritage conservator.

The caution is repeated by Mauro Matteini, who has helped shape conservation methods in the city over the past 40 years. "We first have to maintain what we know we already have," he said. "We are now working on Giambologna's statue of The Rape of the Sabine Women and we need a way to get it monitored cheaply and regularly, so we can clean it before damage is done. But even then there are many questions. Perhaps we should put up a copy, as we have with Michelangelo's David in the same piazza. But then people want to see the real thing."

Seracini's work, now staunchly supported by the city's young mayor, Matteo Renzi, is sponsored by National Geographic, which has now made a dramatic film trailer to trumpet his findings.

A biomedical engineer by training, Seracini took up the challenge of finding Leonardo's original painting in 1975, when he spotted the teasing phrase "Cerca trova", or "The seeker will find", palely inscribed near the top of Vasari's fresco, apparently as a coded invitation to look behind it.

"This had been the most important work ever conceived by Leonardo," said Seracini. "He was the number one artist of his time being asked to paint inside the very symbol of the Renaissance, so he wanted to break the rules and find a way of painting on a wall like an oil painting – to free himself from all limitations of fresco painting, where you have to work quickly before the plaster dries."

Unfortunately the artist's brave experiment failed. Leonardo could not dry the oily paint he was employing without using burning braziers that damaged the paint.

The original commission was a kind of face-off between two great, rival talents. On the opposite wall Michelangelo had been asked to make his own work, but, after drawing a few templates, he was called away to work on the Sistine Chapel in Rome. In 1563 Vasari was asked to cover the same vast wall space in the Palazzo's council hall.

Many of the unanswered questions that swirl around the lives of Renaissance painters, attracting professional and amateur detectives, share the same starting point: Giorgio Vasari. This painter and writer, who lived between 1511 and 1574, was crucial to understanding the story of the fertile Medici era.

"It was such a buzzing time, with the discovery of new techniques and the rediscovery of the classics, too," said Todorow.

Not only did Vasari paint the mural that may cover Leonardo's missing masterpiece, but he also wrote the key text chronicling the life and work of the talents that went before him. It is Vasari who picked up the thread of an old allegation that a young fresco artist, Andrea del Castagno, had stolen out of his studio one night to kill a rival painter, Domenico Veneziano, by bludgeoning him to death, before returning to calmly pick up his paintbrush. Scholars have since disproved the allegation, pointing out that del Castagno died four years before his supposed victim.

Yet Vasari's rendition of a damning epitaph for the painter rings on down the ages: "Envy flared up in him and led to anger, and hence he killed Domitio [sic] Veneziano in an ambush."

For Daniela Murphy, a conservator working on high-profile projects in Florence, Vasari's word is always worth questioning. "I imagine him a bit like a gossip columnist. He was a bit of a creep," she said.

Nevertheless, Vasari's description of the hidden, unfinished Leonardo battle scene is reliable. His account of Leonardo' impressive horses and a central dramatic struggle to win a battle pennant are backed, not just by other eyewitnesses, but by surviving copies, including one by Rubens.

But there is clear doubt about whether Vasari went on to protect the Leonardo by putting up a wall in front before painting his own battle scene. "He was quite capable of whitewashing over the work of other artists he professed to admire, so why would he be so careful here?" wondered Murphy.

Suggestions that the search for Leonardo's work are a distraction from the pressing work of conservation are dismissed by Seracini. He believes such projects are vital for keeping up public interest in great art. "We need to find new ways to captivate the younger generation," he said. "With these mysteries you get their attention. We don't want them herded around museums like sheep."

If historical intrigue is required, there is no shortage in Florence. Across the hall from the scaffolding and National Geographic screening that cloak Seracini's investigations is the small, panelled chamber where Francesco I de' Medici, the Grand Duke of Tuscany, once quietly experimented with poisons and tinctures, before later, so legend has it, disposing of bodies down a hidden water chute that flowed into the Arno.

The infamously treacherous world of the Medicis is strangely mirrored in the complexities and conflicts that face modern research and restoration in Florence. These days, while it is possible to avoid being bludgeoned, dumped in the river, or burned like Savanorola, it is still a perilous business. Although Seracini proceeds according to protocol, he has been condemned by some art historians for interfering with a prominent artwork on the basis of a hunch.

"You need the stubbornness not to be stopped by endless problems and a sense of self-esteem, too, because you will be crushed by the opposition," he said this weekend.

His wider mission, he explained, is to break down the false barriers he detects between art history and science. "The approaches are very different, but you need an interdisciplinary approach. Half my life I have felt that the art historian simply accepts what someone else has said. Some are afraid of these scientific incursions. They might lose their authority."

Less illustrious conservators and restorers, mainly freelancers, must tout for work from a variety of powerful, overlapping authorities. While the Italian ministry of culture has an overview, Florence is divided up into an administrative grid, on top of which the major landmarks are cared for by a powerful, ancient trust or "opera", with a whiff of freemasonry about them.

What is more, there are only a couple of fully recognised training routes for those who want to get their hands dirty on a Giotto, including a period of study at the venerated Opificio delle Pietre Dure.

Murphy, who has recently written a mystery novel, The Restorer, set in 15th-century Florence and among modern-day conservators, is working on the restoration of the central crucifix that hangs over the altar at the Basilica of Santa Croce. She mischievously suggests that two of the more malign faces featured in an Agnolo Gaddi fresco there show just how little has changed. "They are a banker and the man from the opera. This city is exactly the same today, in so many ways," she said.

Her conservation work has been independently funded by a gift to the Opera di Santa Croce, while the extensive work on the surrounding frescoes, including a wall by Giotto, has been financed by an individual Japanese donor. Before National Geographic came along, Seracini's long struggle to find the hidden Leonardo was also supported by one key sympathetic donor, the philanthropist Loel Guinness.

Murphy applauds Seracini's sense of the spectacle in art. "That is what art is, after all. I am absolutely fascinated by the lives of the fresco painters because, in a way, they were the equivalent of filmmakers like Spielberg now, or perhaps 3D special effects experts. They were interested in wowing the people."

During her own work, the famous frescos in Santa Croce have given up their little mysteries, too.

One painter evidently had a habit of painting long, pretty eyelashes on key figures in the fresco. "I can't help thinking he must have been Agnolo's favourite," she said. "I call him the Barbie-doll painter."

Conservators also found a tiny portrait of an early 20th century restorer, Amedeo Benini, in the foliage at the base of one panel. An egotistical flourish like this would be out of the question now, with the sensible emphasis on monitoring and maintenance.

But, while Murphy's work is careful to preserve the true appearance of age, she can appreciate the charming cheek of Benini and the hi-tech drama of Seracini's science. Leonardo, she believes, as the master inventor and showman, would have approved. Seracini is equally convinced: "I am quite sure this is the way Leonardo would like to be found, to be traced, with the newest technology," he said.