Leonardo da Vinci's presumed self portrait has certainly seen better days. Microbiologists often hope to answer key questions - which microbes are present, and what are they doing? - in non-destructive ways. After all, if you’re changing the very system you’re hoping to analyze, how can you be sure that your measurements reflect native conditions? The importance of non-destructive analyses takes on a new dimension when objects of cultural significance are involved. Disruptive techniques won’t merely perturb the natural system, but could destroy a priceless artifact. Leonardo da Vinci’s famous self-portrait, drawn around 1510 using red chalk, is one such work. From the fine, shadowed lines, a wizened man in three-quarter view emerges from the page, casting a solemn stare into the distance. But time has taken a toll: the paper drawing has been infested with “foxing spots” over the centuries, an affliction of reddish-brown spots that has bedeviled collectors and conservationists for years. But what is causing the marks, and could they be removed to preserve this and other works?
Previous researchers have wrestled with this conundrum for decades. A time-series of photographs suggests that the spots were around by 1952, and have not visibly increased in number or intensity since. Some scientists proposed a biological influence, while others favored a chemical one. Subsequent teams in recent years have tried to pinpoint a biological origin of the blemishes, using cotton swabs and a range of media to cultivate the culprit. With little success, researchers were at a loss. Improved sampling and sequencing tools now offer an additional dimension of investigation. Guadalupe Pinar, a senior scientist at the University of Natural Resources and Life Sciences in Vienna, led a team of researchers that recently published molecular and microscopic evidence pointing to prominent fungal involvement. The scientists extracted DNA from the drawing, amplified fungus-specific sequence, and cloned recovered fragments to identify the responsible organisms. These efforts showed a fungal community dominated by several species of the Ascomycota phylum, and, in particular, a previously uncharacterized Acremonium species. Their electron microscopy efforts revealed a zoo of fungal forms: smooth spheres wrapped in filaments, spiky cells congregating on a mysterious particle, and flattened disks with cross-hatched scars. So while biological involvement is clear, Pinar suggests that the foxing spots result from a two-stage, chemically-induced process. First, dust-borne iron particles land randomly on the paper, disrupting the cellulosic structure and generating a nucleation point of damage. With an opening, fungal organisms take hold, burrowing into the paper and waiting out the extended dry, low-nutrient periods by shutting down metabolism. When energy is available, the microbes spit out oxalic acid, which precipitates as calcium oxalate crystals and causes further disruption. For conservationists, the message is mixed. On the one hand, it’s a convincing validation of the idea that future technologies will be better suited to answer difficult questions. If earlier researchers had had their way in 1987, da Vinci’s drawing would have been soaked in ethylene oxide, an intervention that could have caused irreparable damage. On the other hand, modern techniques like microsampling, single-cell genetic analyses, and electron microscopy may be sufficient to address many similar conundrums. Treatment strategies, however, are still far from perfect, but understanding the precise composition of the spots is an important pre-requisite for developing a conservation game plan. Once that is established, work will begin in a race against biology to keep da Vinci from fading further into the centuries-old paper.