Newswise — A team of NIH-funded researchers at the University of Arkansas have demonstrated the novel use of multiphoton microscopy to monitor wound healing in live animals. The scientists measured metabolic changes that occur during healing at the wounds’ surface using autofluorescence imaging. In the future, doctors could use the images to non-invasively diagnose the type of chronic wound and determine the best treatment strategy.

Chronic skin wounds such as diabetic foot ulcers and pressure wounds affect more than 6 million people in the United States, with the cost of treatments mounting to $25 billion each year.  

“Persistent wounds can seriously complicate the health of people with compromised blood circulation or who are immobile for long periods of time,” said Rosemarie Hunziker, Ph.D., director of the program in Tissue Engineering and Regenerative Medicine at the National Institute of Biomedical Imaging and Bioengineering (NIBIB). “What starts as a quality of life issue can rapidly progress to be a major life-threatening medical problem which becomes increasingly difficult to treat due to its complexity.”

Wound healing is a complicated and coordinated process including many cell types. The cells both create and respond to the evolving local environment as the wound heals. Chronic wounds occur when that process fails to progress through the normal healing stages. They are characterized by persistent inflammation, poor blood flow, callus formation, and infection.

There is a lack of non-invasive diagnostic biomarkers to monitor chronic wounds. The current standard of care requires removing a small piece of the wound tissue for laboratory analysis under a microscope, but disturbing tissue around the wound can be disruptive to the healing process.

A team led by Kyle Quinn, Ph.D., assistant professor of Biomedical Engineering at University of Arkansas, has identified a biomarker to track changes in cellular metabolism as wounds move through the healing process. The researchers used multiphoton microscopy to acquire a three-dimensional image of wound structure and its metabolism.

The researchers captured images of wound tissue metabolism using two key molecules in normal cellular metabolism, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). NADH and FAD naturally absorb light from the laser beam of a microscope and thereby emit light, or “auto” fluoresce. Using the emitted light, the researchers measured the concentration of NADH and FAD present in the tissue through a calculation known as the optical redox ratio.

This parameter is especially important since it produces a window into the energy balance available to the cells. “Wound healing is a highly energetic process,” says Hunziker. “So, real time, non-destructive monitoring opens the door to greater overall control.”

“Our long-term goal is to make this clinically useful as a non-invasive diagnostic and monitoring option,” said Quinn. “By shining light on the wound site, we can provide doctors with metabolic and structural information to help guide treatment strategies.”

Because as many as 25 percent of diabetic patients suffer from chronic wounds, the study included both diabetic mice and normal mice, which were controls in the experiment. The researchers recorded images that depicted metabolic activity in wounds over ten days.

As time progressed, the researchers observed metabolic incongruities between the diabetic and normal mice as the wound healed. Due to changes in the optical redox ratio and amount of NADH, Quinn and his team concluded that the dividing cells in the diabetic mice remained at the wound edge and did not migrate over the wound to restore the skin’s protective barrier.

Quinn’s team is looking into other potential biomarkers that could provide doctors with more information on why a wound may not be healing. “Providing quantitative metrics of cell function and wound composition has potential to supplement current standard of care,” said Quinn.

For more about how wounds heal, go to

This research was supported in part by NIBIB (EB 017723) and by the National Institute on Aging (AG 056560), both parts of NIH.

Jones JD, Ramser HE, Woessner AE, Quinn KP. In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing. Communications Biology. 19 Nov 2018.