Novel Bacterial Auto-fluorescence Imaging Device Can Lead to More Targeted Debridement

Acute wounds heal by progressing through a complex, but orderly, series of physiologic and molecular processes. In contrast, chronic wounds, those that fail to heal within 30 days, are characterized as having stalled in this healing progression due to a variety of systemic and local factors. Such factors include high microbial burden and excessive devitalized tissue.1 Within 48 hours of development, Gram positive bacteria from the environment or the patient’s skin flora can infiltrate an open wound2,3; wound healing becomes potentially compromised once bacteria have invaded.

A crucial component of wound management is regular debridement.4 The goal of debridement is to remove all necrotic, fibrous, and devitalized tissue from the wound bed, control infection, and establish a balanced healing environment.4,5 Devitalized tissue in wounds produces a physical barrier to the formation of new tissue and therefore decreases healing rates. If devitalized tissue remains in the wound bed, there is an increase in concealed dead spaces, which can make bacterial colonization in and around the wound more likely. Standard of care remains that unhealthy tissue be sharply debrided to bleeding tissue to: (1) allow for visualization of the extent of the ulcer; (2) detect underlying exposed structures, deep bacterial contamination, or abscesses; and (3) assess the quality of the periwound tissue. Frequent and thorough debridement reduces bacterial bioburden.5 In some cases, although the debridement adequately removes devitalized tissue, the remaining wound bacteria may become problematic.

Bioburden is an all-encompassing term that includes necrotic material, nonviable tissue, wound exudate, and bacteria and other microbes (e.g., fungi). Bioburden tends to accumulate continually in chronic wounds as a result of the underlying pathogenic abnormality caused by systemic conditions such as diabetes or venous disease. The inability to fully resolve these fundamental physiologic issues makes chronic wound bed management with aggressive and complete debridement even more crucial. A point-of-care auto-fluorescence imaging system to aid in bacterial-targeted debridement and bioburden management in the treatment of chronic wounds could prove to be an extremely useful tool to improve patient outcomes.

A novel advancement in wound imaging called the MolecuLight i:X (manufactured by MolecuLight, Inc., Toronto, Ontario, Canada) is now available. This handheld device is an easy-to-use, noninvasive, portable point-of-care fluorescence (Fl) imaging device (Figure 1). The MolecuLight i:X instantly visualizes potentially harmful bacteria on the wound surface and surrounding tissues not otherwise visible with the naked eye. The device emits a violet light (405 nm) that illuminates the wound and surrounding area, exciting the wound tissues and bacteria and resulting in endogenous production of Fl signals3 without the need for additional contrast agents.6 Optical filters built into the device remove noninformative colors, without any digital processing, and one can view the resulting image on the display touch screen in real time.7

The Fl signals (i.e., colors) produced are tissue specific6; endogenous tissue components such as collagen will fluoresce green, while clinically relevant bacteria producing metabolic byproducts like porphyrins and pyoverdine fluoresce red and cyan (blue-green), respectively.8,9 The MolecuLight i:X has been extensively validated in preclinical10 and clinical studies involving patients with chronic wounds.11-15 Clinical trials have shown that endogenous red fluorescent porphyrins emitted from bacteria allow the visualization and location of bacteria present at loads ≥ 104 CFU/g.12

The device has been noted to detect these fluorescent bacterial byproducts on and beneath the surface of wounds, up to ~1.5 mm depth.12 It should be noted that numerous porphyrin-producing bacterial species can colonize on chronic wounds and cause a red fluorescence, but Staphylococcus aureus is the most commonly found bacterial species.11,16 Pyoverdine is unique to Pseudomonas aeruginosa; thus, it is the only bacteria to fluoresce cyan.7,17 The information captured in the images can aid in improved decision making throughout the dynamic wound treatment pathway (e.g., pre-debridement, post-debridement), and in determining the need for antimicrobial therapy.13

The device also contains wound area measurement software that automatically detects the wound border and generates instant, precise wound measurements (wound surface area, length and width). Operators place 2 yellow wound measurement calibration stickers (Figures 2A, 3A, 4A, 5A) in the plane of the wound, 1 on either side, and within the camera’s field of view. A photograph is then taken in the device’s standard imaging mode. By engaging the measure button on the touch-screen, the border is automatically detected and measurements of wound length, width and surface area are displayed on the screen. If preferred, the clinician has the option of using a stylus to outline the wound border manually for measurement.

This case series of 10 patients reports the use of the device’s Fl and wound measurement capabilities to assess wounds and to aid in bacterial-targeted debridement and bacterial burden management in treatment of chronic wounds of the lower extremity.


This case series was observational in nature and no specific inclusion/exclusion criteria were applied to the patients; however, all patients evaluated had an open wound of the lower extremity with a range of chronic wound etiologies, and were > 18 years of age. Patients signed a photo-release consent and were not compensated for participation.

Fluorescence images should always be acquired in a dark environment. A sensor on the device indicated when sufficient darkness for optimal Fl detection was obtained. A rangefinder indicator on the device was used to indicate when the device was at the ideal distance for imaging (8 cm to 12 cm from the wound). The device was used at each patient’s weekly visit to collect wound measurements, standard images, and Fl images to assess wound healing and bacterial burden. Images positive for bacterial Fl (regions of red or cyan) were used to guide debridement to those specific wound regions. Debridement was performed as per standard of care using surgical blade or curette. Data was analyzed to determine the usefulness of the innovative imaging system in ascertaining the effectiveness of debridement, directing the clinical treatment course and influencing the rates of healing in each wound.


Ten patients (5 women, 5 men) were included in this analysis. Wounds had varying etiologies: 4 traumatic wounds, 3 venous leg ulcers, 2 diabetic foot ulcers, and 1 surgical wound. The median patient age was 74.9 years (range, 60-99 years). Wounds were located on the right lower leg (4), the left lower leg (3), the left heel (1), the right heel (1), and the left distal foot/post-transmetatarsal amputation (1). Upon initial presentation to the clinic, the mean ulcer duration was 16.5 weeks (range, 4-32 weeks) with a mean ulcer area of 8.3 cm2 (range, 1.1-35.0 cm2). Using an adaptation of the Wound Infection in Clinical Practice checklist (Table 1),18 all wounds were evaluated as clinically uninfected at the initial assessment visit. All study wounds were considered to display delayed wound healing beyond clinical expectation, but no wounds demonstrated signs of spreading infection. The deepest exposed tissue layer noted on initial presentation was partial thickness (1), subcutaneous (SubQ; 8), and fascia/tendon (1). Most patients had only 1 Fl-directed debridement performed at each visit; a second Fl-guided debridement during a visit was performed on patient 2 at visit 2, patient 4 at visit 2, and patient 6 at visit 1 to remove additional devitalized tissue and red fluorescing material noted within the wound bed.

Case 1

Patient 1 was a 67-year-old woman with a history of trauma to the left lower leg from an automobile accident. The wound duration was 16 weeks at the time of the initial visit. Upon clinical evaluation, there appeared to be no evidence of bacterial contamination or infection. Wound measurement was 1.63 cm2. At visit 1, the wound was noted to have a positive fluorescent signal (Fl+) both in the wound and around the periwound area (Figure 2A, 2B). Debridement did not noticeably change the fluorescent signal at this visit (Figure 2C, 2D). She was treated daily at home with an enzymatic debriding agent and a hydrophobic, bacterial-binding, nonadherent contact layer. The patient was seen weekly in the clinic for evaluation and underwent wound debridement to the SubQ level. At visit 3, it was noted that the wound had a negative fluorescent signature (Fl-) and very scant Fl in the periwound skin (Figure 2E, 2F) and wound size had decreased to 1.16 cm2. However, on visit 5, the periwound area exhibited an alarming red Fl+ signature and the wound measurement increased to 1.40 cm2 (Figure 2G, 2H). This was thought to be early signs of cellulitis, and the patient was placed on doxycycline hyclate (100 mg twice/day for 10 days). At visit 6, the wound size was noted to have decreased to 1.30 cm2 and the fluorescent signal seen previously in the periwound area had resolved (Figure 2I, 2J). It is the author’s opinion that the MolecuLight i:X was able to pick up early cellulitis otherwise not appreciable with the naked eye, allowing for early antibiotic therapy to prevent advancing clinical symptoms and possible need for hospitalization.

Case 2 

Patient 2, a 63-year-old man, presented with a trauma wound to his right lower leg after falling off a ladder. The duration of the wound at presentation was 13 weeks with measurements of 5.10 cm2 and 0.7 cm depth. Clinically, the wound was noted to have a moderate amount of devitalized tissue present, but it did not appear to be overtly infected (Figure 3A). The Fl image showed evidence of red and cyan Fl in and around the wound (Figure 3B). The post-debridement Fl image showed a decrease in cyan Fl but an increase in red (Figure 3C, 3D). He was treated daily at home with an enzymatic debriding agent and a hydrophobic, bacterial-binding, nonadherent contact layer. The patient was seen weekly in the clinic for evaluation and underwent wound debridement to the SubQ level. By visit 5, it is noted that the wound exhibited a marked decrease in red and cyan Fl in the pre-debridement image; the wound size measured 3.77 cm2, and the wound character improved with a decrease in depth to 0.3 cm (Figure 3E, 3F). At visit 5 post-debridement, the wound was Fl- (Figure 3G, 3H).

Case 3

Patient 4 was a 60-year-old woman who presented with a trauma wound of the right lower leg of 28 weeks’ duration. Upon initial presentation, it was noted that the wound (4.76 cm2) had a significant amount of devitalized tissue present without evidence of acute bacterial contamination (Figure 4A). Pre-debridement imaging showed red Fl in and around the wound (Figure 4B). Post-debridement images showed a decrease in the red Fl within the wound, though it still observable red Fl in the periwound area (Figure 4C, 4D). The patient was treated daily at home with an enzymatic debriding agent and a hydrophobic, bacterial-binding, nonadherent contact layer. She was seen weekly in clinic for evaluation and underwent wound debridement to the level of exposed tendon. By visit 3, the wound had decreased in size to 2.21 cm2 and there was decreased evidence of bacterial Fl in the wound and minimal signal seen in the periwound area on the post-debridement Fl image (Figure 4E, 4F). After this visit, the patient was lost to follow-up due to admission into a nursing facility.

Case 4

Patient 6, a 71-year-old woman, presented with a 14-week history of a DFU of the right heel. Upon initial evaluation, the wound measured 1.14 cm2 and was free of acute signs of bacterial infection (Figure 5A). Initial Fl images showed evidence of red and cyan Fl pre-debridement (Figure 5B), with remaining red Fl in the periwound area post-debridement (Figure 5C, 5D). She was treated with a polyhexamethylene biguanide hydrochloride collagen matrix in the clinic and seen for weekly wound evaluation and SubQ tissue debridement. By visit 4, the wound measurement was 0.15 cm2 (Figure 5E, 5F). Following debridement at this visit, the wound was completely Fl- and the periwound area showed very slight Fl+ (Figure 5G, 5H).


Fluorescence imaging with the MolecuLight i:X is an innovative new addition to the field of wound care. The technology allows providers to obtain real-time detection of Fl from bacteria at loads of ≥ 104 CFU/g.11 Clinical studies have consistently shown that the red and cyan signals seen on the MolecuLight i:X images are predictive of moderate to heavy bacterial loads.13,15 It is the authors’ observation that Fl+ found within the wound decreased with debridement at each visit and over the course of therapy. Fl+ found in the periwound area did not, however, decrease significantly with debridement alone; a secondary antimicrobial dressing or oral antibiotics were used to treat patients in these instances. In the cases examined, it was noted that Fl- images correlated to an overall decrease in wound size over the course of treatment. Integration of Fl imaging also enabled bacterial burden-targeted decision making on tissues to remove during debridement. Furthermore, Fl image analysis allowed for bacterial burden-based decision making regarding wound dressings and therapies. Therapies were selected to treat the wound as well as the periwound area.


Current debridement practices have little effect on the periwound tissue and the bioburden found in this region. Routine clinical observations are unable to detect bacterial load and bioburden in the absence of acute bacterial infection and provide no information on bacterial location. Standard clinical practice may not be sufficient to remove bacterial burden and may leave unacceptable levels of bacteria behind (≥ 104 CFU/g). The goal of wound debridement is to facilitate wound healing. This case series noted, over time, with weekly debridement and Fl-directed therapies, bacterial loads decreased within the wound base and the periwound tissues throughout the course of therapy.

There is potential to improve patient outcomes by incorporating Fl imaging into current clinical pathways. By practicing Fl-guided debridement, regions of bacterial burden can be targeted, helping clinicians determine how much and where to selectively remove tissue. As case 1 demonstrated, Fl imaging can potentially identify very early cellulitis before clinical evidence is noted. Early identification and determination of appropriate therapy via Fl imaging may prevent acute cellulitis, abscess, hospitalization, and/or surgical intervention, thus saving health care dollars. In the future, Fl imaging of chronic wounds may usher in a paradigm shift in the wound care community in which more careful attention is paid to the periwound area and treatments are not directed solely to the wound base.

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PROGETTO ITALIANO IN WOUND CARE 🇮🇹. SITO UFFICIALE DI LESIONI TOUR ®️. DAL 2017 Premio Eccellenze Italiane Assotutela 2020-2021.

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