Name: Investigation of a Blue Light LED Device to Suppress Wound Pathogens Using a Collagen-Based Synthetic Skin Model (Video) | JoVE Journal
Uploaded: 2026-02-24T14:05:00.000+00:00
Duration: 8 min 56 s
Description: Explore phototherapy testing using 405 nm blue light LED against wound pathogens on collagen-based synthetic skin. This protocol enables safe, reproducible antimicrobial evaluation without animal models.

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5 minutes

This study was funded (W24-21) by &#34;Applied Research, Innovation and Entrepreneurship Services&#34; (ARIES), Centennial College, Canada. The microbial supplies and BSL-2 facilities were provided by &#34;Applied Biological and Environmental Sciences (ABES), Centennial College, Canada. The authors appreciate the guidance provided by Andrew Baer and Gillian Goring in FT-IR spectroscopy and the prep room staff for their assistance in media preparation and sterilization.

1. Preparation of synthetic skin samples
A collagen-based synthetic skin model (VITRO-Skin) sheet was cut into rectangular swatches measuring 5.0 cm &#215; 2.5 cm under aseptic conditions in a biosafety cabinet. Each swatch was placed into a sterile Petri dish and stored at 4 &#176;C until use. Prior to inoculation, the samples were equilibrated to room temperature and hydrated in a humidity chamber according to the manufacturer&#39;s instructions to mimic the hydration level of human skin.
2. Preparation of microbial cultures
American Type Culture Collection (ATCC) reference strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Enterobacter aerogenes (ATCC 13048), Pseudomonas aeruginosa (ATCC 27853), Acinetobacter baumannii (ATCC 19606), Klebsiella pneumoniae (ATCC 13883), and Candida albicans (ATCC 10231), were used. Single colonies were inoculated into 5 mL Tryptic Soy Broth (TSB) under aseptic conditions. Bacterial cultures were incubated at 35 &#176;C for 24 h, and fungal cultures were incubated at 22 &#176;C for 5 days. Following incubation, turbidity was adjusted to 0.5 McFarland Standard (approximately 1.5 &#215; 10&#8312; CFU/mL) using sterile Phosphate Buffered Saline (PBS) for antimicrobial assays.
3. Inoculation of synthetic skin models
Each hydrated synthetic skin section was inoculated with 100 &#181;L of microbial suspension and evenly spread using a sterile disposable spreader. The inoculated surfaces were air-dried for 15 min in a sterile chamber under ambient laboratory conditions (22-24 &#176;C, 60%-70% RH). Samples were assigned to three groups: untreated control (Group A), light source (blue light LED, 405 nm) irradiation (Group B), and UV-C LED (265 nm) irradiation (Group C). Figure 1 shows the schematic representation of the experiments in evaluating the antimicrobial efficacy of the light source using the synthetic skin model.
4. Light source configuration and irradiation
A 405 nm blue-light LED device was mounted on an aluminum heat sink to prevent heat accumulation and ensure thermally safe irradiation of synthetic skin swatches (Figure 2). Irradiance was measured at the exact treatment plane using a calibrated optical power meter, with the probe height adjusted to account for sensor thickness. The working irradiance was standardized to 30 mW/cm&#178; by adjusting the throw distance of the LED.
Illumination uniformity across the treatment field was verified by measuring irradiance at 16 evenly spaced points, yielding an average of 30.14 &#177; 0.78 mW/cm&#178; (Figure 3). For baseline comparison of germicidal effects, UV-C treatments were performed using a 265 nm LED source delivering 3 mW/cm&#178; at a 20-cm distance for 2 min.
Fluence was calculated as irradiance &#215; exposure time and corresponded to 27 J/cm&#178; for blue light (30 mW/cm&#178; &#215; 900 s) and 3.6 J/cm&#178; for UV-C.
For antimicrobial assays, hydrated synthetic skin swatches inoculated with microbial suspensions were placed aseptically into sterile Petri dishes (lids removed) and positioned directly under the blue-light LED. Samples assigned to the irradiation group (Group B) were exposed continuously until the target fluence was reached (15 min). The 15-min exposure duration (27 J/cm&#178;) was selected based on preliminary time-kill experiments, which demonstrated consistent antimicrobial efficacy across species while representing a practical exposure duration for potential clinical or point-of-care applications. The temperature of each swatch was monitored every 5 min using a non-contact infrared thermometer to confirm that no significant heating occurred during illumination. Temperature monitoring revealed no measurable increase in surface temperature (&#62;2 &#176;C) during blue-light irradiation, confirming that the observed antimicrobial effects were non-thermal.
5. Antimicrobial efficacy of blue light LED and UV-C against wound pathogens
Following treatment, each synthetic skin swatch was transferred into a sterile 15 mL tube containing 9.0 mL PBS (pH 7.4). The tubes were vortexed gently for 1 min to dislodge surface-adherent microbes. Serial ten-fold dilutions were prepared, and 100 &#181;L aliquots were plated on Tryptic Soy Agar (TSA) for bacteria and Sabouraud Dextrose Agar (SDA) for fungi. Plates were incubated (bacteria at 35 &#176;C for 24 h; fungi at 22 &#176;C for 5 days), and colony-forming units (CFUs) were enumerated. Microbial reduction was quantified using the following equations:
Log Reduction = log10(A) - log10(B)
Percent Reduction = ((A - B)/A) &#215; 100
Where A = CFU count from control; B = CFU count from treated samples.
Negative controls consisted of media-only plates and PBS to verify sterility, while untreated inoculated swatches were used for comparing the treatment efficacy. Positive control plates consisted of inoculated ATCC microbial suspensions to confirm culture viability. All experimental steps were performed under aseptic conditions.
6. Time-dependent antimicrobial efficacy of blue light irradiation
To assess the fluence-dependent effect, blue light exposure durations were set to 2 min, 5 min, 10 min, 15 min, 20 min, and 30 min. Antimicrobial activity was evaluated by CFU enumeration as above, allowing analysis of dose-dependent relationships.
7. Blue light LED efficacy in an aerosolized chamber simulating nosocomial conditions
A customized acrylic aerosol chamber, under controlled environmental conditions, was used to simulate nosocomial airborne exposure (Figure 4). Aerosolized microbial suspensions (~1.5 x 106 CFU/mL) were introduced using a nebulizer, and synthetic skin swatches were exposed for 30 min. The blue light was directed at the swatches throughout exposure (10 &#181;W/cm&#178;, 20 cm distance). In the control experiment, identical exposure conditions were applied, but the BL-LED device remained turned off. Microbial cell densities within the aerosol chamber were monitored using fall-out activity measurements, in which the agar plates were placed inside the chamber prior to exposure. Viable microbial cells adhered to the synthetic skin swatches (2 cm x 2 cm) were quantified using standard plate count analysis as described above. This low irradiance (10 &#181;W/cm&#178;) was selected to mimic real-world ambient lighting conditions in healthcare settings, where continuous high-intensity irradiation is not feasible.
8. Optical transmission and embedded-colony testing
<ol>
	<li>Optical transmission through synthetic skin substrate 
	To quantify blue-light penetration through synthetic skin, irradiance was measured using a calibrated optical power meter positioned at the treatment plane. BL intensity (405 nm, 30 mW/cm&#178;) was recorded with and without hydrated synthetic skin swatches placed horizontally between the LED and the detector. Transmission (%) was calculated as the ratio of transmitted irradiance (with skin) to incident irradiance (no skin). 
	Transmission in aqueous conditions was evaluated using a UV-Viz spectrophotometer. A hydrated synthetic skin strip was inserted vertically into a standard polystyrene cuvette filled with deionized water. The cuvette filled with water alone served as the blank. Absorbance at 405 nm was recorded, and percent transmission was calculated using %T = 10(-A) &#215; 100.</li>
	<li>Functional validation using embedded-colony assay 
	Surface- and embedded-colony microbial reduction assays were performed as a functional correlate of transmission, where swatches were inoculated on one surface but irradiated from the opposite (bottom) side to simulate microbes residing beneath the skin surface. CFUs were quantified as described above in the antimicrobial efficacy section.</li>
</ol>
9. FT-IR spectroscopy for synthetic skin degradation after blue light and UV-C exposure
To assess potential chemical and structural changes to the collagen-based synthetic skin matrix due to light exposure, Fourier Transform Infrared (FT-IR) spectroscopy was performed. Synthetic skin swatches (2 cm &#215; 2 cm) were hydrated per manufacturer instructions, identically to swatches used in the microbial reduction assays. Hydrated samples were exposed to either 405 nm blue light (30 mW/cm&#178;) or 265 nm UV-C (3 mW/cm&#178;) for 15 min (standard exposure, 27 J/cm&#178;) or 60 min (worst-case stress condition). After irradiation, samples were gently blotted to remove surface moisture and immediately analyzed by FT-IR.
10. ATR-FTIR measurement
Spectra were acquired using an IR spectrophotometer equipped with a compatible analysis software. All samples were analyzed using the Attenuated Total Reflectance (ATR) iD7 module. To isolate spectral changes attributable to irradiation, (1) Direct spectral comparison was performed between untreated controls and irradiated samples. (2) Baseline-subtracted comparisons were performed by using the untreated control swatch as the background reference before scanning the irradiated samples, allowing visualization of only the new or altered peaks.
This dual approach reduces topographical noise and improves the detection of subtle changes in amide and fingerprint regions. Hydration level, swatch thickness, and orientation were standardized across all samples to reduce spectral variability. Each exposure condition (control, BL-15 min, BL-60 min, UV-15 min, UV-60 min) was scanned in triplicate.
11. Statistical analysis
All experiments were performed using independent biological triplicates unless otherwise stated. Data are presented as mean &#177; standard deviation (SD) or as mean with 95% confidence intervals (CI), as indicated in the figure legends. Normality of data distribution was assessed using the Shapiro-Wilk test. Pairwise comparisons between two groups (e.g., blue light vs. UV-C, surface vs. embedded colonies) were performed using unpaired two-tailed Student&#39;s t-tests. For comparisons involving more than two conditions or time points, one-way analysis of variance (ANOVA) followed by appropriate post-hoc testing was applied. The specific statistical test used for each comparison is indicated in the corresponding figure legend. A p-value &#60;0.05 was considered statistically significant.

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The authors have no conflicts of interest.

This study demonstrates the significant antimicrobial potential of a 405 nm Blue Light LED (BL-LED) device against a panel of clinically relevant wound pathogens tested on collagen-based synthetic skin models. The BL-LED exposure at a fluence of 27 J/cm&#178; achieved up to a 3.5-log reduction in microbial load (Figure 6), exceeding the antimicrobial performance reported in several earlier studies6,7,17</...

Wound infections pose a persistent challenge and significant burden on the global healthcare system and affect the quality of life of patients. Wound healing is often complicated by microbial infections that delay the closure of cutaneous wounds, resulting in lesions and chronic wounds predominantly in elderly and diabetic populations. Conventional wound management strategies rely on antibiotics, antiseptics, chemicals, and natural bioactive agents that primarily suppress wound-causing organisms. While these traditional methods are effective in speeding up the healing process by reducing inflammatory cells and enhancing the number of fibroblasts1, they are associated with many side effects. Many topical antimicrobials used in wound dressings are generally not skin-friendly and may lead to undesirable effects such as skin dehydration, tissue irritation, and cytotoxic effects on cells required for the wound healing process2. Antimicrobial resistance (AMR), which is widely reported due to the overuse of antimicrobials3, mainly systemic antibiotic use, threatens the long-term viability of existing pharmacological treatments. Considering these limitations of existing clinical practice, the need for safer and sustainable alternatives for wound care management is emphasized, particularly the non-contact and pain-free methods like phototherapy. 
Phototherapy, harnessing the visible spectrum of light, has progressively gained attention as a promising non-invasive and non-pharmacological intervention for various dermatological and therapeutic applications. Its applications extend from general skin rejuvenation4 and cosmetic enhancements to targeted treatments for conditions like acne and anti-aging therapies. Among the visible wavelengths (400-760 nm), blue light, typically ranging from 400 nm to 480 nm, has garnered substantial scientific and clinical interest due to its multifaceted therapeutic potential5. In contrast to ultraviolet (UV) radiation, particularly the UV-C band (100-280 nm), which is widely employed for surface disinfection due to its germicidal properties but carries well-documented risks of DNA damage, carcinogenesis, and skin cancer, blue light is classified as non-ionizing radiation. This fundamental difference renders blue light inherently safer for human tissues5, as it does not possess the energy to directly damage cellular DNA or induce mutagenic effects. 
The antimicrobial activity of blue light has been reported against both Gram-positive6 and Gram-negative7 bacteria. Some clinical trials have been conducted to validate the use of blue light therapy against common infections or disorders8, for example, the suppression of Propionibacterium acne that causes acne vulgaris9, and the treatment for stomach infections10 caused by Helicobacter pylori. However, clinical trials investigating the antimicrobial efficacy of wound infections are not reported. A growing number of recent in vitro studies demonstrate that violet-blue light (400-420 nm) is capable of inactivating multiple ESKAPE pathogens while maintaining minimal cytotoxicity to mammalian cells11. Recently, blue light within the 410-430 nm range has been shown to modulate fibroblast activity and collagen deposition, promote cell proliferation, regulate inflammatory responses, and generate reactive oxygen species (ROS) via endogenous porphyrin excitation that disrupts bacterial cells12, making it a promising candidate for wound-healing applications.
However, the complexity of wound healing, determined by multiple variables including individual variance, type of wounds, and physiological and pre-existing pathological conditions, poses significant challenges for research. Direct in vivo testing of wound therapies remains complicated due to biological variability, ethical considerations, and the complexity of wound environments. Animal models, though commonly used, often fail to accurately represent human skin physiology and are limited by stringent regulatory requirements13. Wound healing studies involving clinical trials are rare and often rarely representative due to small sample size, necessitating large statistical samples, robust controls, and stringent ethical approvals. To overcome these barriers, in vitro models that replicate human skin properties have emerged as effective platforms for initial screening14. Collagen-based synthetic skin systems, such as VITRO-Skin, mimic human skin in pH, moisture retention, and surface texture, and are ideal for testing external interventions under controlled conditions15. Although several studies have shown broad-spectrum antimicrobial effects of blue light, most have used broth cultures, agar plates, or animal models, with limited application to skin-like substrates.
Building upon the demonstrated potential of blue light and the advantages of in vitro models, the current study aims to test the efficacy of a Light Emitting Diode (LED) device operating at a peak wavelength of 405 nm for suppressing common wound pathogens. We introduce a novel evaluation methodology for assessing wound healing efficacy based on a simplified microbiome model applied to collagen-based synthetic skin. It also compares antimicrobial efficacy and the impact on the skin topography of Light Emitting Diode-Generated Blue Light (LED-BL) against germicidal UV-C fluence. Although this method lacks the complexity of biological systems, it adopts a pragmatic approach that allows for highly controlled and reproducible experiments that circumvent the variability and ethical complexities associated with in vivo studies and clinical trials, thereby accelerating the preliminary evaluation and initial screening of similar novel therapeutic interventions. The central innovation of this work lies in applying a collagen-based skin model as a reproducible, controllable platform for evaluating blue light antimicrobial efficacy, accompanied by optical penetration, uniformity, and FTIR-based safety analyses.

<table><tbody><tr><td>&lt;em&gt;Acinetobacter baumannii&amp;nbsp;&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATCC 19606</td><td></td></tr><tr><td>Blue Light - Light Emiiting Diode&amp;nbsp;</td><td>Violumas</td><td>N/A</td><td>BL-LED is a prototype thus does not have a Catalog number&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Candida albicans&amp;nbsp;&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATCC 10231</td><td></td></tr><tr><td>&lt;em&gt;Enterobacter aerogenes&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATCC 13048</td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATCC 25922</td><td></td></tr><tr><td>Fourier Transform InfraRed&amp;nbsp; (FT-IR) Spectrophotometer Nicolet iS5</td><td>Thermo Scientific</td><td>IQLAADGAAGFAHDMAZA</td><td>OMNIC Software is used for Analysis</td></tr><tr><td>FT-IR Nicolet iS5 iD1 Direct Beam Transmission Module (Base Adaptor)</td><td>Thermo Scientific</td><td>IQLAADGAAGFAJAMAYX</td><td></td></tr><tr><td>FT-IR Nicolet iS5 iD7 Diamond Attenuated Internal Refraction Module</td><td>Thermo Scientific</td><td>IQLAADGAAGFAJAMBFN</td><td></td></tr><tr><td>Incubator&amp;nbsp;</td><td>Percival Scientific&amp;nbsp;</td><td>9330.01.06L</td><td></td></tr><tr><td>IR theromometer</td><td>Thermo Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>06-664-254</td><td></td></tr><tr><td>&lt;em&gt;Klebsiella pneumonia&amp;nbsp;&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATCC 13883</td><td></td></tr><tr><td>Phosphate-Buffered Saline</td><td>Thermo Fisher Scientific&amp;nbsp;&amp;nbsp;</td><td>AM9624</td><td>PBS Catalog number (Thermo Fisher Scientific)&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATCC 27853</td><td></td></tr><tr><td>Sabouraud Dextrose Agar</td><td>BD Difco&amp;nbsp;</td><td>DF0109-17-1</td><td>SAB Catalog number (BD Difco)</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&amp;nbsp;&lt;/em&gt;</td><td>ATCC The Global Bioresource Center</td><td>ATCC 25923</td><td></td></tr><tr><td>Tryptic Soy Agar</td><td>BD Difco</td><td>DF0369-17-6</td><td>TSA Catalog number (BD Difco)</td></tr><tr><td>Tryptic Soy Broth</td><td>BD Difco&amp;nbsp;</td><td>DF0370-17-3</td><td>TSB Catalog number (BD Difco)</td></tr><tr><td>UV-LED</td><td>Violumas</td><td>N/A</td><td>UV-LED is a prototype thus does not have a Catalog number&amp;nbsp;</td></tr><tr><td>UV-VIZ Spectrophometer (Genesys 10 UV Scanning)</td><td>Thermo Scientific&amp;nbsp;</td><td>335907P</td><td></td></tr><tr><td>Vitro-Skin</td><td>MEDELINK</td><td>SKU24000</td><td>Vitro-Skin</td></tr></tbody></table>

investigation of a blue light led device to suppress wound pathogens using a collagen-based synthetic skin model

Investigation of a Blue Light LED Device to Suppress Wound Pathogens Using a Collagen-Based Synthetic Skin Model

Conventional wound management is increasingly challenged by the rise of antimicrobial resistance and the cytotoxic side effects of traditional agents. Phototherapy, particularly using blue light (BL), offers a promising non-invasive and non-contact alternative. This study evaluated the antimicrobial efficacy and safety of a 405 nm BL-LED device against a broad spectrum of medically important wound pathogens using a collagen-based synthetic skin model, emphasizing the novelty of applying this platform for light-based antimicrobial testing. This synthetic skin provides a more realistic environment, mimicking the structure and topography of human skin. Light uniformity mapping showed consistent irradiance across a 10 cm diameter (30.14 &#177; 0.78 mW/cm2). The device achieved a substantial fluence-dependent microbial log reduction of up to 3.5 at 27 J/cm2, demonstrating efficacy considerably higher than that reported in similar studies. Its broad-spectrum activity was confirmed against ESKAPE pathogens and Candida albicans, a significant advantage for managing polymicrobial wound infections. A key finding was the differential susceptibility among species, with Klebsiella pneumoniae exhibiting the highest susceptibility while Staphylococcus aureus proved most resistant. This is likely due to variations in microbial envelope structure and photosensitive molecule content, which affect the reactive oxygen species (ROS). Optical transmission experiments and surface versus embedded-colony analyses revealed no statistical significance, suggesting the potential application of BL to inhibit pathogens beneath the skin. Infrared spectroscopy confirmed that BL exposure, even at high fluences (108 J/cm2), caused no detectable chemical or conformational degradation of the collagen matrix, unlike the damaging effects observed with germicidal UV-C control. Finally, a unique evaluation in an aerosol chamber demonstrated that BL treatment reduced microbial deposition on surfaces by over 95%, highlighting its potential for mitigating nosocomial infections. These findings demonstrate the suitability of skin-mimicking substrates as an accessible alternative for evaluating BL in preclinical testing, thereby eliminating the need for animal models.

Antimicrobial efficacy of blue light LED and UV-C against wound pathogens 
The blue light LED device (405 nm), operated at an intensity of 30 mW/cm² for 15 min, demonstrated significant antimicrobial efficacy against all tested wound pathogens on synthetic skin models (Figure 5). Among the tested species, Klebsiella pneumoniae showed the highest susceptibility to blue light, with a mean log reduction of 3.4 in viable cell counts. Conversely, Staphylococcus au...

A 405 nm blue light LED device demonstrates antimicrobial efficacy against a broad range of wound pathogens when evaluated on a collagen-based synthetic skin model. This simplified in vitro approach offers a reproducible and ethically viable alternative for early-phase evaluation of light-based antimicrobial therapies.

Conventional wound management is increasingly challenged by the rise of antimicrobial resistance and the cytotoxic side effects of traditional agents. ...

Our research investigates how a 405-nanometer blue light can safely suppress wound pathogens using a collagen-based synthetic skin model. Most studies use broth cultures, agar plates, or animal models. We introduce realistic, reproducible skin mimicking platform for phototherapy testing.To begin, obtain the desired bacterial and fungal strains. Pick a single isolated colony and aseptically inoculate it into five milliliters of tryptic soy broth. Once all strains are inoculated, incubate the cultures.After the desired incubation time, adjust the turbidity using sterile PBS to the 0.5 McFarland standard for antimicrobial assays. Next, take previously prepared collagen-based hydrated synthetic skin sections and inoculate each section with 100 microliters of microbial suspension. Using a sterile spreader, distribute the suspension evenly across the surface.Then air dry the inoculated samples in a sterile chamber under ambient conditions. Assign the samples to three treatment groups for varying light exposure. For irradiation, mount the blue light LED device onto an aluminum heat sink.Use a calibrated optical power meter to measure irradiance at the exact treatment plane. Adjust the probe height to account for sensor thickness. Now adjust the throw distance of the LED to standardize the working irradiance to 30 milliwatts per square centimeter.Place the inoculated skin swatches from group B aseptically into sterile Petri dishes without lids. Position the dishes directly under the blue light LED and irradiate for 15 minutes to achieve a target fluence of 27 joules per square centimeter. Similarly, irradiate group C under UVC light for two minutes to achieve a target fluence of 3.6 joules per square centimeter.Following irradiation, transfer the treated synthetic skin swatch into a sterile 15-milliliter tube containing nine milliliters of PBS at pH 7.4 and vortex it gently to dislodge surface-adherent microbes. Now prepare tenfold serial dilutions of the suspension. Plate 100 microliters from the selected dilutions onto the agar plates, and incubate the plates.At the end of the incubation period, count the number of colonies and record the number of colony-forming units. Calculate microbial reduction using the presented equations. To simulate nosocomial airborne exposure, use a customized acrylic aerosol chamber under controlled environmental conditions.Place sterile agar plates inside the chamber to monitor microbial fallout activity. Then position the sterile skin swatches inside the chamber. Using a nebulizer, aerosolize two milliliters of microbial suspension into the chamber and expose the swatches to continuous blue light at 10 microwatts per square centimeter from a distance of 20 centimeters for 30 minutes.For the control, repeat identical chamber conditions with the blue light device turned off. Quantify viable counts from the swatches by standard plate count analysis. For FTIR analysis, obtain synthetic skin swatches and hydrate them as per the manufacturer's instructions.Divide the hydrated swatches into four sets on a sterile surface. Expose two sets of swatches to 405-nanometer blue light at an intensity of 30 milliwatts per square centimeter. Expose one set for 15 minutes and the other for 60 minutes.Similarly, expose two sets of swatches to 265-nanometer UVC light at an intensity of three milliwatts per square centimeter, with one set exposed for 15 minutes and the other for 60 minutes. After irradiation, gently blot the samples to remove surface moisture. Analyze the samples using FTIR spectroscopy to assess chemical and structural changes.Configure the FTIR instrument in ATR mode and prepare the crystal surface for sample placement. Place the swatch onto the ATR crystal for spectral acquisition. Perform direct spectral comparisons between untreated control and irradiated samples.Next, use the untreated control swatch as a background reference and scan the irradiated samples to obtain baseline subtracted spectra. The blue light LED device demonstrated significant antimicrobial efficacy against all tested wound pathogens on synthetic skin models. Average log reductions reveal species-dependent differences in susceptibility, with Klebsiella pneumoniae demonstrating the greatest sensitivity to blue light and Staphylococcus aureus showing comparatively lower, though still significant reduction.UVC irradiation achieved higher microbial reductions across all tested species. Blue light and UVC irradiation demonstrated broad spectrum antimicrobial activity against clinically relevant bacterial pathogens, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. Exposure to blue light caused a time-dependent reduction in all three wound pathogens, with Escherichia coli and Candida albicans inactivating rapidly, and Staphylococcus aureus showing a slower progressive decline.Blue light efficacy was further assessed under simulated airborne contamination conditions. Continuous exposure significantly decreased microbial deposition on synthetic skin surfaces relative to untreated controls for every species evaluated. Baseline-corrected ATR-FTIR spectra indicated that UVC treatment reduced the intensity of amide peaks and enhanced carbon-oxygen stretching vibrations, reflecting collagen breakdown.In contrast, blue light exposure did not induce any significant spectral or chemical changes. This method evaluates phototherapy light sources by testing antimicrobial efficacy while preserving collagen integrity and safety on skin-like surfaces. The key challenge in this protocol is maintaining uniform irradiance and accurate fluence control of the light sources to ensure reproducible antimicrobial results.This protocol can be extended to biofilm testing, oxidative stress analysis, next-generation light device optimization, and screening of similar novel therapeutic interventions.

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Source: de Vries, C. R., et.al. A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection. J. Vis. Exp. (2020)This video demonstrates the delayed inoculation of luminescent Pseudomonas aeruginosa into a mouse bearing two full-thickness dorsal skin wounds covered by a transparent film dressing. This facilitates biofilm formation and the establishment of chronic infection. Regular bioluminescence monitoring enables quantification of bacterial burden and tracking of infection...

Developing a Mouse Model of Chronic Pseudomonas aeruginosa Skin Wound Infection 

Source: Wang, Y., et al. In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging. J. Vis. Exp. (2017)This video demonstrates the procedure for treating burn wound infections caused by bioluminescent, drug-resistant bacteria in mice using blue light therapy, followed by bioluminescence imaging to monitor treatment efficacy.

Blue Light Therapy for Drug-Resistant Bacterial Infections in Burn Wounds in a Mouse Model

Source: Hunt, A. M. A., et al. Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model. J. Vis. Exp.(2017).This video demonstrates the procedure for assessing infection clearance and healing progression in a wounded diabetic mouse using bioluminescent and microscopic images.

Bioluminescence Imaging to Track Bacterial Infection and Wound Healing in a Mouse Model

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In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

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Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

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Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

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A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

Explore phototherapy testing using 405 nm blue light LED against wound pathogens on collagen-based synthetic skin. This protocol enables safe, reproducible antimicrobial evaluation without animal models.

Investigation of a Blue Light LED Device to Suppress Wound Pathogens Using a Collagen-Based Synthetic Skin Model

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