Raman spectroscopy can be utilized for advanced immunological characterization of different immune cells. Macrophages, important immune cells found in almost all tissues, develop different functional phenotypes. Raman spectroscopic fingerprints of cells point to differences in lipid composition and allowed a reliable differentiation of the different phenotypes using statistical models. Raman analysis of mixed cell populations isolated from the small intestine intraepithelial lymphocyte compartment from a mouse model of T cell-deficient mice with and without T cell transfer revealed two major subpopulations: one was made up of larger cells (e.g., epithelial cells or myeloid cells/macrophages), and one revealed typical spectral features of T cells. Quantification of the latter from the Raman data was in excellent agreement with flow cytometry data.

Role of antibodies in the worsening of COVID-19 disease is not obvious and its action in fatality cases is under debate. Our results using micro-Fourier-Transform Infrared reflectance spectroscopy (micro-FTIR) indicated that overall discrimination of healthy and COVID-19 individuals and severity prediction can be sucessfully implemented using for blood serum samples case (specificity =87.5 % ; sensisity = 100%.) Considering the minimal and reagent-free sample preparation procedures combined to fast (few minutes) outcome of FTIR we can state that this technology is very suitable for fast screening of immune response of individuals with COVID-19. It would be an important tool in prospective investigations, helping investigate the physiology of the asymptomatic, oligosymptomatic, or severe individuals and measure the extension of infection dissemination in patients.

We present novel implementations of intelligent Raman spectroscopy for determining infectious pathogens across the cycle of infection including 1) contaminated sources of food and water; 2) culture and drug co-incubation independent diagnosis and susceptibility testing from patient samples, and 3) indirect detection of causative agents in non-sample producing disease conditions leading to negative results in gold standard diagnostics. We span identification of major infectious agents including TB and non-productive pneumonia across liquid, dried and vapor sample conditions. Our approaches push the limits of Raman spectroscopy in addressing various facets of infectious disease burden through creative integration of the optics with chemistry, fluidics and machine learning approaches.

We present a novel method of quantifying nitric oxide (NO) concentration, using ratiometric fluorescent nanosensors, during treatment with room-temperature plasma sources. Plasmas, which are ionized gases, generate copious amounts of reactive species and is widely used for different antimicrobial applications. These nanosensors comprise NO-responsive dye and perovskite nanocrystals encapsulated within hydrophobic biocompatible polymers, silica, and poly-lactic-co-glycolic acid (PLGA). They display dual emission at 510 nm, which is used as reference, and 586 nm, which is sensitive to NO concentration. These nanosensors can be useful for quantifying the NO production from different plasma sources and help with dose optimization.

Necrotizing soft-tissue infections (NSTIs) are aggressive and deadly. A major obstacle to prompt NSTI management is the lack of a definitive diagnostic test, causing treatment delays and increasing mortality. NSTI-affected tissues undergo prominent blood vessel thrombosis invisible to the naked eye that can be rapidly visualized using perfusion-based fluorescence imaging. To validate fluorescence imaging for rapid NSTI detection, we are developing a preclinical mouse model of NSTI using light-producing strains of causative bacteria that have biological equivalence to wild type. Co-registration of our wide-field fluorescence imagery with spectrally distinct, bacterial bioluminescence provides validation of infection presence and spatial extent.

This study assesses the sensitivity of label-free fluorescence lifetime imaging (FLIm) for detecting low-grade gastrointestinal inflammation in mice. A FLIm probe was developed for in vivo colon imaging, enabling complete colon scanning. Using the probe, low-grade inflammation induced by streptomycin was imaged, showing decreased fluorescence lifetime at wavelengths related to metabolic activity, indicating a shift to glycolytic metabolism in inflamed tissue. The potential of validating FLIm maps with spatial transcriptomics was explored. These methodologies provide a basis for further experimentation to establish FLIm as a tool to quantify colon epithelial metabolism over time and its relevance to monitor colorectal inflammatory disease.

Novel dual imaging-antimicrobial photodynamic therapy probe SeNBD-van is a promising alternative to conventional antibiotics in the treatment against gram-positive bacteria. It successfully labels and identifies gram-positive bacteria and is capable of selectively killing such bacteria, even those with existing multi-resistances to antibiotics in as little at 20 min. It maintains its high efficacy even in complex environments such as biofilms and skin models, demonstrating great value and potential as an alternative to current methods of management of infections.

Saccharibacteria Nanosynbacter lyticus type strain TM7x exhibits a remarkably compact genome and an extraordinarily small cell size. This obligate epibiotic parasite forms a symbiotic relationship with its bacterial host, Actinomyces odontolyticus, strain XH001. Due to its limited genome size, TM7x possesses restrained metabolic capacities, predominantly staying outside its host cells to sustain this symbiotic lifestyle. To comprehend the evolutionary strategies of Saccharibacteria, a thorough understanding of the physical interaction between TM7x and XH001 is imperative. Unexpectedly, we serendipitously found the formation of intrabacterial lipid droplets, a feature scarcely observed inside prokaryotic cells. Additionally, the binding with TM7x led to a substantial alteration in the membrane fluidity of the host cells. Employing label-free non-invasive Raman spectroscopy, we unveiled numerous phenotypical differences between the host cells alone and the TM7x/XH001 co-culture, especially in regard to saturated fatty acids.

Conventional imaging techniques target this problem by using specific antibody markers. Although such markers allow decent specificity, they are often limited in the field of application, especially for in vivo use, which limits the potential for clinical translations. In contrast to that, label-free optical technologies, like multiphoton microscopy (MPM), can generate highly resolved 3D images from unstained samples, by exploiting natural optical contrast. Label-free MPM can show epithelial damage and immune infiltration in unstained colon samples. Here, we imaged a mixture of T cells and neutrophils with label-free MPM. In order to obtain ground-truth images, we simultaneously recorded images of a Cd4+ specific fluorescent marker for T cells. A deep neural network was then trained for the segmentation of T cells and neutrophils based on such label-free MPM images. Upon training, this model can then be used to detect both cell types without relying on specific fluorescent markers, that were used to obtain ground truth. In the future, the augmentation of label-free MPM by such computational specificity could have great potential for in vivo endomicroscopy.

Sepsis is responsible for over 50% of hospital deaths. Changes in mitochondrial redox state can be indicative of cellular and organ function. Therefore, a method to evaluate mitochondrial utilization of oxygen continuously is critical. Cytochrome-c oxidase (CCO) is a mitochondrial enzyme that participates in oxidative phosphorylation and interacts with near-infrared light, potentially yielding an optical indicator of cellular metabolism. In this work, we use a computational framework to study the feasibility of utilizing photoplethysmography (PPG) signals for detecting CCO when HbO2 and Hb are present. We use a 3D Monte Carlo model of light absorption and transport in tissue to generate optical readouts in the form of temporal PPG signals corresponding to different physiological states. Furthermore, a machine learning model is trained to predict the CCO redox state from PPG signals containing different CCO and hemoglobin species concentrations.

Escherichia coli is responsible for infections in the community and in hospital settings. Photodynamic inactivation (PDI) is an alternative for the treatment of E. coli infections. However, the internalization of photosensitizers (PS), a key element for PDI efficient performance, is still not fully understood. Here, we evaluated thermal and temporal effects on PS uptake in E. coli suspensions. PS internalization estimates were performed by UV-visible absorption spectroscopy and showed influences of temperature and time in PS internalization. We believe that finding ideal conditions for PS uptake in E. coli can improve the PDI process.

We developed a wearable antimicrobial photodynamic therapy (aPDT) device that can sterilize multidrug-resistant (MDR) wound infections safely and effectively at the point of care. This aPDT platform was developed by leveraging advanced optical device fabrication techniques and evaluated by a combination of in vitro, ex vivo, and in vivo bacterial wound infection models. Throughout all the wound infection models, this aPDT platform is capable to inactivate at least 90% of MDR pathogens in the wound infections, showing potential to generalize the application of aPDT in the clinic for MDR infection prevention and treatment for chronic wounds.

Photodynamic nasal decolonization is a prophylactic technology utilizing a photosensitizer activated by light to induce photochemical reactions, which rapidly and effectively eliminate pathogens without harming human tissue. Eliminating nasal-borne pathogens before surgery significantly reduces risk of infection and pathogen transmission. We report an exploratory study performed in 35 healthy volunteers in which nasal microbial population responses were characterized over time, following photodynamic nasal decolonization (Steriwave®, Ondine Biomedical Inc., Vancouver, BC, Canada). Plate culture and 16s sequencing of nasal swab samples revealed a highly significant reduction in the number of viable bacterial cells and species immediately following treatment. Over time, the microbial population returned to its initial diversity level (24 hours) and quantitative number (48 hours). This is consistent with the design intent for a powerful, non-selective bactericidal modality allowing return to microbiome baseline less than 1-week post-treatment, thereby reducing the potential for long-term microbiome disturbances.

The pandemic SARS-CoV-2 exacerbated the number of intubated patients, because of the worst form of the disease cause respiratory failure. Endotracheal tubes are placed to facilitate gas exchange and intubated patient has a major risk (5-40%) of developing Ventilator Associated Pneumonia, where half of this number happen to die. Functionalization in polymeric materials, associated to photodynamic therapy, is a strategy which has been applied with success to avoid and decontaminated biofilms in medical devices. This study developed a curcumin-functionalized ET by applying photodynamic therapy inactivate different bacterial biofilm species associated with PAV. Cytotoxicity assays hasn’t revealed any toxic effect in mammal cells and the respiratory system model support that the functionalized tube when irradiated can act such as a photodynamic barrier to avoid the infection spread and reach the lungs. In vivo studies were performed in pigs intubated and ventilated for 24h and the animals intubated with the functionalized tube and irradiated showed a significant reduction of biofilm development, without adverse effects in oral, tracheal and pulmonary mucosa.

Bacterial biofilms are a pervasive issue in orthopaedic surgery causing up to 80% of infections. Antimicrobial photodynamic therapy (aPDT) is a proposed technique for eradication of biofilms, clinical translation requires optimization of treatment parameters. This study assessed the effectiveness of three light spectra in activating photosensitizing porphyrins to kill a dual species biofilm of E. Coli and E. faecalis grown in a microfluidic device. Blue-red, amber, or blue-amber-red light sources were compared at either 30J/cm2 or 60J/cm2 doses given to activate endogenously produced porphyrins after one hour incubation with 10 or 20% 5-ALA in a saline solution. Changes in biomass 24 hours after treatment were measured using confocal microscopy and OCT to determine treatment effectiveness.

Transfusion-transmitted infections (TTIs) is a major public health risk for ex vivo platelet concentrates (PCs) stored for transfusion. Current pathogen reduction technologies (PRTs) use chemicals and/or UV light, which are known to impede quality and efficacy of the treated products and potentiate adverse events in transfusion recipients, while mitigating TTIs. Relative to UV light, visible 405-nm violet-blue light is safer and recently studies were initiated to demonstrate the violet-blue light’s potential at a dose of 270 Jcm-2 for reducing microbial contamination in human plasma and platelets, while preserving the survival and recovery functions intact in a SCID mouse model.

Antibiotic resistance has posed a grand and rising threat to the global health. Blue light, specifically 400-430 nm range, has been shown as an attractive antimicrobial alternative considering its drug/agent-free nature, broad-spectrum antimicrobial effect, and no reported resistance. However, its clinical applications have been hampered by several major bottlenecks. Here, we present our translational development towards clinical application of blue light for managing wound infections via innovations in establishing a safe, effective treatment regimen built upon bacteriostatic and long-term illumination strategy (with therapeutical window identified between minimum inhibitory irradiance, MII, and maximum permissible irradiance, MPI), a wearable LED array-based device prototype, an in vivo testbed of free-moving rats with skin wound infections, and its integration with standard wound care procedures. These concepts, devices, safety, and effectiveness have been validated in vitro, ex vivo, and in vivo. This work paves a solid yet encouraging foundation for our follow-up clinical study on contaminated/infected wounds.

Tympanostomy tube otorrhea (TTO) is a frequent complication associated with tympanostomy tube (TT) insertion in young children. Despite the standard treatment using topical antibiotics, TTO is often persistent or recurrent due to biofilm development on TT. In this study, we developed a novel antimicrobial blue light (aBL; 405nm) activatable Optical-TT, which emits aBL from the entire TT surface to kill major otopathogens in biofilms on TT. Our preliminary results showed that the Optical-TT activated by aBL led to over 2.5-log10 CFU reduction in 120 h-old H. influenzae films and 48 h-old P. aeruginosa biofilms formed on TT after 30 J/cm2 aBL and in 48 h-old S. aureus biofilms formed on TT after 60 J/cm2, implying the potential of aBL-activable Optical-TT in the management of TTO. Animal studies using a chinchilla model of TTO are currently underway.

Biofilms often persist in wounds because the extracellular polymeric substances (EPS) can protect embedded bacteria against host phagocytes. Treatment options for wound biofilms are limited due to antibiotic resistance and tolerance. Wound debridement remains a routine practice to disrupt the resilient biofilms by mechanical forces, but it is painful and must be done by well-trained professionals. Blue-light (BL) treatment represents a rising alternative for managing wound infections because it can directly inactivate a number of bacterial species and prevent biofilm formation. However, established biofilms remain recalcitrant to BL-mediated eradication. To enhance BL’s efficacy on mature biofilms, we investigated whether BL could directly and sufficiently disassemble the extracellular matrix when combined with other elements.

Antimicrobial resistance is a global health problem that stems from the indiscriminate use of antibiotics, resulting in the emergence and spread of resistant pathogens. Bacterial persistence is a phenomenon where cells survive prolonged exposure to a bactericidal antibiotic despite being genetically susceptible Photodynamic inactivation (PDI) combines the use of photosensitizers and specific light to generate reactive oxygen species that can inactivate bacteria. Studies have shown that the combination of PDI and antibiotics has synergistic effects in treating infections. The use of PDI combined with antibiotics is a promising approach for the treatment of persistent infections. This work consists of applying doses of bactericidal antibiotics such as amoxicillin in strains of Staphylococcus aureus to evaluate their time-dependent death, isolate and culture persistent cells for application of subdoses of PDI observing their effects on bacteria with the persistence phenotype.

The purpose of this study was to elucidate the bactericidal effect of femtosecond pulsed laser operating at 206 nm or 222 nm wavelength. We experimentally investigated bactericidal capacity against Gram-negative and Gram-positive pathogenic bacteria (Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Methicillin resistant Staphylococcus aureus). The settled bacteria culture on a Luria-Bertani agar plate was irradiated with 206 nm or 222 nm UVC light with irradiation dose of 1, 5 or 25 mJ/cm2. We used a pulse repetition rate (PRR) of 10 kHz during our irradiation. We find that the pulsed far-UVC light with wavelength of 222 nm and 206 nm was significantly germicidal on Gram-negative and Gram-positive bacteria. Both wavelengths were as efficient in killing bugs as 255 nm UVC. Direct absorption of 206 nm or 222 nm UVC irradiation led to a generation of reactive oxygen species.

In the inactivation of pathogens by ultraviolet light, the inactivation rate has been reported to decrease with increasing irradiation dose, commonly known as the tailing effect. In this study, we compared the inactivation effects between CW-LED and femtosecond pulsed laser for developing a new method to suppress such tailing effect. We found that femtosecond pulsed lasers suppressed the tailing effect and inactivated pathogens by more than one order of magnitude in comparison with CW-LED. The results of this study provide a new method for the practical application of bacterial inactivation by ultraviolet light.

Preventing and managing infections is a key aspect of clinical care. UVC light has well-known germicidal properties, but can also cause DNA mutations in human cells. Recently, reduced mutagenic impact was reported in tissue exposed to deep-UV (DUV) light (180 – 230 nm), which remains bactericidal. For future clinical applications, delivering DUV light onto tissue via optical fibres is of interest. We detail a laser direct-write process for inscribing scattering points in silica optical fibre for efficient side-scattering of transmitted DUV light. We report on the germicidal efficacy of the illuminated fibre for inhibiting bacteria growth, determined by in vitro feasibility assays.

This study investigated the potential of an innovative combinational therapy utilizing antimicrobial blue light (aBL) and cold atmospheric plasma (CAP) against Staphylococcus aureus, a clinically challenging gram-positive bacterium. For planktonic S. aureus, sequential treatment (1-3 min CAP followed by 270 J/cm2 aBL) demonstrated an increase of 3- to 6- log10CFU reduction compared to aBL alone, while simultaneous treatment (concurrent 90 J/cm2 aBL and 25 min CAP) led to an increase of 7-log10CFU reduction. For biofilms, a remarkable CFU reduction of 6.5- to 7- log10 was observed for sequential treatment (3-5 min CAP followed by 432 J/cm2 aBL) and a 3-log10CFU reduction for simultaneous treatment (concurrent application 108 J/cm2 aBL and 30 min CAP). These findings highlight the unprecedented efficacy of the aBL + CAP combinational therapy in enhancing the potency against S. aureus, offering a promising approach to overcome antibiotic resistance.

The rise of antibiotic-resistant infectious diseases is becoming an alarming problem for public health to the difficulty of treatment, resulting in a high number of deaths annually. Photodynamic inactivation (PDI) has shown promise as an effective method, which is based on the use of a photosensitize, when activated by light, generates reactive oxygen species (ROS), causing damage to the metabolic and structures of target cells, resulting in their death. This study aims to analyze the action of curcumin acting as a photosensitizer and the effect on the susceptibility of different antibiotics to Staphylococcus aureus resistant to methicillin (MRSA). The experimental groups and the analyzes were established from the central composite planning (10 µM, 10 J/cm²). The results demonstrated that PDI reduces the concentration of antibiotics needed to act against MRSA compared to the results of the control group. Combining both treatments enhances the action against target cells, enabling successful results.

This study investigated the potentiation of KSeCN for both antimicrobial blue light (aBL,405nm) and antimicrobial photodynamic inactivation (aPDI) against different species of Gram-negative bacteria. For planktonic (108CFU/ml) E.coli, with KSeCN treatment (50mM concentration and 60J/cm2 aBL) demonstrated an increase of 4- to 5-log10 CFU reduction compared to aBL alone. E. coli, with KSeCN treatment (50mM,100uM Verteporfin and 20J/cm2 aBL) demonstrated an increase of 4- to 6-log10 CFU reduction compared to aPDI (100uM concentration of Verteporfin and 20J/cm2 aBL, 30min incubation time) alone. K. Pneumoniae and A. Baumannii show similar results. For biofilm (106 CFU/mL), an obvious CFU reduction of 5- to 7-log10 could be seen with KSeCN treatment (50mM and 100J/cm2 aBL) compared to aBL alone. CFU reduction of 6.5- to 8-log10 could be seen with KSeCN treatment (50mM,100uM Verteporfin and 40J/cm2 aBL) compared to aPDI alone (100uM concentration of Verteporfin and 40J/cm2). Confocal results (live/dead) made these findings solid which highlight the KSeCN function to potentiate aBL and aPDI against G-bacteria, offering a novel approach for antibiotic resistance infections.