This PDF file contains the front matter associated with SPIE Proceedings Volume 12627, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

Cutaneous melanoma is one of the most lethal types of skin cancer. Non-invasively distinguishing malignant melanoma from benign tumours has always been a challenge. In this paper, an OCT and co-localized Raman spectroscopic system was built and Raman spectroscopy was used to measure skin samples of suspected melanoma lesions and their surrounding healthy areas non-invasively and in vivo. The Raman spectral signal intensities of the lesions increased at 1320 cm^-1 and 1650 cm^-1, while the content of carotenoids decreased, compared to that of the healthy skin samples. The results of the Shapley analysis values showed that the spectral peaks at 1320 cm^-1 and 1650 cm^-1 had a more significant effect on the differentiation of lesions from normal skin. This result can be used to guide the diagnosis of melanoma based on Raman spectroscopy.

Dermatologists are starting to make use of Computer-Aided Diagnosis based on deep learning algorithms, which can provide them with an objective judgement during evaluation of equivocal lesions. DL algorithms can be trained to classify skin lesions with datasets of diverse nature like traditional RGB, clinical and dermoscopic images, or more experimentally, with images from other modalities, such as multispectral imaging. In this work, we have evaluated and customized the different DL approaches that exist in the state of the art to classify a dataset of +500 images acquired on skin lesions. The images were acquired with a staring multispectral imaging prototype in the visible and near-infrared ranges. The best results were obtained for a customized model VGG-16 that combined 3D convolutional layers, 3D maxpooling layers and dropout regularization, leading to an overall accuracy of 71%.

Real-time physiological monitoring faces inevitable challenges arising from physical activity accompanying body motion and contact pressure variations, disturbing photoplethysmography (PPG) based monitoring. Opto-physiological modelling (OPM) underpins the radiative transfer theorem (RTT) to reveal the essence of light trans-illumination beyond the standard Beer-Lambert law driving PPG technologies. The principles of OPM have been well established through a new multiwavelength optoelectronic patch sensor (mOEPS) that overcomes drawbacks of present PPG sensors caused by gravity, imbalance, skin tone, thermoregulation, and contact force. A protocol engaging six healthy subjects has been implemented to obtain high-quality pulsatile signals using mOEPS system, and corresponding perfusion indices were computed. Comparative results with two selected clinical grade pulse oximetry probes are presented. The outcomes demonstrate the capability of the mOEPS system to provide real-time and any time physiological monitoring across different variations of skin types (I – VI, Fitzpatrick scale). Upcoming mOEPS validation work against gold-standards will be performed to validate a prospective wearable system for clinical grade monitoring and assessment over continuous physiological statuses.

MicroRNAs are small ~22 nucleotide RNA sequences that are guided to the 3’ untranslated region (UTR) of protein-coding target mRNA sequences. One particular microRNA, miR155, plays a remarkable role in the immune system, where it is essential for mounting appropriate immune responses. However, its dysregulation has been identified in multiple inflammatory disorders such as Multiple Sclerosis (MS), arthritis, psoriasis and colitis. More specifically, miR-155 has been found to be elevated in the serum and brain lesions of MS patients. Importantly, therapeutic inhibition of miR-155 can inhibit progression of the MS disease model. One of us has identified that macrophages are major contributor to miR-155 elevation in the MS disease model, whilst its inhibition specifically in macrophages can limit the disease. Here macrophages were isolated from the femur and tibia of wild-type (WT) mice and mice with a knock-out (KO) of the gene regulating miR-155 production, and were cultured in-vitro and stimulated with lipopolysaccharide (LPS) to simulate an immune response. Cells were then prepared for spectral analysis by FTIR imaging with a Perkin-Elmer Spotlight 400 imaging microscope. After pre-processing the dimensionality of spectra were reduced using principal components analysis, kernel-PCA and universal manifold application and projection (UMAP) and classified using a support vector machine algorithm, delivering a classification performance approaching F1~0.89. Spectral features differentiating WT and KO classes were observed across the fingerprint region with no single spectral marker being the sole source of differentiation of the downstream molecular events. This study exemplifies the challenge in spectral discrimination of the complexity of molecular events in ex-vivo models of immune dysregulation.

Heart Failure with Preserved Ejection Fraction (HFpEF) is a complex cardiovascular disease that represents a major clinical challenge. Its development is often the result of the contribution of a wide plethora of severe comorbidities such as obesity, diabetes, and hypertension. The complexity of this clinical picture makes HFpEF’s progression mechanism mostly unknown and its diagnosis challenging and often belated. In this work, by FTIR absorption and Raman scattering techniques, we performed an ex-vivo investigation of the cardiac ventricles of rats to detect biochemical alterations due to the progression of HFpEF and its related comorbidities. In addition, a new sampling technique was adopted (tissue print on a CaF₂ disk) to characterize the extracellular matrix. By the analyses of tissues and tissue prints, FTIR and Raman spectroscopies were shown to be highly sensitive and selective in detecting changes in the chemistry of the heart due to the set of pathological conditions.

Multi-modal spectroscopic analysis of biological systems may offer an improved overall non-invasive biophotonic metric of the status of the system, further enhancing the diagnostic and prognostic capabilities of these technologies. In the present study macrophages were extracted from wild-type mice and mice with a knock-out of the gene regulating miR-155, which has been observed to occur in patients with various autoimmune disorders, including multiple sclerosis (MS). Macrophages were stimulated in-vitro to produce an immune response and were then screened spectroscopically with FTIR and Raman spectroscopy (at 532nm and 660nm). Low, medium and high level data fusion strategies for classification of response to stimulation and miRNA regulation were piloted, using downstream principal components analysis-support vector machine classifiers to test the impact of these strategies on classification performance. These techniques allowed the development of a combined highlevel data-fusion, classification pipeline with a high level of classification accuracy (F1<0.9), with reduced variability in performance. Our proposed spectroscopic assay-data fusion strategy may provide an adjunct to clinical screening and diagnosis of various autoimmune disorders whose aetiology is associated with genetic dysregulation.

Oral cancer (OC) is one of the most common oral malignancies. Despite significant advances in medical devices, the five-year survival rate of OC remains low. Current technologies based on tissue pathology are insufficient to diagnose OC at early stages. Molecular sensitive technique such as optical spectroscopy, on the other hand, has the potential for early-stage diagnostics and non-invasive tissue interrogation. Raman spectroscopy (RS), for instance, is a powerful vibrational spectroscopy that allows highly sensitive detection of low concentration analytes, as well as molecular fingerprints of bio samples to be studied non-invasively. Additionally, higher spatial resolution, narrow peaks, better sensitivity and minimal sample preparation makes RS a potential tool for analysing oral cancer in a clinical setting. In this study, we will validate the potential of Raman spectroscopy (RS) and surface enhanced Raman spectroscopy (SERS) for oral cancer diagnostics. Patients having biopsy and histopathological examination were involved in this study. Ex vivo measurements were performed on saliva specimen using SERS while in-vivo analysis was performed by RS. Integration of in vivo tissue and ex vivo sample analysis could potentially improve early-stage OC detection, and hence the overall survival rate of OC.

Alzheimer’s disease (AD) is characterized by the presence of extracellular deposits of amyloid-beta peptides (known as AD plaques). Its assessment is usually achieved post-mortem, requiring chemical pre-treatment via an antibody or indirect labelling. Label-free imaging techniques, like auto-fluorescence, spontaneous Raman (SpR) and stimulated Raman (SRS) imaging could be performed on tissue in its native state to study the biomolecular composition of AD plaques and contribute to a better understanding of the disease. Here we present imaging results of human brain amyloid core plaques. We show blue and green autofluorescence emission localized at the same plaque position while Raman spectroscopy revealed the presence of carotenoids at the same spot. For identifying the underlying carotenoids, first carotenoid reference spectra in hexane solution and then adsorbed on aggregated Aβ42 peptides were recorded. From the six carotenoids measured, lycopene matched closest with the Raman peak positions observed in the measured AD plaque. Furthermore, we used SRS to investigate the presence of a lipid halo around plaque locations as reported in literature for transgenic AD mice.

Several Raman spectroscopy studies, over the past few decades have demonstrated, its utility as an adjunct screening tool in oral cancer diagnosis. A screening/diagnostic test should mandatorily have high sensitivity to detect minuscule tumour load. Therefore, in this study, role of tumour load on efficacy of Serum Raman Spectroscopy (SRS) in the Hamster Buccal Pouch (HBP) model was evaluated. Serum samples were collected in a longitudinal manner over 14 weeks from DMBA induced oral carcinogenesis in HBP model. The tumour load i.e., number of tumours on the treated HBP, ranged from 1- 8. Raman spectra of sera samples were recorded using Confocal Raman microscope, WITec alpha300R with 532 nm excitation laser (30 mW power) and 600 g/mm grating, in the spectral range 400-4000 cm^-1. Multivariate analyses of averaged serum Raman spectra, generated from pre-processed spectra of each sample which were grouped on basis of tumour load (low and high), in a 5-model system, stratified the distinct phases of oral carcinogenesis (week 0: healthy, week 1-3: inflammation, week 4-7: hyperplasia, week 8-11: dysplasia, and week 12-14: moderate to well-differentiated squamous cell carcinoma). As expected, week 0 symbolising healthy condition clearly distinguished from all other DMBA treated intervals. Misclassifications (biochemical homogeneity) were highest at week interval 1-4 irrespective of the tumour load; majorly with week 0. Another evident observation is after a drop in classification accuracy at weeks 1-4, a gradual increase in classification is noted at later intervals, attributable to progressive DMBA treatments and corresponding development of oral tumours. The findings suggested near equivalent sensitivity for both low and high tumour loads and demonstrate that tumour load has no effect on efficacy of SRS, emphasizing the clinical utility of the technique in oral cancer screening/diagnosis.

Food waste during all stages of the supply chain and at the consumer is an emerging problem. Non-destructive methods to determine the freshness of packaged food products could play an important role in the reduction of this problem. In this study, we developed a chemical sensor, i.e. a sensor foil, capable of detecting amines as food spoilage indicators in reaction vessels and model packaging units by fluorescence spectroscopy. To obtain the foil, a phosphorylated porphyrin was adsorbed to silica and then extruded in polyethylene. The reactivity of the foil was tested with single amines in reaction vessels to demonstrate the behavior under ideal conditions. The sensor foil was applied in model packages containing salmon or cheese to show that fluorescence spectroscopy can be used to detect the emission of spoilage indicators. Lastly, model experiments with cod filets were carried out to obtain data to test the capability of determining the freshness by machine learning algorithms.

The use of light for sterilization is very well known in the scientific literature. However, the recent pandemic outbreak and the antimicrobial resistance question drew attention to this topic: to design new light sources for preventing viral epidemic spread is of utmost importance, as an alternative use of chemicals and drugs. Here we present the preliminary ex vivo studies aiming at verifying the potential of new UVC light sources as barriers to the spread of airborne viruses and bacteria. The emitted light is at very short wavelengths (around 220 nm): optical penetration in biological media is limited to a few micrometers, thus preventing the possible damages to the skin and the cornea; the absorption of RNA/DNA shows a minimum at 230 nm, increasing at shorter wavelengths. In this study we optimized a UVC commercial excimer lamp to design a light barrier. The sterilization efficacy has been tested in vitro in cultured Staphylococcus aureus, Pseudomonas aeruginosa and in Sars-Cov-2. The results point out a strong antimicrobial effect (<99.9% bacteria reduced) at ∼15 mJ/cm² (corresponding to 1 minute treatment time @0.25 mW/cm²). The designed prototype can thus be proposed as a light barrier for preventing contamination, reducing the risks for human beings.

Titanium and its alloys are extensively utilized in dental and medical applications as implant materials. These implants have undergone continuous development and refinement to leverage the unique properties of titanium and its alloys. However, a persistent challenge is the detrimental impact of bacterial colonization on treatment effectiveness, which can potentially lead to implant failure. To address this issue, researchers have explored surface modifications to influence bacterial adhesion and enhance treatment outcomes of titanium dental implants. In this study, titanium metal-based material samples, namely Plain, Line Type 1, and Line Type 2, were subjected to laser cutting technology to achieve 10.95 mm diameters. These samples had equal thickness (0.50 mm height) but varied in terms of shaped lines and line spacing on the titanium plate surfaces. The primary objective of this research was to develop an optimal pattern or structure on the surface of titanium plates that could effectively minimize bacterial adhesion and thereby enhance the longevity of the implants.

Handheld gamma cameras with coded aperture collimators are under investigation for intraoperative imaging in nuclear medicine. Unlike other types of collimators, a coded aperture offers the possibility of 3D imaging of small spherical sources in the near field. However, due to the non-linear system model this option has rarely been investigated. We hypothesize that a deep learning approach is capable of 3D localization of spherical sources with an accuracy that is required for sentinel lymph node biopsy. In order to generate a sufficient amount of training data and densely sample the field of view Monte Carlo simulations are computationally too expensive. Thus, a fast and accurate simulation framework called ConvSim is presented. It computes the detector image from a 3D volume distribution of gamma sources in front of the gamma camera described as a voxel cube. By slice-wise convolutions with the corresponding point spread functions (PSF) a simulation time of few seconds is reached. For spherical sources non-linear near field effects are considered additionally. Comparing the generated detector images of exemplary spherical sources at different distances to Monte Carlo simulations yield a good correspondence with a multi-scale structural similarity index (MS-SSIM) between 0.65 and 0.91. Even though ConvSim entirely ignores the photon energy, the simulation framework serves as a fast and accurate alternative to time-consuming Monte Carlo simulations. For the future, the framework might be useful for investigating new types of coded apertures or the reconstruction of extended sources. ConvSim is available at https://github.com/tomeiss/convsim.

The ‘Translational Biophotonics: Diagnostics and Therapeutic’ sessions are part of the European Conferences on Biomedical Optics, providing a platform for translational research in biomedical optics and biophotonics. This paper complements our invited talk at the conference held in Munich, Germany, June 25-29, 2023. We briefly describe the Longfellow Project as a new model of innovation through collaboration across academia and industry that we have developed at Massachusetts General Hospital Research Institute, Boston, USA, since 2015. We illustrate the implementation of the model by sharing the translation journey of a novel concept in corneal refractive surgery. Invented and currently in development at the Wellman Center for Photomedicine at Massachusetts General Hospital, Boston, USA, in collaboration with international academic institutions and industry partners in Luebeck, Jena, and Munich, Germany, the procedure consists of injecting a viscous filler into a femtosecond laser-created corneal pocket to increase the refractive power thereby minimally invasive treating hyperopia and presbyopia. Our intent is not to provide a full review of the work in the field but to show through one concrete example what it takes to move a discovery in the lab along the translational pathway to clinical practice.

The development of effective cancer therapies is required to improve outcomes in colorectal cancer (CRC) treatment. Photodynamic therapy (PDT) has been proposed as more beneficial over common therapeutic approaches and is currently used to treat numerous cancer indications. PDT relies on a selective photosensitizer (PS) that gets activated by laser light in the presence of molecular oxygen, thereby inducing tumour death. To date, zinc phthalocyanine tetrasulfonate acid (ZnPcS₄) - mediated photodynamic therapy (PDT) has shown strong cytotoxic effects in twodimensional (2D) cell culture. Three-dimensional (3D) structures such as multicellular cancer tumour spheroids (MCTS) are being recognised to accurately mimic similarities to the in vivo tumour tissue architecture. In this study, the cytotoxicity assessment of ZnPcS₄ against MCTS established from human colorectal carcinoma (Caco-2 cell line), under irradiation at 673nm at a fluence of 10 J/cm2 was evaluated. Spheroids were cultured in 96-well non adherent plates, using low attachment technique. Post treatment, MCTS morphology was characterized. Cytotoxic responses on spheroids were assessed post ZnPcS₄-based PDT using lactase dehydrogenase-cytotoxicity assay and quantitative assessment of cell death mechanism using Annexin V/PI Staining protocol was conducted. ZnPcS₄ in combination with laser irradiation exerted a dose dependent increase in LDH level patterns. In addition, an induction of apoptotic cell death mechanism was observed. These findings suggest that ZnPcS4 exhibits considerable potential as a photosensitizer for photodynamic therapy against multicellular cancer tumour spheroids

Melanoma is a dreadful skin malignancy caused by genetic mutations in melanocytes. The inherent unresponsiveness of melanoma cells to conventional therapies leads to uncontrollable tumour growth and alarming fatalities rates. Photodynamic therapy (PDT) is a novel therapeutic option for the eradication of malignant tumours and, compared to traditional therapies, is minimally invasive and exhibits increased efficacy. Currently, most PDT experiments are still conducted on two-dimensional (2-D) monoculture, which may not sufficiently mimic the physiological conditions and the three-dimensional (3-D) architecture of native tumours. Therefore, 3-D cell cultures serve as excellent models to replicate tumour tissue in terms of structural and functional properties. Commercially available A375 melanoma cells were cultivated as monolayers and 3-D tumour spheroids for this study. A375 cells were treated with zinc phthalocyanine tetrasulfonic acid (ZnPcS₄) at varying doses (0.125-20 μM) and photoactivation was achieved using a 673 nm diode laser at a fluency of 10 J/cm². Photoactivated ZnPcS₄ resulted in a dose-dependent reduction in cell proliferation, and increased cytotoxicity as determined by adenosine 5′-triphosphate (ATP), and lactate dehydrogenase (LDH) assay respectively. Morphological changes also confirmed the phototoxic effect of ZnPcS4, while cell death pathways were detected via annexin V-FITC-PI. The half-maximal inhibitory concentration (IC₅₀) of ZnPcS₄-mediated PDT on 3-D tumour spheroids was higher than that of monolayers. In conclusion, 3-D cell cultures are unresponsive to PDT compared to traditional monolayer cell cultures and can therefore provide more realistic data and reduce significant discrepancies between in vitro and in vivo studies for better prediction in clinical responses.

Globally, multidrug resistance (MDR) in breast cancer has become the major cause of morbidity and mortality among women. This study was designed to overcome resistance, reduce dose-dependence in photodynamic therapy (PDT) and evaluate cell death mechanisms induced by green synthesized silver nanoparticles (AgNPs) in combination with pheophorbide-a mediated PDT on superlative, and most architectured three-dimensional (3-D) doxorubicin (DOX) resistant MCF-7 breast cancer cells with overexpressed p-glycoproteins in vitro. In addition to the aforementioned scope, the combination of green NPs with PDT has been reported to yield a good disease prognosis which in most cases is accompanied with manageable adverse effects. Briefly, MDR MCF-7 breast cancer cells were cultured in a 96 well plate to form 3D tumor spheroids and later treated with optimized concentrations of AgNPs and pheophorbide-a in monotherapy. After 24 h treatment, 3-[4,5-dimethylthiazole-2- yl]-2,5- diphenyl tetrazolium bromide (MTT) assay was performed to determine the 50% inhibitory concentration (IC₅₀) for both experimental models. Morphological changes were observed by using an inverted light microscope, viability by MTT assay, and cell death analysis by Annexin VFITC-PI staining. Taken together, the results from this study displayed a dose-dependent decrease in cell viability which was accompanied by significant morphological changes. Furthermore, Annexin V-FITC-PI assay showed apoptosis as the most prominent cell death mechanism induced by PPBa-mediated PDT and AgNPs. Taken together, the findings from the present study highlight the advantages of green nanotechnology in cancer therapy.

Microscope integrated real time 4D MHz-OCT operating at high scanning densities are capable of capturing additional visual contrast resolving depth and tissue. Even within a plain C-scan en-face projection structures are recognizable, that are not visible in a white light camera image. With advanced post processing methods, such as absorption coefficient mapping, and morphological classifiers more information is extracted. Presentation to the user in an intuitive way poses practical challenges that go beyond the implementation of a mere overlay display. We present our microscope integrated high speed 4D OCT imaging system, its clinical study use for in-vivo brain tissue imaging, and user feedback on the presentation methods we developed. In neurosurgery the de-facto standard contrast agents used for visibly highlighting brain tumors are Fluorescin and ALA, both of which come with certain caveats. As part of a clinical study we developed a microscope integrated real time 4D MHz-OCT system, operating as high scanning densities, with the intent of creating visual tissue contrast without the use of such contrast agents. Advanced post processing methods to classify tissue can be derived from static properties such as light absorption and morphology, and from dynamic properties, such as perfusion and elastography. However we also noticed that even in a plain C-scan en-face projection structures of interest could be recognized, that were not visible in the corresponding white light camera image. As part of a clinical study so far we collected data from 20 patients, used it for machine learning based classifiers and developing data presentation modalities for eventual use in a surgical environment. We present the challenges in implementing our microscope integrated high speed 4D OCT imaging system, a selection of the imaging data we collected so far during brain tumor surgeries, and the avenues toward presenting processed data to the surgeon.

Ultrasonic aspirators are commonly used for volume reduction of neurosurgical tumours. Bleeding occurs occasionally during ultrasonic debulking since ultrasonic aspirators do not coagulate affected vessels. Usually bipolar forceps are used for haemostasis, however requiring a change of instrumentation by the surgeon. Thulium laser emitting at a wavelength of 1940 nm in a strong water absorption band are suitable for tissue and blood vessel coagulation with subsequent haemostasis. Therefore, such laser system was combined with an ultrasonic aspirator by adapting the light transmitting multimode fiber tip to the distal tip of the ultrasonic aspirator. The thulium laser showed very good haemostasis during tumour debulking. Instrumental changes to bipolar forceps were reduced, surgeon’s feedbacks were convincingly positive.

This work shows the application of LSCI for mapping the cerebral vessels of a laboratory animal, and also presents the time-frequency processing of the registered signal. Thus, we expand the capabilities of the existing LSCI approach and demonstrate spatial mapping of blood flow fluctuations.

The ability to fabricate plasmonic structures on the distal facet of an optical fiber has led to a diverse range of minimally invasive sensors. However these applications have been hindered by the inherent turbidity of the fiber and complex transmission properties of the nanostructures. We propose to use a wavefront shaping technique to pre-shape light prior to transmission through the nanostructed fiber to control the coupling between the guided modes of the fiber and the plasmonic nanostructures. We show that the sensing resolution of a plasmonic fiber optic can achieve a sub-cellular spatial resolution in biological applications. In this work, a broad range of plasmonic structures are explored as candidates for spatially resolved plasmonic sensing including periodic nanostructures for extraordinary optical transmission and sub-diffraction beam formation as well as nanoislands fabricated by a solid-state dewetting procedure for surface enhanced Raman spectroscopy.

Skin is the largest organ of the body, responsible for thermoregulation and barrier against external agents, including bacteria. Wounds cause loss of skin integrity and can also lead to severe pathologies. For these reasons, efficient treatment of skin wounds is necessary, especially when they become chronic or appear in subjects with comorbidity. Blue LED light photobiomodulation is successfully used in wound management. Although the mechanism of action is still unclear, many studies have been conducted, and some target molecules of blue light are unanimously recognised. Among them, Cytochrome C oxidase is included. For this reason, mitochondria represent a target organelle for blue light radiation. Mitochondria are involved in redox signaling and in maintaining the balance of reactive oxygen species (ROS), essential for several vital functions such as calcium homeostasis. Therefore, studying the effects of blue light on mitochondria may be helpful in identifying a therapeutic dose exploitable in clinical practice. Whit this purpose, primary cultures of human dermal fibroblasts were obtained, and a blue LED light device (410-430 nm in emission, 1 W optical emission power) was used. Three doses of blue light (4, 21, 42 J/cm²) were applied once. Electron microscopy was used to reveal mitochondrial morphology before and after irradiation, while confocal microscopy was used to reveal ROS concentration. Our results demonstrated that blue light stimulates ROS dose-dependently, and mitochondria are subject to morphological changes. Future studies will be devoted to determining whether the change in morphology is also related to changes in function.

The diagnostics depth of tumour during photodynamic diagnosis (PDD) is dependent on the wavelength of the excitation light, as there is the attenuation of light by absorption and scattering. The use of 505 nm excitation to extend the diagnostics depth of deeply located tumour has been reported. However, the fluorescence emission intensity during fluorescence observation of the tumour is affected by the photobleaching of the photosensitizer. The photosensitizer photobleaching is accompanied by the formation of its photoproduct. This study has investigated the potential use of fluorescence emission from protoporphyrin IX (PpIX), followed by the combined use of the fluorescence emission from PpIX and its photoproduct, for the fluorescence observation of deeply located tumours. We have introduced the concept of fluorescence photoswitching, where the excitation of the photoproduct formed during the initial PDD, can lead to fluorescence intensity higher than that obtained by PpIX excitation. An increase in the fluorescence detection intensity and time for tumour detection may be achieved with fluorescence photoswitching. Preliminary experiments on fluorescence photoswitching were performed for PpIX in solution and ex vivo. Fluorescence photoswitching was observed in both forms of PpIX investigated, with a maximum of 94 % initial PpIX intensity obtained with the excitation of the photoproduct. It is expected that a higher fluorescence emission can be attained with the optimisation of the irradiation conditions. Although further investigations are required, this work has demonstrated the potential of fluorescence photoswitching for extending the fluorescence observation time for the PDD of deeply located tumours.

This study contains analysis of existing approaches for detecting photosensitizer-free laser-induced singlet oxygen, including their ability to quantitatively measure the relationship between a dose of laser radiation and the amount of singlet oxygen produced.

Squamous Cell Carcinoma (SCC) is a common type of skin cancer usually detectable in body areas exposed to UV rays of sun such as head, neck and arms. Among the methods that are proposed in order to treat cancer is Photodynamic Therapy (PDT), an innovative therapy. Critical to the effectiveness of the treatment is a chemical molecule, the photosensitizer, which via excitation produces reactive oxygen species that destroy tumor lesions. Silicon phthalocyanines have drawn the attention of the scientific community since the presence of silicon ensures the increased production of reactive oxygen species. However due to their structure, they are insoluble in aqueous solutions. Silicon phthalocyanine dichloride, SiCl₂Pc, is a hydrophobic second-generation photosensitizer showing aggregation in waterbased solutions. In order to overcome hydrophobicity, in this study, the encapsulation of SiCl₂Pc in β-cyclodextrin and hydroxypropyl-β-cyclodextrin using the kneading method was proposed. Their action as photosensitizers was assessed by photophysical, photochemical and in vitro photobiological studies against squamous cell model skin cancer A431. The Dynamic Light Scattering (DLS) method was used to determine their size, polydispersity index and z-potential, while their structural characterization was performed by Infrared Spectroscopy (FT-IR). The results of photodynamic treatment showed that the encapsulation of SiCl₂Pc into cyclodextrins improved its aqueous solubility enhancing its photodynamic action (50% cell viability for β-CD–SiCl₂Pc, 57% for HP-β-CD–SiCl₂Pc and 67% SiCl₂Pc after irradiation for 3 min with 15 mW/cm²).

When transitioning onto cardiopulmonary bypass (CPB) during cardiac surgery, blood flow to the brain is maintained by controlling the CPB flow rate and mean arterial pressure (MAP). CPB flow rates are based on patient body mass, and a MAP target of 60 mmHg is based on clinical experience and guidelines for CPB. However, studies have shown that up to 20% of the population has limited cerebral autoregulation and that conditions such as hypertension can exceed an individual’s autoregulatory limits, leaving room for potential adverse cerebral events. Therefore, maintenance of adequate cerebral blood flow (CBF), oxygen delivery, and metabolism during surgery plays a critical role in reducing the risk of neurological complications. Given its sensitivity to tissue oxygen saturation (StO₂), near-infrared spectroscopy (NIRS) is frequently used for intraoperative neuromonitoring modalities; however, StO₂ is not a direct marker of CBF, or the energy demands of brain tissue. CBF can be measured by diffuse correlation spectroscopy (DCS) and the unique absorption features of cytochrome c oxidase (oxCCO) offers a means of assessing oxygen metabolism. In this study, an in-house built hyperspectral NIRS/DCS system was used to continuously monitor changes in the redox state of oxCCO (ΔoxCCO), StO₂, and CBF in fifteen patients when transitioning onto CPB, with the purpose of evaluating the relationship between MAP on pump and brain blood flow and metabolism. Results demonstrated a nonsignificant ΔoxCCO (-0.13 ± 0.12 μM) in those patients with MAP > 70 mmHg, while a significant decrease in ΔoxCCO (-0.69 ± 0.17 μM) was found for patients for whom their MAP dropped to < 50 mmHg when placed on CPB. These results indicate that ΔoxCCO monitoring has the capability of providing real-time assessment of the effect of MAP on brain health during cardiac surgery, which could help reduce the incidence of cerebral complications.

Separable spectral unmixing designates techniques that allow to decompose spectra as a linear or non-linear combination of spectra of the targets (endmembers) collected. These techniques allow quantitative measurements but several drawbacks limit its use with standard optical devices like RGB cameras. We propose a new method for estimating endmembers and their proportion without calibration of the acquisition device with the analysis of periodic events in the signal. We evaluated the performances of the method for identifying functional brain areas during neurosurgery using RGB imaging. Results were consistent with clinical gold standards. This work can allow a widespread use of spectral imaging in the industrial or medical field.

This study presents an artificial intelligence algorithm to classify the heterogeneity and homogeneity of ex vivo mouse liver tumor phantom using dynamic thermal imaging. The algorithm suggests that the phantom’s temperature response to a 1340 nm collimated narrow laser beam results in a thermal pattern that may help separate tumor tissue from healthy tissue. The algorithm can classify homogeneity and heterogeneity with an accuracy of 98.6%.

In the treatment of various retinal pigment epithelium (RPE) related retinal diseases, selective retina therapy (SRT) is highly demanded, as SRT intends to selectively damage the RPE while sparing the neurosensory retina (NSR) and the choroid. A gentle method for removing diseased host RPE cells is still missing regarding RPE stem cell therapy. Cell therapeutics for age-related macular degeneration are often implanted regardless of host RPE status in the target zone, which may result in RPE multilayering. Here, we study a novel laser for selective large-area RPE removal without damaging the surrounding tissue prior to RPE implantation to promote subretinal integration. Therefore, pigmented rabbit eyes were exposed to laser pulses of 8 μs in duration (wavelength, 532 nm; top-hat beam profile, 223 × 223 μm²). Postirradiation retinal changes were assessed with color fundus photography, fluorescein angiography, indocyanine green angiography, and optical coherence tomography (OCT). Here we present the histological outcome of four animals after laser treatment. Following euthanization, the eyes of the animals were processed for histology, sectioned in 5 μm paraffin sections and stained with hematoxylin and eosin. Particular emphasis was given to an OCT vs light microscopy comparison. Our results reveal that RPE can be removed selectively using laser pulses of 8 µs duration in the green spectral range without damaging the NSR. Therefore, this regime proves to be applicable in the sense of SRT.

Color vision screening tests can give an information about cataract progression. Aim of or study was to observe the color vision sensitivity changes before and one week after cataract surgery using Farnsworth D15 and Hardy Rand and Rittler test. The changes were noted with D15 test, but not with HRR.

The treatment of retinal pigment epithelium (RPE) related diseases, utilizing minimally invasive laser techniques like selective retina therapy (SRT) is highly demanded. However, due to the strong inter- and intraindividual variability of RPE absorption, as well as optical transmission, laser microsurgery requires reliable real-time feedback-controlled dosimetry (RFD) to prevent unwanted retinal overexposure. The formation of microbubbles around the strong absorbing melanosomes inside RPE cells, has been identified as the leading mechanism of RPE cell damage during low microsecond laser pulse exposure. Their formation and collapse cause measurable optoacoustic (OA) transients. In the presented experiment OA transients are compared to fringe-washouts in simultaneously recorded optical coherence tomography (OCT) M-scans directly following RPE laser irradiation. Ex-vivo porcine RPE-choroid-sclera explants were exposed to laser pulses of 8, 12, 16 and 20 μs duration and pulse energies ranging from 15 to 100 μJ (wavelength: 532 nm, exposure area: 120 × 120 μm²). Simultaneously, time-resolved OCT M-scans were recorded (central wavelength: 840 nm, scan rate: 77 kHz). Post irradiation, RPE cell damage was quantified using a calcein-AM viability assay and correlated with OA transients and fringe-washouts in OCT M-scans. The results show that the detection of fringe-washouts in OCT M-scans linearly scales with OA transients and correctly identifies the destruction of RPE cells. Furthermore, the findings indicate that the optical detection is more sensitive for SRT dosimetry than OA, because OCT reacts to fast dynamic changes of the scattering structure, which possibly are related to minute cell collapses yet indiscernible by OA transients.

LC-OCT is a recently developed technology for the diagnosis and monitoring of skin pathologies. 3D LC-OCT images with isotropic cellular resolution allow visualization of skin layers and tissue characterization at the cellular level. A dermoscopic imaging system has been coupled to LC-OCT, allowing localization and coverage of lesions, together with characterization of their margins. Finally, segmentations of the epidermal layers and keratinocyte nuclei performed using artificial intelligence allow diagnosis and follow-up of the treatment of skin pathologies in a non-invasive and quantitative way. These tools could help improve the accuracy of clinical diagnosis, allowing early detection of malignant skin tumors.

Calcium phosphate glasses offer an exclusive combination of optical, bioresorbable, and enhanced thermo-mechanical properties, making them an attractive material for fabricating resorbable biomedical devices. In the present study, we report the in vitro dissolution test of a multimode (MM) phosphate fiber and a hollow fiber in phosphate buffered saline (PBS) solution. The power transmission change with the MM fiber’s invitro dissolution is also presented. Then, using the same respective glass compositions of the MM fiber and hollow fiber, we report the realization of a novel bi-functional microstructured optical fiber with a MM core for light delivery and a microfluidic channel for drug delivery. The multistage fabrication process involves the techniques of extrusion, rod-in-tube, and stack-and-draw. The core was tested for light guidance and the channel for liquid delivery. The proposed approach illustrates the vast potentiality of phosphate glassbased micro-structured fibers that could be used as a theranostic device to be implanted at specific areas inside the body without needing an explant procedure.

Profilometry is the technique that estimates the surface height and tomography of a sample. This technique is crucial to correct the intensity variations and angle changes when optical properties of a sample are estimated and its calculation depends on the surface profile. Two systems have been evaluated in this work: an Optical Coherence Tomography (OCT) system and a Spatial-Frecuency Domain Imaging (Hyperspectral-SFDI) system made up of a rotating mirror hyperspectral camera and a Red-Green-Blue (RGB) projector. The estimation of the height map with the OCT system based on the high contrasted backscattering of the air-sample interface whereas, while with the HSI-SFDI system the Phase Shifting Profilometry (PSP) technique has been implemented employing different frequencies. This work compares both approaches and evaluates the differences between them.

Bladder cancer was the 10th most prevalent cancer worldwide in 2020. Currently, the gold standard for diagnosing bladder cancer is a cystoscopy followed by a transurethral resection of the bladder tumour. The tumour invasion and grade are needed to determine the treatment plan. However, a transurethral resection is an invasive procedure, needs planning and has complication risks. Therefore, finding an alternative option to determine tumour invasion and grade is necessary. That would also enable other treatment options for bladder cancer such as laser fulguration, chemo-resection and active surveillance. Optical Coherence Tomography (OCT) has the potential to aid in the diagnosis of bladder cancer

Chronic venous insufficiency (CVI) ranks among the most common health care issues worldwide. The current diagnosis of CVI is done by clinical examination and duplex ultrasound, which can only detect visible physical changes and deeper vascular structures whereas the superficial cutaneous vasculature cannot be resolved. There is indeed a lack of information that can potentially be extracted from the cutaneous microvasculature of patients affected by CVI. In this work, we designed and applied an optical coherence tomography angiography (OCTA) system, which is customized for lower extremity imaging of patients. Featuring fast imaging speed, large field of view, high spatial resolution, and most importantly non-invasiveness, this OCTA system was successfully applied in CVI and venous leg ulcer patient imaging. Using the OCTA results acquired from a cohort of 27 human subjects, we can clearly distinguish the vascular patterns uniquely associated with various stages of CVI. The findings of this study give an unexplored indicator to the disease of CVI and venous leg ulcer. With more patients to be recruited, we believe that OCTA imaging results for CVI can be used as a powerful tool in CVI screening and diagnosis.

Recently, anticancer treatments were discovered to induce cell senescence other than death, a critical phenotype driving tumor recurrence. This calls for the development of safe, precise, and rapid tools to reveal critical therapy-induced senescence (TIS). Here, we present label-free multimodal nonlinear optical (NLO) microscopy as a powerful technique to spot early TIS. We home-built a microscope including different NLO modalities: Stimulated Raman Scattering (SRS), forward and epi-detected Coherent Anti-Stokes Raman Scattering (CARS and E-CARS), and Two-Photon Excited Fluorescence (TPEF). The infrared laser source outputs synchronized narrowband 780 nm pump pulses and 950-1050 nm tunable Stokes pulses, so to match the CH-stretching region of the Raman spectrum. Thanks to the co-registration of these NLO signals from label-free TIS cells and controls, we unveiled quantitative all-optical traits of early-stage TIS, monitored over 72 hours of treatment. TPEF from metabolic coenzymes combined with E-CARS from cardiolipin and cytochrome C indicated an shrinking of mitochondrial networks. CARS and SRS revealed lipid vesicles accumulation in cytoplasms. Nuclei enlarged irregularly, visualized via subtraction of SRS signals of proteins and lipids, and CARS from deoxyribose. We believe our results will strongly influence anticancer pre-clinical studies and translated clinical applications, constituting a quick, non-invasive, and accurate aid to expose TIS manifestation in tumors.

Chronic persistent middle ear infections (otitis media) can lead to cholesteatoma, an ingrowth of multi-layered keratinizing squamous epithelium, which requires complete removal in a surgical procedure. However, intraoperative detection is difficult and may result in remaining inflammatory tissue, leading to recurrence. Fluorescence lifetime imaging microscopy (FLIM) is a technique to measure and image the fluorescence lifetime (FLT) of fluorescent molecules. To date, there is no commercial system available to monitor FLT of intrinsic fluorophores during middle ear surgery. In this work, we performed FLIM on cryopreserved human middle ear tissue samples: two non-inflammatory biopsy specimens (external auditory canal and middle ear mucosa), otitis media and cholesteatoma. The thin-sliced tissue samples were examined with FLIM using excitation wavelengths of 375 nm and 473 nm. Results showed different FLTs among the various tissue types. Differences in the FLTs were observed in the 500-575 nm emission range with the 473 nm excitation. This could either be related to the presence and amount of enzymes in the cells or also refer to the structural diversity in the human middle ear tissues and the respective content of, for instance, collagen, elastin and keratin. Additional series of experiments are needed for more detailed analyses on the possible sources of the emission signals. These initial measurements should provide an overview of the occurring endogenous autofluorescence and the FLTs of different human middle ear tissue, in order to distinguish tissue types.

The perfusion in cutaneous flap transplants needs to be monitored in order to detect vascular problems as early as possible. This can avoid tissue hypoxia and therefore, necrosis of the transplant. Since free flap transplant failures tend to happen more commonly during surgery than at a later onset, a non-contact real-time imaging device would be most advantageous. As hyperspectral imaging (HSI) is a new emerging modality to asses free flap perfusion contactless, this study aimed to investigate whether perfusion data can be interpreted appropriately using HSI, especially in regard to the individual skin tone. Further factors that might alter these HSI-interpretations, such as aging, BMI, different sexes or smoking habits, were also considered. Therefore, a prospective feasibility study was conducted, including 101 volunteers from whom images were taken on 16 different body sites. Skin pigmentation classification was performed using the Fitzpatrick skin type classification questionnaire and the individual typology angle (ITA) acquired from the images. Perfusion indices provided by the camera software were correlated to the possible influencing factors. The results show that a dark skin tone related to a high amount of melanin may influence the HSI-measurements and thus changes the HSI-derived perfusion indices. In addition, certain physiological influencing factors such as age, BMI and sex alter the tissue composition and qualities, thus showing measurement peculiarities within these groups. In conclusion, hyperspectral imaging can be used for perfusion assessment for people with lighter skin tone levels. Further developments are appreciated especially regarding skin pigmentation and the interpretation of indices of greater skin tone levels.

Uncontrolled therapy depth is the significant challenge for achieving an adequate removal of superficial lesions especially for mucosal tissue in the gastrointestinal (GI) tract. Although various treatment methods are available providing some successful applications, an effective approach that limit thermal damage to the epithelium layer is of great interest to therapeutic endoscopy technology studies. In this study, we aimed to propose an endoscopic approach to limit the depth of laser-induced thermal injury by trapping mucosal tissue in the recessed area by using negative pressure. This study includes a Monte-Carlo-based computer modeling for numerical analysis and a protype design for ex vivo animal model which followed by a histological study to assess thermal tissue damage to investigate proposed approach. The laser wavelength used in the studies is 1.5 µm, while the diameter of the laser beam is 0.4 mm and laser power of 0.4 W. The findings suggest that with further refinement in design and preclinical trials, this approach may hold promise for laser-induced thermal therapy in gastrointestinal (GI) tract interventions.

Brillouin and Raman microspectroscopy (BRamS) is a scattering technique that simultaneously assesses the mechanical and chemical properties of tissues with micrometric resolution. It has gained increasing attention in the biomedical field over the last decade and has been successfully used for both single-cell studies and whole-tissue characterization under physiological and pathological conditions. In addition, it is non-destructive, non-contact, and does not require labeling, offering the potential for future in vivo applications. The close interdependence between morphology, biochemistry, and mechanics is particularly relevant in the case of musculoskeletal tissues, where the complex structure is well-designed to ensure exceptional mechanical performance. The ability of tissues to resist and adapt to the mechanical and chemical stresses to which they are subjected depends to a large extent on maintaining the correct arrangement of all their components, starting from the microscopic level. In several common degenerative diseases, such as osteoarthritis (OA), the tissue architecture is destroyed by inflammatory processes, resulting in a rearrangement of its entire structure, leading to a complete loss of function and, often the need for prosthetic replacement. In this case, the use of minimally invasive techniques to explore the lesions could become a valuable resource for the surgeon in formulating a more precise diagnosis and, therefore, in providing more appropriate treatments. Here we discuss some of the results obtained by our group in characterizing human musculoskeletal tissue and detecting OA lesions in joints using BRamS.

Complete removal of the oral cancer is very crucial and depends upon the early-stage detection and regular screening. Oral squamous cell carcinoma (OSCC) is the main cancer among oral cancer and shows a high fatality rate. Early-stage screening could be possible with the help of optical techniques and can lead to low fatality. Present manuscript deals with two modalities fluorescence microscopy and fluorescence spectroscopy of OSCC, precancerous and normal tissues. A GRIN lens based micro-endoscope is used for the fluorescence microscopic imaging and spectroscopy of the oral cancer tissue and precancerous tissues. Fluorescence technique provides the chemical compositions of normal, precancerous and cancerous tissue as the microenvironment is changed in each case. Fluorescence microscopic imaging is done using micro-endo-spectroscopic system which gives quantitative fluorescence contrast with simultaneous fluorescence spectroscopy. Integration of both modalities in single study shows potentiality for OSCC screening. A significant change in fluorescence contrast of OSCC, precancerous and normal tissue is observed with their adjacent fluorescence spectra. In fluorescence spectra a red-shift is recorded for OSCC, precancerous and normal tissue. Fluorescence imaging gives the statistical quantitative parameters based on the fluorescence contrast and fluorescence spectroscopy gives the information about molecular transitions present in cancerous and normal tissue.

Single photon detection offers enhanced measurement through observation of timing dynamics on the picosecond to nanosecond scale. This has been widely exploited across many fields, including biological or medical technology. However practical application to clinic is often limited due to acquisition limitations of single point detectors combined with practical and cost limitations to employing larger numbers of detectors in combination. However, recent advancements in CMOS single photon avalanche diodes (SPADs) enable massively multiplexed time correlated single photon counting (TCSPC) in a compact format suitable for practical application. This work describes our efforts to employ this technology to enhance a range of technologies, including fibre optic spectroscopy, endoscopic imaging, and widefield clinical imaging. We have demonstrated multiple applications to improve signal to noise in fibre optic probes with this technology. This includes: improved Raman spectroscopy with single fibre optic probes through time resolved separation of unwanted background; better disambiguation of fluorescently labelled bacteria through fluorescent lifetime measurements in both spectroscopic and endoscopic imaging modalities; and fibre optic fluorescence spectroscopy of tissue autofluorescence indicating disease state. Further we detail a time resolved widefield imaging system applied to the observation of diffuse photons transmitted from fibre optic light sources placed deep within tissue (porcine and human cadavers), where transiting photons are observed from outside the body. This technology is now being translated to initial clinical investigation for locating inserted medical devices.

Objective dose control is currently not possible for laser treatments at the retina. Especially in the case of subvisible irradiations, the assessment of the intended temperature effects by the visual control of the doctors is impossible. Due to the large individual differences in light transmission of the eye and absorption at the RPE, for the same laser power the achieved temperature varies at the RPE. Therefore, an opto acoustic technique to measure the temperature of the retina during laser treatment was developed and applied in a clinical study. A conventional 532 nm cw laser was used during a standard treatment. A microcontroller-based control module was optically coupled between the treatment laser and the slit lamp. This control module is able to measure and automatically control the RPE temperature rise in real time at a rate of 3 kHz and regulates the laser power in such a way that a target temperature specified by the physician is reached within several ms and kept constant until the end of the irradiation time of 100 ms. In the clinical study on patients with CSCR, a target temperature of 51°C was set. So far 7 Patients were treated. Target temperature could be reached and kept constant until the end of irradiation time of 100 ms. A wide range of applied laser power (35 to 95 mW) was used and show the need for active control during retinal laser treatment. It was demonstrated that temperature-controlled retinal laser therapy can be applied safely in patients.

Three-dimensional (3D) cultured skin with vascular networks is a useful skin substitute that enables rapid perfusion after grafting. However, the efficiency of the medium supply to thick cultured skin is limited, resulting in a reduction in viability. In this study, we applied photobiomodulation (PBM) to control the viability of 3D skins during cultivation. We compared the effects of PBM with illumination by a light-emitting diode (LED) array at four different peak wavelengths (440 nm, 523 nm, 658 nm and 823 nm); PBM was applied once during cultivation and the viability of the 3D skins was evaluated. The results showed that PBM with 823-nm light significantly improved the viability of the skins, while PBM with other wavelengths was not effective. Based on this result, we applied PBM at 823 nm every 24 hours during cultivation, and we found that the viability of the 3D skins with daily PBM was significantly higher than that with single PBM.

Bringing laser-induced thermal therapy to gastroenterology and accepting it as a traditional method continues to be an essential topic of discussion. This discussion highlights coagulation parameters such as laser power, surface scanning speed, beam diameter, and irradiation duration. In addition, the parameters form a large matrix that must be optimized for successful treatment, including minimal damage to surrounding tissues. In this study, we aimed to propose a guide map representing the results of a simulation algorithm developed to provide information about the coagulation parameters of laser-induced thermal therapy of esophageal mucosal tissue. The simulation algorithm is based on the Monte-Carlo method for light transport in tissue, the time-dependent finite difference method for heat transfer, and the Arrhenius damage integral. This study includes validation experiments performed in ex vivo sheep esophagus, including histological analysis, light microscopy imaging, and block-face scanning electron microscopy investigations. The laser wavelength used in the studies is 1.5 µm, providing an optical penetration depth of around 0.5 mm in soft tissue, while the diameter of the laser beam on the tissue surface is 0.9 mm. The simulation algorithm evaluated the photothermal coagulation area in a tissue model with a volume of 4 x 4 x 4 mm3 for laser power up to 0.5 W and a surface scanning speed range of 0.5 mm/sec to 8 mm/sec. Direct comparison of simulation results with ex vivo studies showed significant overlap in laser energy per unit area for successful mucosal coagulation. The findings suggest that the proposed simulation approach can serve as a complementary guide tool for laser-induced photothermal therapy for superficial treatments and as a ground algorithm for future preclinical and clinical trials.

The main objective of this work is the development of a multispectral illumination and data processing pipeline that enables the visualization of tissue oxygenation in real time for minimally invasive interventions.

A numerical analysis of multispectral transvaginal imaging probe for a rapid diagnosis of cervical cancer is performed. The analysis over the positioning and placement of a circular array of LEDs and camera for balanced illumination over the nonuniform cervical tissue is performed.

Multispectral imaging (MSI) devices are optical diagnostic tools that can be used for the non-invasive monitoring and characterization of various kinds of pathologies, including skin conditions such as wounds and ulcers, due to the capability of such technology to track alterations of structural and physiological parameters (e.g., oxygenation and haemodynamics) from changes in the optical properties of the investigated tissue across a large number of spectral bands. In this work, a novel, compact and transportable MSI device based on spectral scanning and diffuse reflectance imaging is going to be presented. The apparatus is composed of light emitting diodes (LEDs) as light sources and a CMOS camera, making it a very compact, manageable, user-friendly, and cost-effective system. The wavelengths of the LED sources, that are located in the visible-NIR portion of the spectrum, have been specifically selected to target and monitor alterations of oxygenation and haemodynamics that can provide biomarkers of monitoring wound healing in chronic ulcers. The calibration of the MSI system is going to be illustrated, discussing the calibration procedure and results obtained with Monte Carlo-based, digital phantoms and liquid optical phantoms. Both types of phantoms mimic the properties of biological tissues and allow to introduce variations in a controlled manner. The proposed MSI system is also going to be tested on patients affected by chronic skin ulcers in order to assess its efficacy and accuracy.

Vibrational spectroscopy is a powerful probe of molecular structure and its advantages for biomedical and biophysical research, with a special emphasis on proteins, lipids and nucleic acids, are widely recognized in the literature. It is well-known that infrared and Raman spectroscopic techniques are complementary for the structural analysis of any molecule. Although they differ in selection rules, both techniques are rapid, non-destructive and generally do not need special protocols for sample preparation. Fourier-transform infrared (FTIR) microspectroscopy, in particular, allows for fast biochemical imaging of many biological tissues, however, the application of FTIR for the assessment of heart and kidney lesions induced by cardiovascular diseases has been poorly explored.

The paper is aimed at comparing blood microcirculation parameters of conditionally healthy volunteers and patients undergoing rehabilitation after COVID-19 to identify possible blood flow dysregulation that may result from the disease. A system of wearable laser Doppler monitors was used to conduct the study. The study demonstrated an increase in overall oscillatory blood flow activity in the group of patients compared to the control group, with especially pronounced differences in the neurogenic, respiratory and cardiac ranges. It had been shown that optical non-invasive technologies have the potential for further application in the research related to COVID-19.

This study presents a hyperspectral optical imaging system for the diagnosis of infantile hemangiomas. Pilot studies were conducted in the clinic on infants. As the main results, the parameters of blood flow and saturation of the areas with hemangiomas were calculated using a previously developed neural network approach. The results indicate the possibility of using this system to monitor the effectiveness of hemangiomas therapy.

Diagnosis of degenerative collagen-related skin diseases is a complex process that requires histopathological evaluation of various pathological alterations, which in addition are not unambiguous in themselves. Fluorescence spectroscopy has proven to be valuable tool for tissue differentiation, whereas the biomedical application of tissue polarimetry is establishing as a valuable diagnostic modality. In this work we present the evaluation of experimental results of histology tissue slides from three collagen-related skin diseases: psoriasis, lupus and scleroderma, through fluorescence spectroscopy and Muller polarimetry.

The detection of bacterial deoxyribonucleic acid (DNA) can be helpful to quickly identify a bacterium. A rapid SERS detection of bacterial DNA using microwave synthesized silver (Ag) nanoparticles (NPs) was reported. The microwave synthesizer technique offers quick synthesis of Ag NPs providing control pressure and uniform heat. The NPs based SERS can detect the genomic content of Escherichia coli (E. coli) NC_000913.3 and Pseudomonas aeruginosa (P. aeruginosa) NZ_CP012001 up to 50 ng/μL and 40 ng/μL respectively.

Understanding tumors and their microenvironment is essential for successful and accurate disease diagnosis. Hyperspectral imaging in VIS-NIR spectral region was used to image benign and malign skin tumors in the head and neck regions of human volunteers. The images were analyzed using five tissue indices (Dawson’s melanin and corrected erythema indices, Huang’s and Ishimaru’s skin oxygenation indices, and tissue water index) to extract tissue parameters important for understanding tumor physiology and morphology. Two examples are presented, one of a benign papillary nevus, and one of a basal cell carcinoma (BCC). The indices show that the nevus has substantially higher melanin index, whereas the BCC has increased erythema index, both oxygenations, and water index. The indices can help with determination and classification of tumors, and provide information about the processes present in the tumorous and healthy tissue.

This work considers the measurement of FAD fluorescence intensity as a method for the safety, simple and real-time detection of pathological cells and informative value of this approach. FAD participates in essential processes such as fatty acid oxidation, the Krebs cycle and other redox reactions. According to literature, cells in different physiological states have different levels of FAD intensity in green-blue spectrum. Hence, it is highly relevant to determine the physiological state of cells by the difference in FAD signal intensity. The study was realized with skin fibroblasts as a model object. On the first stage of experiments 20-days cells cultured on a pre-marked coverslips were divided into two subgroups on the basis of the autofluorescence signal intensity. The first subgroup included cells with a high autofluorescence signal (presumably senescent or pathological), and the second – cells and low one. During subsequent experiments after 24 hours necrotic cells were analyzed in a culture using Hoechst 33342 and propidium iodide in two subgroups separately. According to the results, over 50% of cells with high autofluorescence intensity were identified as necrotic, that can subsequently be used for early diagnosis of various pathologies states. Thus, this study, with its advantages such as non-invasiveness, high sensitivity and biosafety, shows the possibility of early diagnosis of various diseases by measuring the fluorescence signal of FAD and finding cells with high fluorescence levels, which are mostly necrotic.

Measuring cell growth on adhesive substrates is critical for understanding cell biophysical properties and drug response. Traditional optical techniques have low sensitivity and vary in reliability depending on cell type, while microfluidic technologies rely on cell suspension. In this study, a new platform has been developed that is able to measure the weight and growth of individual cells in real−time. The platform can determine the growth rates of cells in just 10 minutes and map the growth of cell populations in short intervals. It can also identify differences in the growth of different subpopulations within a larger group. The platform was used to study the growth of MCF−7 cells and the impact of two intracellular metabolic processes on cell proliferation. The platform demonstrated the negative effect of serum starvation on cell growth and the role of a particular enzyme, ornithine decarboxylase (ODC), in cell proliferation. It was also able to show the ability of an external factor, putrescine, to rescue cells from the inhibitory effects of low osmolarity. In addition to measuring intracellular processes, the platform can determine the response of cancer cells to drug treatment. It showed the susceptibility of MCF−7 cells to a particular drug, difluoromethylornithine (DFMO), and the ability of a resistant subpopulation to survive in the presence of the drug. The platform’s ability to quickly measure cell growth in small samples makes it a potential tool for both research and clinical use.

The development of rapid diagnostic kits is very critical for the early diagnosis and treatment of infectious diseases. In this study, a lightweight and field-portable biosensor that uses a plasmonic chip based on nanohole arrays integrated into a lens-free imaging framework was presented for label-free virus detection in field settings. A high-efficiency CMOS camera was used in the biosensor platform to observe the diffraction field patterns of nanohole arrays under uniform illumination from a spectrally-tuned LED source, which is specifically configured to excite the plasmonic mode supported by the nanohole arrays. The portable biosensor presented reliable labelfree detection of H1N1 viruses and produced accurate results at medically relevant concentrations. A low-cost and user-friendly sample preparation kit was developed in order to prepare the surface of the plasmonic chip for analyte binding, e.g., virus-antibody binding. A Python-based graphical user interface (GUI) was also developed to make it easy for the user to access the biosensor hardware, capture and process diffraction field images, and present virus information to the end-user. The portable biosensor platform employs nanohole arrays and lens-free imaging for highly sensitive virus detection with an LOD of 10³ TCID₅₀/mL. It is accurate and efficient, making it suitable for diagnostic use in resource-limited settings where access to advanced equipment may be limited. The presented platform technology could quickly adapt to capture and detect other different viral diseases, e.g., COVID-19 or influenza by simply coating the plasmonic chip surface with an antibody possessing affinity to the virus type of interest.

The study of lipid components in human cells is of fundamental importance in many cellular functions, such as cell adhesion and migration, formation of membrane domain, DNA damage response, senescence, ageing autophagy, and apoptosis. For this reason, we investigated the different phospholipids and sphingolipids components of human cells by using Fourier Transform Infrared (FT-IR) spectroscopy that allows lipids detection and their characterization in biological samples. Commercial samples of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), ceramide (Cer), ceramide 1-phosphate (C1P), sphingosine 1-phosphate (S1P) and sphingomyelin (SM) were used for collecting spectra using ATR acquisition mode. The infrared spectra of different lipids show the contribution of various functional groups from hydrocarbon chains and polar head groups. The present analysis of these spectra contributed to a better understanding of the characteristics of infrared spectra of single lipid components that can be considered a preliminary step in the FT-IR characterization of lipids extracts from human cells affected by pathologies or exposed to different external agents.

Efficient point-of-care (POC) molecular diagnostic techniques can be designed using highly selective hybridization of G-quadruplex-based DNA-nanomachine (G-4DNM) with specific analytes that produce readable outputs. We propose the design of G-4DNM that can unwind an analyte’s secondary structures, solving the problem of nucleic acid analytes’ recognition at ambient temperatures. S.aureus is a pathogen responsible for a number of nosocomial infections. For detection, we chose a specific gene that is constitutively expressed. S.aureus was analyzed by G-4DNM equipped with 3 long analyte binding arms to tightly bind and unwind single-stranded analytes. Only when all arms bind the analyte a G-quadruplex (G-4) structure is formed. This structure can form a complex with hemin, which exhibits peroxidase activity. In the presence of the G-4/hemin complex, the chemiluminescence (CL) of luminol molecules, activated by hydrogen peroxide, is enhanced. We evaluated the CL kinetics for several minutes, comparing them with the background signal using a highly sensitive photon counting head. The results indicated that S.aureus was recognized with high selectivity and sensitivity up to attomolar concentrations in a quartz cuvette. Our research provides the basis for rapid and affordable POC diagnostics. In the future, this system may become a full-fledged lab-on-a-chip for the detection of marker-sequenced nucleic acids.

Current medicine might be greatly enhanced by the ability to in vivo control and monitor neurons using opsins/phytochromes expressed in neural cells. The fundamental challenge with non-invasive neural cell activity regulation is a high absorption of visible light into biological tissues. This drawback could be mitigated by the photoconversion of phytochromes in spectral ranges with higher tissue transparency. In this study, we first demonstrated two-photon Pr→Pfr conversion of monomeric phytochrome at 1.2 μm wavelength. We did a comparison of linear and nonlinear conversion of truncated DrBphP bacterial phytochromes. This work provides a structured understanding of the optical properties of the dimer and monomer of phytochrome as well as their potential for use in optogenetics.

Sensing using optical fibers is quite an established technology and is increasingly used in the field of bio-medical sensing applications owing to its small size, light weight, immunity towards electromagnetic interference, biocompatibility, sensitivity, and the ease with which it can be integrated with standard catheters leading to a designated point of inspection. Fiber Bragg gratings (FBGs), due to their ease of multiplexing, inherent sensitivity towards strain, and thereby pressure, can be suitably designed to make a novel pressure sensor for diagnosing and monitoring angiogenesis in brain tumors and for assessing vascular lesions inside coronary arteries. However, standard FBGs have a poor pressure sensitivity of 4pm/MPa (0.5fm/mmHg), which is insufficient to detect a few mmHg blood pressure changes. By utilizing the mechanical properties of modified FBGs with an elastomeric material coating, it is possible to improve the transduction mechanism of effectively translating pressure to strain and increase the resolution and sensitivity by two orders of magnitude (53.4 times) compared to standard FBGs. These modified FBGs could then be used to monitor respective pressure indices, i.e., Intracranial Pressure (ICP) and Instantaneous wave-free Ratio (iFR), by integrating them with catheters or endoscopes and using appropriate signal-processing algorithms. Moreover, a simulation of the modification of the blood vessel flow with respect to the secondary vessel formation is done to study the impact of different blood vessel formations during angiogenesis on pressure, thereby co-relating flow patterns to angiogenesis.

Rare diseases place a high burden on society as they are often associated with significant disability, potential years of life lost, high rate of hospitalization and admission to long-term care, high cost of illness and immense mortality rate. Effective screening approaches followed by verification with genetics analysis would be crucial for early diagnosis and treatment in order to prevent complications and decrease the disease burden. This study proposes development and clinical validation of rare skin diseases early screening System for identification of rare disease groups followed by multimodal spectroscopic dermoscopic evaluation. For image acquisition an imaging prototype was used utilizing 526 nm, 663 nm and 964 nm multispectral LEDs for diffuse reflectance imaging and 405 nm LEDs for autofluorescence excitation, as well as Nuance multispectral imaging camera with spectral range 450 – 950 nm. Spectral reflectance and autofluorescence images were analyzed to determine informative/efficient parameters suitable for identification and diagnostics of rare diseases.

Oral cancers form a significant health burden in developing countries like India. Studies have indicated that early detection of oral cancers can improve prognosis and increase survival rates to up to about 80%. Tobacco is one of the strongest risk factors for oral cancers. Visual inspection, followed by incisional biopsy and histopathology of suspicious lesions discovered during clinical examination is the current gold standard for diagnosis. Due to its invasive nature, tissue biopsy is an unsuitable screening tool for mass populations who are at risk for developing oral cancer. Development of a rapid, non-invasive, label free method which will serve as a screening tool and enable detection of malignant and premalignant lesions in early stages is of immense importance for better disease management. Several minimally invasive Raman spectroscopy-based studies on blood, saliva, urine and exfoliated cells, have demonstrated the utility for the detection of a variety of cancers, including oral cancers by stratifying healthy and oral cancer subjects. Urine has been successfully explored to study a number of diseases including cancers, and its collection is non-invasive and requires no expert medical practitioner. Since tobacco consumption is one of the major etiological factors for oral cancers, development of screening tools which can stratify tobacco habitués and oral cancer subjects is warranted. Such a tool can be used for mass screening and enable early detection of oral cancer. In this study, urine analysis using Raman spectroscopy has been explored to stratify tobacco habitués and oral cancer subjects.

This manuscript analyzes the optical properties of dystrophic mice legs, which have been obtained by Spatial Frequency Domain Imaging (SFDI). We used a custom-built SFDI system with spectrometric capabilities so that wavelength-resolved absorption (μ_a) and scattering (μ′_s) coefficients can be calculated. Samples were measured sequentially at ten different frequencies to find their frequency-dependent diffuse reflectance. Additionally, the Monte Carlo method has been applied to generate a Look-Up Table (LUT) to speed up the estimation of the optical parameters, with the GPU-accelerated version of Monte Carlo for Multi-Layered tissues (MCML), CUDAMCML. We found that the diffuse reflectance (Rd) has a different behavior in terms of the wavelength (λ), which gave rise to different values of µa and μ′_s in terms of λ. Given that the μ_a is related to the chemical composition, the differences in wavelength could be used to quantify the presence of chemical components in the samples and, the μ′_s, which relates to the internal structure, can be utilized to identify dystrophy centers inside the mice leg.

Gingival crevicular fluid (GCF) is an exudate originating in the epithelium lining of the gingival sulcus. GCF analysis can represent a simple and noninvasive tool for monitoring periodontal and bone remodeling during the orthodontic tooth movement (OTM). Gingival crevicular fluid (GCF) samples were investigated by using Fourier transform infrared (FTIR), Raman, and Surface-Enhanced Raman (SERS) micro-spectroscopies. Samples were collected at different stages of orthodontic treatments. Vibrational spectra were acquired on the collected samples for characterizing the biochemical changes that are present during the OTM. The attention was focused on the amide I band region (1500 to 1800 cm^-1) of the spectra acquired by using FTIR, Raman, and SERS spectroscopy. Deconvolution procedures were adopted for analyzing this spectral region by means of Gaussian–Lorentzian functions for infrared spectra and Lorentzian ones for Raman spectra. This analysis evidences the different contributions of subcomponents of the amide I band and their changes. These alterations can be attributed to changes in the secondary structure of GCF proteins or to the formation of amyloid aggregates induced by the mechanical stress associated to orthodontic treatments. The results of the present investigation confirm that vibrational spectroscopies can be usefully employed for monitoring orthodontic treatments.

The interrogation of polarisation state of light is a developing technique in biomedical imaging. As polarised light interacts with structural changes in tissue, it has seen use in differentiation between (pre)cancerous and noncancerous tissue, for example. In biomedical imaging rapid diagnostics using minimally invasive techniques is desirable. Endoscopy is already very prevalent in medicine and therefore miniaturisation of polarimetry systems onto endoscopic platforms is a natural development. Flexibility of such a device allows navigation to more complicated parts of the body. All polarimetric systems consist of a polarisation state generator (PSG) and a polarisation state analyser (PSA) which need to be integrated into such a system. A rigid endoscope capable of imaging a full 4×4 Mueller matrix has been developed by Qi et al. This endoscope achieves the polarisation state generation a rigid rotating sheath. Partial polarimetric endoscopy which captures a 3×3 Mueller matrix has also been demonstrated and is easier to achieve since it does not require quarter wave plates in the generator or analyser. Clancy et al and Qi et al both demonstrate a rigid polarimetric endoscopy using a stereo endoscope and a standard rigid endoscopy, respectively. Integration of polarisation state analyser and generator into the tip has been demonstrated using complex mechanical designs. However questions have been raised regarding the electromagnetic compatibility of such a system due to the presence of motors in the tip. Forward et al present a flexible 3×3 fibre based probe that uses diced polarisers orientated at the horizontal, vertical, and -45 degree positions to generate and acquire the necessary polarisation states. This work presents an imaging probe designed to enable in-vivo polarimetry measurements using a micro camera on the tip as a sensor. A 3×3 Mueller matrix image of crossed linear polarisers, captured using a micro camera is demonstrated. This device demonstrates the potential of micro camera sensors in providing 2-dimensional polarimetry data in a flexible endoscopic system. For a device to be used in a clinical setting it needs to be capable of providing data rapidly when it is needed, as well as being navigable to the target location. A fibre optically illuminated endoscope with micro-camera sensor allows for rapid switching of illumination fibres using backend illumination systems as well as rapid acquisition of data. Optical fibres enable the probe to be rigid or flexible depending on application, and the camera at the tip ensures consistent image quality regardless of application area. An idealised system and its potential future of polarimetry in translational biophotonic devices is also discussed.

Breast cancer has become the most diagnosed cancer globally, replacing lung cancer in 2020, with 2.3 million new cases and an estimated death of 685000 women. It is predicted that by the year of 2040 there would be an increase to around 3 million new cases and 1 million deaths worldwide. This calls for techniques for better and faster diagnosis, and in understanding the different biomarkers and the resulting metabolic alterations aiding the development and progression of the tumour. Obesity is associated with metabolic alterations that have shown to increase the risk of cancer and worsen its prognosis. It is associated with dysfunction of adipose tissue that alter the lipid metabolism resulting in excessive accumulation of adipose tissue at sites other than where they are classically found. Tumour cells depend on their microenvironment for nutrients, oxygen and for proliferation. The tumor microenvironment in breast cancer constitutes adipocytes, fibroblasts, endothelial cells, immune cells, and components of the extracellular matrix. The effects of adipocytes on the tumor prognosis are predominant as the breast is composed of abundant fatty tissue. Hence it is important to investigate this effects on molecular levels for understanding the communication between the adipocytes and the tumoral cells which is supporting the proliferation of the cancer. The current diagnostic technique of cancer includes a three step procedure including imaging (such as MRI, Ultrasound imaging), clinical examination and histopathological assessment of biopsy sample if a lesion is suspected to be malignant. However, histopathological assessment observes the morphologic abnormalities in the sample sections and is limited to provide information on the biochemical alterations likely to occur within the tissue even before the morphology is modified. Vibrational spectroscopy has demonstrated its potential to provide diagnostic information. Additionally, vibrational spectroscopy can facilitate the prediction of the biochemical progression for different diseases in a rapid non-destructive manner. Raman spectroscopy is an inelastic scattering process which has incredible potential in biological sample analysis. This technique is capable of rendering information on the vibrational modes of molecules, thus giving access to the biochemical information needed about the sample of interest. Raman spectroscopy is also less time consuming compared to conventional methods of tissue analysis, because the hassle of sample preparation is minimum or not required. The goal of this project is to study the alterations of lipid metabolic pathways in the tumour microenvironment and the impact of obesity in development and progression of breast cancer using vibrational spectroscopy.

Portable Raman and surface-enhanced Raman scattering spectroscopy (SERS) systems have gained great interest in biomedical science, especially when combined with microfluidics, since it allows the application of this sensitive technology in low-resource settings, providing fast results at a low-cost level. Moreover, these portable systems are also more versatile and user-friendly than microscope-based approaches. In this work, we propose the design of a complete portable SERS equipment for the analysis of biofluids. The system was optimized and tested using the Raman reporter 4- mercaptobenzoic acid (4-MBA) and, as a proof of concept, we performed a calibration curve using another Raman reporter: 1-naphtalenethiol (1NAT), being able to detect concentrations as lower as 2*10^-4 M. The results presented here show the potential of the system to detect different and relevant biomarkers, and even be part of more complex systems like Point-of-care or Lab-on-a-chip devices.

Skin and subcutaneous tumors are common in companion animals, that can be difficult to diagnose and treat. Raman spectroscopy shows high diagnostic accuracy in identifying malignant tumors and benign lipomas in dogs and cats. However, the traditional single-point raster scanning approach is not ideal for a large-field-of-view Raman imaging due to its time-consuming nature when scanning areas larger than a square centimeter. Additionally, focusing the excitation spot can lead to high levels of light fluency (J/cm²), potentially causing damage to tissue biomolecules. Furthermore, the resulting raster-scan image often lacks sufficient spatial resolution to effectively compare it with tissue morphology findings. In this study, we focused on implementing EMCCD camera-based Raman imaging to accurately capture Raman spectral band signatures and overcome autofluorescence interference in veterinary cancer samples ex vivo. By utilizing the tunable band-pass filters set-up, our system enables large-field-of-view imaging of specific Raman bands, such as the 1437 cm^-1 band or 1652 cm^-1 band in biological tissue, proposing a more efficient, accurate and safe approach for Raman imaging in the veterinary field.

We propose an image acquisition and processing pipeline to quantitatively characterize the morphology of the transverse tubular network of left ventricular cardiomyocytes. The approach will be an asset in quantifying the degree of structural order of this network in the context of pathophysiological modifications.