Thermal therapies are energy-based interventions that provide a minimally invasive treatment option for cancer patients who may not be surgical candidates. Being these therapies based on the increase of tissue temperature to achieve tumor necrosis, it is important to assure complete tumor destruction and simultaneous safety by minimizing collateral thermal damage. Thus, the availability of accurate tools for monitoring tissue temperature changes during the treatment and its control represents an important clinical need. This work presents an overview of two main optical tools (i.e., fiber optic sensors and hyperspectral imaging) which are under investigation for thermometry purposes in tumors and organs undergoing thermal therapies.

High-resolution optical in vivo and in vitro skin biopsies with subcellular resolution and metabolic information can be provided by multimodal multiphoton tomography. We report on the use of an ultracompact air-chilled 50/80 MHz femtosecond fiber laser for two-photon autofluorescence excitation, generation of higher harmonics, fluorescence lifetime imaging, and confocal reflectance imaging. The 18cm long laser head is placed into a 360° imaging head without requiring an optical arm. The tomograph operates efficiently with a total energy consumption of only 235 watts. This energy efficiency enables portability, allowing operation with batteries even in remote areas, with the added convenience of recharging via foldable flexible photovoltaics. Multiphoton and confocal reflectance images of in vivo human skin are presented. Furthermore, rapid, wide-field ("mosaicking"), and deep skin imaging of label-free fresh skin biopsies in a clinical environment is demonstrated.

Surgical resection of skin cancer implies safety margins delineation: currently, surgeons have no diagnostic aid to narrow or widen such margins if necessary. A promising approach is the use of optical methods, which can be used non-invasively and offer real-time diagnostic assistance.

This study presents the results of classification of autofluorescence (AF) and diffuse reflectance (DR) spectra obtained in vivo on skin Basal Cell Carcinomas (BCC) and Squamous Cell Carcinomas (SCC), Actinic Keratoses (AK) and Healthy skin (H) of 140 patients. The bimodal spectroscopic instrument used in this study uses five LEDs for fluorescence excitation at wavelengths peaks between 365 and 415 nm, and a xenon lamp featuring 350-800 nm emission range to obtain AF and DR spectra for four source-detector distances (from 400 to 1000 μm).

The classification (C vs H, H vs AK) was done by support vector machine, discriminant analysis, and multilayer perceptron. Final accuracy of two-class classification tests for almost all pairs of classes was more than 80%. This study presents a comparison of the performance of these combination of methods with the standard clinical procedure.

One in three people living with diabetes are affected by diabetic foot ulcers (DFUs) which, if left unmonitored and untreated, reduce quality of life, and may lead to foot amputation. Regular foot screening is suggested by relevant national guidelines. Yet, compliance with screening is poor, mostly due to the inconvenience of the screening process itself. In this work, we describe a preliminary version of a simple low-cost device intended for self-monitoring of foot circulation, to identify the areas of poor perfusion expected to be at the root of the formation of DFUs. This device is based on multispectral imaging of the feet in the visible and near-infrared ranges. The device is tested on the hands and feet of a single subject, where impairment of circulation has been simulated through a brief ligature of a finger/two toes. Multispectral images are captured, and a simple machine-learning-based classifier correctly identifies areas of low perfusion on the hand and shows promising data on toes. Albeit the device and the classifier are still susceptible of significant improvement, this is indicative of the fact that the multispectral images contain relevant information on tissue perfusion.

Stereotactic interstitial photodynamic therapy using 5 aminolevulinic acid is a more upcoming approach for the treatment of malignant gliomas, whose treatment is remaining a major challenge in brain tumor therapy. The therapeutic outcome of 16 patients who underwent 5-ALA iPDT for newly diagnosed glioblastomas, are presented. In addition to the basic survival analysis, MRI data was analyzed concerning image changes after iPDT and the possibility to use these changes as prognostic factor for therapy response. Overall the iPDT showed a progression-free survival (PFS) of 16.4 months and an overall survival (OS) of 28.0 months. A PFS longer than 2-years was seen for 43.8% of iPDT patients. In contrast to this complete tumor resection with consecutive chemoradiation shows 8.9% 2-year PFS. Standard MRI-related prognostic factors of the tumor resection like necrosis-tumor ratio, tumor volume and post-treatment contrast enhancement are not useful for iPDT prognosis. This shows that the MRI interpretation has to be different compared to conventional tumor therapy. The survival results showed that iPDT is a potential treatment option especially for tumors, where standard therapy is not possible. Further studies are needed.

Laser Speckle Contrast Imaging is a well-established technique able to produce relative blood flow maps contactless and without using dyes. It relies on the statistical analysis of dynamic speckle images, observed when a coherent light is used to illuminate a medium that contains moving scatterers. The local speckle contrast is related to the movements of the scatterers. Multiple exposure speckle imaging (MESI) is a variant of the technique that takes advantage of multiple exposure data to retrieve more quantitative flow maps by accounting for the unwanted and superimposed contribution of static scatterers. Yet, in MESI, a model is adjusted pixelwise to the experimental data requiring long computation times and an a priori guess on the flow regimes. These issues hindered so far, the translation of MESI to clinical applications though some studies have already demonstrated its potential. Here we propose an alternative method based on Convolutional Neural Networks to analyze MESI data. The proposed CNN architecture has been trained and validated using experimental data acquired on calibrated microfluidics flow phantoms. Then, the trained network was applied to analyze MESI data acquired in vivo in mice brain. In addition to be model-bias-free, we have found that the CNN approach infers flow maps much faster than the classical pixelwise regression approach. This new approach is promising for the clinical translation of MESI.

Diffuse gliomas account for more than fifty percent of primitive brain tumors and are challenging to remove because tumor margins are not distinguishable from healthy tissues to the naked eye. To help neurosurgeon in localizing tumoral areas, 5-ALA induced fluorescence of protoporphyrin IX (PpIX) is currently used through surgical microscopes. Various methods based on single wavelength excitation have been proposed to tackle sensitivity issues. New methods based on multiple excitation wavelengths, aim at improving the expert-based estimation models for detection of the tumoral areas. We previously demonstrated^1,2 using a digital phantom the improvement of classification by our method, which does not have any a priori on other fluorophores. In the present work, we perform the comparison of the separability between healthy and tumoral categories on real clinical data between a state-of-the-art model described in³ and our model.^1,2 We demonstrated a reduction of the fit residual by 95% in comparison with the reference model.³

Oral squamous cell carcinomas represent a significant number of cancers diagnosed globally. Of these cancers, surgical resection of the primary tumor is the standard treatment. Conventional methods of assessing completeness of resection are time-consuming, laborious, and cannot be used to evaluate the entire margin of a resected tumor. As such, widefield fluorescence molecular imaging is being explored as an intraoperative technique to guide resections. The widely used single-view, wide aperture techniques have had high success in identifying positive margins (those with thickness < 1mm), but limited success in identifying close margins (1-5 mm). Here a dual aperture fluorescence ratio is presented as a means of improved detection of close margins, with evidence that this technique may be highly useful for future intraoperative fluorescence molecular imaging applications. Monte Carlo simulations are conducted to assess the technique’s ability to minimize optical property heterogeneities across regions with varying absorption and scattering characteristics.

Cancer continues to be a significant global health issue in the 21st century, presenting substantial risks to the health and well-being of individuals worldwide. Although there have been improvements in comprehending the molecular processes of the disease and creating treatments that specifically target it, a considerable portion of patients continue to encounter difficulties in attaining favorable results. Conventional two-dimensional (2D) cell cultures have been extensively used in cancer research. However, their inability to accurately mimic the intricate characteristics of tumors limits their effectiveness in predicting how anticancer treatments would perform in clinical settings. In order to overcome these restrictions, three-dimensional (3D) cell culture models, specifically multicellular spheroids, have arisen as promising tool for investigating cancer biology and therapeutic response. This work analyzes the development and growth dynamics of spheroids obtained from four distinct cancer cell lines: 9L-GFP, U251-RFP, A431, and FaDu. The stability and growth features of these spheroids were evaluated by culturing them using different cell counts and dilution ratios. Confocal microscopy was used to observe the formation of spheroids and measure their sizes for a duration of seven days. The results of our study reveal clear variations in growth patterns and stability profiles across the investigated cell lines. Notably, the 9L-GFP cell line demonstrates exceptional stability and continuous growth. The statistical analysis demonstrated that spheroids exhibited the most stability when the cell count was 25,000 cells and the dilution ratio was 1:3, as indicated by the high R-squared values. These findings highlight the significance of adjusting the number of cells and dilution ratios to ensure consistent and replicable spheroid formation. In summary, our study emphasizes the capacity of 3D spheroid models as effective instruments in cancer research and medication development, providing vital information about tumor biology and therapeutic responses in a context that closely resembles the physiological conditions.

There is a need for a noninvasive diagnostic method for early detection of basal cell carcinoma which is the most common type of cancer in the general population. Basal cell nevus syndrome is a rare autosomal dominant disorder that increases predisposition to basal cell carcinoma, with a lower average age of onset and a higher number of lesions. Autofluorescence and autofluorescence photobleaching imaging is a potential approach to early diagnosis and determining whether an aggressive form of basal cell carcinoma is present earlier, however, the mechanism is still not fully understood. Investigation of basal cell nevus syndrome associated basal cell carcinoma autofluorescence intensity and autofluorescence photobleaching kinetics could assist in early detection and assessment of basal cell carcinoma in general.

An imaging device with 405 nm LED illumination at power density 7 mW/cm² was used for cutaneous autofluorescence excitation. Autofluorescence photobleaching was detected by imaging under continuous irradiation for 20 seconds. It was found that on average basal cell carcinoma in patients with basal cell nevus syndrome has a lower autofluorescence intensity at the first second of excitation, as well as smaller decrease in intensity after 20 seconds of irradiation compared to sporadic basal cell carcinoma. This may show that basal cell carcinoma in patients with basal cell nevus syndrome have a different composition of endogenous fluorophores than in sporadic cases which could be investigated in further research.

Spectral imaging – acquisition of images within specific spectral intervals - is a powerful tool for optical diagnostics, able to provide objective data on various clinical parameters, e.g. abnormal content of biomolecules in pathologic tissues. Performance of diagnostics depends on the spectral selectivity of imaging; from this point, ultra-narrowband spectral line imaging appears well-suited for diagnostic applications. Two prototype devices for triple laser line imaging have been developed and tested in laboratory and clinical environments. Large area or whole-body skin spectral imaging device comprises vertically movable high-resolution camera coupled with a specific illumination unit - side-emitting optical fiber spiral that emits simultaneously three laser spectral lines at the wavelengths 450 nm, 520 nm and 628 nm. In the other device, conventional white broadband endoscopic illumination has been replaced by a combined three spectral line white illumination from a low power RGB laser-fiber system attached to the lighting channel of intranasal endoscope. Both prototypes undergo clinical validation; their design details and preliminary test results are reported and discussed.

Diagnosing skin cancer, such as basal cell carcinoma, requires a biopsy, which is a time-consuming and expensive process. Therefore, it is essential to explore alternative diagnostic methods that are both more efficient and effective. We developed a handheld optical coherence microscopy (OCM) imaging device to achieve high-resolution optical biopsies in real-time. The instrument uses a variable focus liquid lens that allows fast shifting of the focus inside the sample, resulting in high-resolution lateral images throughout an extended axial imaging range. Our instrument can produce images with an axial resolution of approximately 5 μm, currently limited by the light source employed, and better than 2 μm transversal resolution images. The acquisition, data processing, and display of the 3D volumes are performed in real time, primarily enabled by the Master-Slave approach employed to produce the optical biopsies. The acquisition rate of the current camera used in the spectrometer is limited to 70 kHz. Our benchmarking shows that the real-time operation of the instrument can be sustained even at over 250 kHz solely by utilizing the computing power of the CPU, with no need to employ graphic cards or FPGAs. The instrument’s capability is showcased through images featuring various samples, such as an IR card and skin.

Aortic diseases, which are among the leading causes of death worldwide, are extremely complex. These diseases, including aneurysm, atherosclerosis, and aortic stenosis, cause both morphological and chemical changes in the affected tissues. Understanding their nature, to improve diagnostics and disease progression monitoring, often presents a challenge due to the disease development over the years. Typically, CT scans with contrast agents are used for diagnosis, which are sometimes invasive for the patient providing only morphological information. Therefore, employing techniques that aid in the early diagnosis of these diseases would be of interest.

In this work, ex-vivo tissue samples of 32 human aortic rings including aneurysm, atherosclerosis, aortic stenosis, aortic insufficiency, and bicuspid aortic valve diseases were obtained from surgical interventions. Healthy aortic specimens were considered as controls when excised from transplant donors undergoing non-aortic related pathologies.

The aim is to co-register measurements from HSI (HyperSpectral Imaging) and OCT (Optical Coherence Tomography) imaging modalities, obtaining maps of chemical composition and morphological structure, being able track changes at each point of the tissue sample in approximately 100 cm² of the inner aortic wall. These samples have been imaged ex-vivo using wide-field HSI, in the SWIR (1000-1700 nm) ranges, and OCT. OCT was used to generate attenuation coefficient maps of tissue specimens. Additionally, HSI was used to estimate elastin, collagen, lipid and water content of the samples.

An inversely proportional relationship has been observed between the aorta’s diameter and their attenuation coefficient. Furthermore, an increase in the mean squared error of the spectral fitting has been noted in pathological samples. This study underscores the potential of integrating HSI and OCT for the advanced characterization and early diagnosis of complex aortic diseases, highlighting their critical role in improving patient outcomes.

Optical imaging is a non-invasive technique that is able to monitor hemodynamic and metabolic brain response following neuronal activation during neurosurgery. However, it still lacks robustness to be used as a clinical standard. In particular, the quantification of the biomarkers of brain functionality needs to be improved. The quantification relies on the modified Beer Lambert law, which needs a correct estimation of the optical mean path length of traveled photons. Monte Carlo simulations are used for estimating the optical path length, but it is time-consuming, especially when modeling a patient’s brain cortex. In this study, we developed a neural network based on the UNET architecture for a pixel-wise and real-time estimation of optical mean path length. The neural network was trained with segmentation of brain cortex as input and mean path length data as target. This deep learning approach allows a real time estimation of the optical mean path length. The results can be beneficial and useful within the framework of our EU-funded HyperProbe project, which aims at transforming neuronavigation during glioma resection using novel hyperspectral imaging technology.

This study aims to integrate real-time hyperspectral (HS) imaging with a surgical microscope to assist neurosurgeons in differentiating between healthy and pathological tissue during procedures. Using the LEICA M525 microscope’s optical ports, we register HS data and RGB, in an efforts to improve margin delineation and surgical outcomes. The CUBERT ULTRIS SR5 camera with 51 bands and 15 Hz is employed, and critical calibration steps are outlined for clinical application. Experimental validation is conducted on ex-vivo animal tissue using reflectance spectroscopy. We present the preliminary validation results of the performance comparison between the designed hyperspectral imaging microscope prototype and diffuse reflectance spectroscopy conducted on animal tissue.

Today, the scientific literature agrees on the importance of light as a regulator of our biological clock and its impact on physiological and neuroendocrine functions as well as visual health. Refining our understanding of the effects of light in terms of levels, spectrum and temporality requires the collection of real time, continuous data as close to the subject's ocular surface as possible. In addition, most sensors typically provide visible light data integrated over a range of wavelengths and already weighted by a sensitivity curve, limiting further analysis and translation of measured data for simultaneous assessment of multiple physiological parameters. To the best of our knowledge, there is currently no device that fulfils all these conditions, is aesthetically acceptable and does not interfere with the wearer's daily life.

We have therefore developed instrumented eyewear that incorporates visible light sensors, wear sensors and an on-board memory that enables continuous data flow recording every 30 seconds for 36 days. The integration of the sensors and the design of the eyewear make it possible to have a light quantification device that is acceptable and wearable every day, whether in terms of aesthetics, comfort, or use.

Our visible light sensors have been specifically selected to provide spectral irradiance and illuminance received at the ocular surface. Their positioning, integration and pre-laboratory calibration ensure accurate, repeatable measurement over the full dynamic range of daily irradiance received by the eye. This instrumented eyewear is designed for use in all studies requiring rigorous assessment of the lighting environment.

Photodynamic therapy (PDT) is a therapeutic modality that combines light, a light-activated drug, and the molecular oxygen of the medium to disrupt cells. Due to the low penetration of light in biological tissue and its absorption by pigmented cells, such as melanoma, the effective application of PDT is usually limited to superficial and non-pigmented lesions. The utilization of ultrasound-based therapies, such as Sonodynamic therapy (SDT) and Sono-photodynamic Therapy (SPDT), poses a possible solution to overcome these limitations. Besides the greater penetration of acoustic waves in the tissue, the propagation of ultrasonic waves can form gas/vapor bubbles in the medium and cause its subsequent oscillation and implosion, known as acoustic nucleation and cavitation. The implosion of oscillating bubbles can generate sonomechanical and sonochemical effects, such as the production of reactive oxygen species (ROS) by pyrolysis and sonoluminescence, leading to tumor death. This study aims to obtain, in a first approximation, the profile of light and ultrasound penetration in skin and melanoma cells, as well as the increase in temperature generated by the implosion of cavitated bubbles. For this purpose, the Monte Carlo eXtreme (MCX) and k-wave toolbox for MATLAB were used to obtain the beam profile of light and ultrasound, respectively, in skin and melanoma. For obtaining the inertial cavitation threshold and the peak of temperature due to the bubble’s implosion, the Keller-Miksis (KM) equation was solved in the software Wolfram Mathematica. While the results were obtained from simplified models, they corroborate the greater penetration of ultrasound (US) in skin and melanoma when compared to light. While light at a wavelength (λ) equal to 630 nm reaches less than 3 mm within the tissue, US waves at the frequency of 1 MHz and acoustic pressure equal to 0.32 MPa can penetrate 15 mm, and the acoustic intensity is smoothly reduced in melanoma. Furthermore, the implosion of the cavitated bubbles can generate a local increase in temperature up to 1000 K, which can be responsible for the generation of ROS. Despite the need for more refined models to describe these phenomena with greater precision, they demonstrate that the utilization of SDT and SPDT can be a good approach for the effective and noninvasive treatment of melanoma.

In light microscopy, achieving uniform lighting throughout the field of view is essential to obtain clear images. However, thicker samples commonly suffer from image blur due to out-of-focus illumination, notably raised in fluorescence microscopy because dye molecules are activated across all planes, including those out of focus. Confocal-type fluorescence microscopy efficiently addresses this obstacle by reducing out-of-focus light, thereby permitting high-quality imaging in thick tissues. By focusing the light and detector on a diffraction-limited point and monitoring the material point by point, confocal microscopes may obtain full pictures with optical sectioning abilities for 3D reconstruction. With numerous varieties offering distinct advantages and limits, confocal microscopes serve as important tools for accurate imaging and analysis across different sectors. This article gives a concise review of confocal fluorescence microscopy.