Conference Agenda

On the tenth anniversary of the first demonstration of daytime radiative cooling, we will both provide an introduction to, and survey, the state of the field, highlighting the role of advanced in optical and nanoscale materials in enabling recent progress. We will also look ahead to new frontiers and challenges in radiative cooling materials and technological applications enabled by radiative cooling.

Self-adaptive thermoregulation, the mechanism living organisms use to balance their temperature, holds great promise for decarbonizing cooling and heating processes. This functionality can be effectively emulated by engineering the thermal emissivity of materials to adapt to background temperature variations. Yet, solutions that marry large emissivity switching (Δϵ) with scalability, cost-effectiveness and design freedom are still lacking. Here, we fill this gap by introducing infrared dipole antennas made of tuneable thermochromic materials. We demonstrate that non-spherical antennas (e.g. nanorods) made of vanadium-dioxide can exhibit a massive (~200-fold) increase in their absorption cross-section as temperature rises. Embedding these antennas in polymer films, or simply spraying them directly, creates free-form thermoregulation composites, featuring an outstanding Δϵ~0.6 in spectral ranges that can be tuned at will. Our research paves the way for versatile self-adaptive heat management solutions (coatings, fibers, membranes, and films) that could find application in radiative-cooling, heat-sensing, thermal-camouflage, and other.

Radiative cooling involves decreasing the temperature of a body by emitting infrared radiation. When the heat loss from the emitting surface exceeds the heat gain, e.g., from the sun or the atmosphere, a passive net cooling effect occurs without the need for electricity or other power sources. Integrating radiative cooling materials with other renewable energy technologies represents a promising frontier in sustainable energy systems. In this study, we explore the strategic utilization of the net cooling effect resulting from radiative cooling materials to enhance the efficiency of photovoltaic panels, as they are susceptible to performance degradation with temperature variations. Our investigation focuses on the integration of these materials with photovoltaic cells and explores the possibility of integrating them in to other renewable energy technologies such as thermoelectric generators, addressing critical challenges, including thermal management, efficiency optimization and operational stability.

We present preliminary results on the fabrication of patterned surfaces by Direct Laser Interference Patterning and the characterization and theoretical interpretation of their infrared emissivities. The upgraded experimental method is capable of studying the full directional emission of samples under a controlled atmosphere at high temperatures. The effects of surface patterning can be quantitatively studied and modeled using a numerical method based on rigorous coupled-wave analysis (RCWA), a technique usually employed for periodic surfaces. The results show that laser interference patterning is capable of modifying the infrared emission of metallic materials, and that these changes can be accurately measured and numerically reproduced.

Conventional methods for producing superhydrophobic passive daytime radiative cooling coatings are considered inexpensive, however the majorities use fluorinated polymer. In this work a novel method, fluorine-free, developed for synthesizing porous structures coating by using low density polyethylene is presented. The method has precise control over the thickness and microstructure. Moreover, the materials and the machinery required to produce the coatings are minimal in comparison to the conventional methods, hence lowering the cost of production. The coating, poly-cool, exhibits superhydrophobic properties as well as radiative cooling capabilities with solar reflectance of 97.4% and emissivity of 91% within the atmospheric window. The coating consists of highly porous polyethylene and PDMS nanoparticles. The high porosity of polyethylene helps improve the reflectivity of solar irradiance whereas the PDMS nanoparticles tune the emissivity of the coating to match that of the atmospheric window (8 to 13m).

The performance of radiative coolers are directly affected by their constituting materials’ optical properties and micro-structures. Here, we investigate the effects of optical properties and microstructure on radiative cooling performance, namely solar reflectance and emittance at 9-13 µm region. The aim is to provide guidance to reach the ideal radiative cooler. Inspected phenomena are included but not limited to Mie resonance, light scattering, effective medium theory.

Daytime radiative cooling has emerged as a promising solution for continuous passive cooling. While significant progress has been made in material developments, questions persist regarding the potential large-scale deployment of this technology. Among the potential applications, two predominant uses of daytime radiative cooling materials are under study: passive roof cooling and water-cooling panels. Passive roof cooling involves the deposition of radiative cooling material on the roof’s surface to passively cool the building by reflecting incoming solar rays. The second application employs outdoor radiative cooling panels to passively cool water, which can be used to assist cooling systems. For instance, cooled water could sub-cool the cooling refrigerant, thereby improving the air conditioner's energy efficiency.

In the present study, a numerical assessment to compare the benefits of both application schemes for radiative cooling materials is conducted. For each application, the model predicts the energy savings achievable by each integration mode. Preliminary results indicate that prioritizing roof cooling applications would be most effective in tropical regions. The deployment of water cooling panels for refrigerant sub-cooling appears to be more beneficial in mid-latitude temperate zones. These findings suggest adjusted approaches based on climatic considerations for the optimal deployment of daytime radiative cooling technologies worldwide.

Remote sensing in thermal infrared bands (TIR) is largely dominated by cumbersome dispersive-type hyperspectral imagers, which usually require expensive and cryo-cooled quantum detectors to make up for their low optical throughput. Here, we present a compact and low-cost TIR hyperspectral camera based on the Fourier-transform approach. It combines an uncooled bolometer detector and a common-path birefringent interferometer made of calomel (Hg2Cl2). It features high optical throughput, an interferometric contrast greater than 90% even for incoherent radiation, spectral resolution tunable up to 4.5 cm-1, robust and long-term interferometric stability. Retrieving in a few minutes the infrared spectrum in all pixels of the TIR image, it could constitute a valuable tool for evaluating radiative cooling materials' spatial and spectral properties over extended areas. We test the capabilities of the instrument by measuring the emissivity map of different butterfly wings, which provide a natural example of radiative cooling.

The observation of organelles dynamics and macromolecular complex interactions inside living cells and tissues requires minimally invasive imaging strategies. In this context, photocontrollable fluorescent proteins (FPs) play a crucial role as tags in optical super-resolution microscopy and functional live cell imaging. To this end we have previously shown that reversibly switchable FPs enable fast (1 Hz for a 50 x 50 µm2) and gentler (< 1 kW/cm2 illuminations) nanoscopy (Masullo et al Nat. Comm 2018). Additionally, irreversibly photoconvertible FPs can achieve photolabeling with high spatiotemporal precision. Nevertheless, their photophysical complexity poses challenges in expanding such techniques toward multiplexing and in vivo imaging. Here, we explore novel photoswitching mechanisms for fluorescent proteins in the red and near-infrared region of the spectra and assess their compatibility with live cell imaging at the nanoscale (Pennacchietti et al, Nat. Meth, 2018). Finally, we present strategies to combine the spectral and photophysical fingerprint of distinct photocontrollable FPs to achieve multiplexing in live cell imaging at the nanoscale and photolabeling studies (Pennacchietti et al, Nat Comm, 2023).

Image Scanning Microscopy (ISM) enables good signal-to-noise ratio (SNR), super-resolution and high information content imaging by leveraging array detection in a laser-scanning architecture. However, the SNR is still limited by the size of the detector, which is conventionally small to avoid collecting out-of-focus light. Nonetheless, the ISM dataset inherently contains the axial information of the fluorescence emitters. We leverage this knowledge to achieve computational optical sectioning without sacrificing the conventional benefits of ISM. We invert the physical model to fuse the raw dataset into a single image with improved sampling, SNR, lateral resolution, and optical sectioning. We provide a complete theoretical framework and validate our approach with experimental images of biological samples acquired with a custom setup equipped with a single photon avalanche diode (SPAD) array detector. Furthermore, we generalize our method to other imaging techniques, such as multi-photon excitation fluorescence microscopy and fluoresce lifetime imaging. To enable this latter, we take advantage of the single-photon timing ability of SPAD arrays, accessing additional sample information. Our method outperforms conventional reconstruction techniques and opens new perspectives for exploring the unique spatio-temporal information provided by SPAD array detectors.

Simultaneous field- and aperture-plane (back-focal plane, BFP) imaging enriches the infor-mation content of fluorescence microscopy. In addition to the usual density and concentration maps of sample-plane images, BFP images provide information on the surface proximity and orientation of molecu-lar fluorophores. They also give access to the refractive index of the fluorophore-embedding medium. However, in the high-NA, wide-field detection geometry commonly used in single-molecule localisation microscopies, such measurements are averaged over all fluorophores present in the objective’s field of view, thus limiting spatial resolution and specificity. We here solve this problem and demonstrate how an oblique, variable-angle, coherent ring illumination can be used to generate a Bessel beam that - for supercritical exci-tation angles - produces an evanescent needle of light. Scanning the sample through the this evanescent needle enables us to acquire combined sample-plane and BFP images with sub-diffraction resolution and axial localisation precision. Background, resolution and polarisation considerations will be discussed.

We present the use of wavefront coding (WFC) combined with machine learning in a light sheet fluorescence microscopy (LSFM) system. We visualize the 3D dynamics of sperm flagellar motion at an imaging speed up to 80 volumes per second, which is faster than twice volumetric video rate. By using the WFC technique we achieve to extend the depth of field of the collection objective with high numerical aperture (NA=1) from 2.6 μm to 50 μm, i. e., more than one order of magnitude. To improve the quality of the final images, we applied a machine learning-based algorithm to the acquired sperm raw images and to the point spread function (PSF) of the generated cubic phase masks previous to the deconvolution process.

The ability to generate a structured illumination pattern in microscopy is a fundamental need of numerous optical microscopy techniques, such as Structured Illumination Microscopy (SIM) and HiLo microscopy. However, existing pattern generating techniques, such as using diffraction gratings or SLMs, are either slow, bulky, or very alignment-sensitive, making the widespread acquisition of such techniques harder. We present an integrated, monolithic device, fabricated in a glass substrate via Femtosecond Laser Micromachining, capable of generating and translating a highly stable structured pattern on top of the sample plane of a microscope, enabling a widefield microscope to perform SIM.

Carcinogenesis involves DNA methylation which is a primary alteration in DNA in the development of cancer before genetic mutation. Because the abnormal DNA methylation is found in most cancer cells, the assessment of DNA methylation using terahertz radiation can be a novel optical method to detect and control cancer. The methylation has been directly observed by terahertz time-domain spectroscopy and this epigenetic chemical change could be manipulated to the state of demethylation using resonant terahertz radiation. Demethylation of cancer cells is a key issue in epigenetic cancer therapy and our results demonstrate the feasibility of the cancer treatment using optical technique.

Controlled temperature elevation within biological tissues, known as hyperthermia, holds promise as a therapeutic treatment. Its efficacy depends on several factors including timing, pulsing, and repetition. Recent research indicates the potential of heat-based therapies not only for cancer treatment but also in tissue regeneration. The usage of photothermal agents, such as gold nanoparticles, enables precise spatio-temporal heat generation, known as photothermal therapy (PTT). Hydra vulgaris, with their unique regenerative capabilities, serve as valuable models for exploring the effects of nanoparticles on tissue regeneration. AuNPs thanks to their plasmonic properties can induce physiological responses in the animals under near-infrared (NIR) irradiation, ranging from cell ablation to programmed cell death or thermotolerance. By tuning the NIR irradiation and the AuNPs dose, the capability of treated polyps to regenerate the missing heads under photostimulation will be dissected, at whole animal, cellular and molecular levels and compared to exposure to external macroscopic heat sources.

Coronaviruses are characterized by spike (S) glycoproteins, which are the largest structural membrane proteins and the first involved in the anchoring of the host receptor angiotensin-converting enzyme 2 (ACE2) through the receptor binding domain (RBD). Its secondary structure is of great interest for shedding light on various aspects, from functionality to pathogenesis, finally to spectral fingerprint for the design of optical biosensors. The aim of this work is the characterization of the whole monomeric SARS-CoV-2 S protein, its constituting components, namely RBD, S1 and S2, at serological pH (7.4) and the S1 alterations induced to chemical/physical environmental modifications by measuring their amide I absorption bands through Attenuated Total Reflectance Infrared spectroscopy (ATR-IR).

Nature provides various organisms with ordered or quasi-ordered dielectric nanostructures that enable several animals, plants, and protists to manipulate light, optimizing inter- and intra-species communication, camouflage, or solar light harvesting. In particular, diatom microalgae possess nanostructured silica cell walls, known as frustules, which efficiently interact with optical radiation through multiple diffractive, refractive, scattering, waveguiding, and frequency down-conversion mechanisms. These properties contribute to diatoms’ efficiency in photosynthesis, UV tolerance, and possibly influence the phototaxis mechanisms of motile species. In our study, we utilized several imaging, spectroscopic, and numerical techniques to explore the optical functionalities of individual frustule components in the pennate, motile diatom Pleurosigma strigosum. We discuss the implications of frustule photonic properties on the living cell, and envision the exploitation of these properties in multifunctional, bio-derived photonic devices.

Thiophene-based materials (TMs) have emerged as promising candidates in the field of photodynamic therapy (PDT) as photosensitizers agents owing to their remarkable electron transport properties, which facilitate efficient energy transfer processes crucial for PDT. In detail, TMs exhibit favourable optical characteristics, making them suitable candidates for the absorption and conversion of light energy into reactive oxygen species (ROS), thereby inducing cytotoxic effects in targeted cells. Recent studies have explored natural carriers, including proteins and phages, for enhanced cell uptake and permeation of photosensitizers, thereby enabling the induction of apoptosis across various cell lines. Despite the remarkable potential of this approach for PDT purposes, clinical translation necessitates in vivo models to validate these innovative tools. Here, we investigated the nanosafety and in vivo efficacy of these phototheranostic agents using the tissue-like animal model Hydra vulgaris. The transparency, softness, structural simplicity, and ethical neutrality of Hydra collectively render it an exemplary model for such inquiries. These features facilitate rapid screening of cytotoxicity and the effectiveness for photodinamic purposes.

Bioresorbable photonic implants are emerging as potential material choice for interstitial theranostic and monitoring applications. They gradually dissolve within the physiological environment in a clinically relevant period, eliminating the need for extraction surgeries. In the present study, we tested the suitability of in-house fabricated bioresorbable optical fibres based on calcium phosphate (CaP) glass for diffuse correlation spectroscopic (DCS) and diffuse fluorescence tomographic (DFT) applications. The results represent the potential of bioresorbable fibers for the monitoring of interstitial microvascular blood flow and the spatial distribution of fluorescent photosensitizer drugs that are administered prior to therapies. Together or separate, the continuous monitoring of these parameters can have significant implications in planning, optimizing and in predicting or monitoring the outcomes in interstitial photodynamic therapy (PDT).

Single-cell analysis without immune-specific labelling is essential across research fields, but conventional flow cytometers (FCMs) struggle with label-free analysis. We introduce a novel microfluidic scanning flow cytometer (µSFC) designed for label-free analysis within a simple microfluidic chip. Our system outperforms traditional FCMs for label-free analysis but its signal-to-noise ratio (SNR) limits the minimum detectable size. We present three modifications to enhance SNR and improve the smallest detectable particle size: additional neutral optical density filtering, a lower noise-equivalent-power photoreceiver, and laser spot size reduction. These improvements enable reliable characterization of particles as small as 3 µm. Experimental results validate the correlation between angular profile oscillations and particle size. While reliable detection down to 1 µm is achieved, further refinement is needed. The simplicity and low setup of the µSFC make it promising for integration into multi-parametric single-cell analysis systems, facilitating comprehensive cellular characterization for diagnostic and point-of-care applications.

Imaging flow cytometry (IFC) integrates flow cytometry with optical microscopy, enabling high-throughput, multi-parameter analysis of single cells. Current 3D IFC systems face limitations related to instrumental complexity that might lead to optical misalignment or mechanical instabilities in day-by-day operation. We propose a fully integrated optofluidic platform combining reconfigurable photonic circuits and cylindrical hollow lenses for structured light sheet microscopy in a microfluidic channel. The components are fabricated using femtosecond laser irradiation and chemical etching, ensuring a high level of integration that allows durable alignment and mechanical stability.

Statistical analysis of properties of single microparticles, such as cells, bacteria or plastic slivers, has attracted increasing interest in recent years. In this field flow cytometry is considered the gold standard technique, but commercially available instruments are bulky, expensive, and not suitable for use in Point-of-Care (PoC) testing. Microfluidic flow cytometers, on the other hand, are small, cheap and can be used for on-site analysis. However, in order to detect small particles, they require complex geometries and the aid of external optical components. To overcome these limitations here we present an opto-fluidic flow cytometer with an integrated 3D in-plane spherical mirror for enhanced optical signal collection. As result the signal-to-noise ratio is increased by a factor of 6, enabling the detection of particle sizes down to 1.5µm. The proposed optofluidic detection scheme allows the simultaneous collection of particles fluorescence and scattering - using a single optical fiber - which is crucial to easily distinguish particle populations with different optical properties. The devices have been fully characterized using fluorescent polystyrene beads of different sizes. As a proof of concept for potential real-world applications, signals from fluorescent HEK cells and Escherichia coli bacteria were analyzed.

This project aims to produce a microfluidic device capable of separating 6 µm and 20 µm diameters particles by inertial sorting. This Lab-on-Chip (LoC) was designed with a trapezoidal cross-section for better fluid control and effective particle manipulation at the microscopic level, as demonstrated by COMSOL simulations. The device was manufactured on a substrate of Polymethyl Methacrylate (PMMA) by femtosecond laser technology and then assembled using an innovative geometry-preserving Isopropyl alcohol-based procedure. The LoC was test with spherical plastic microparticles of two diameters (6 µm and 20 µm) suspended in distilled water. The separation efficiencies were (98.2 ± 1.6) % for 20 µm diameter particles and (70.0 ± 1.8) % for 6 µm diameter particles in good agreement with the simulation results. Finally, after a microfluidic channels’ acetone vapors treatment, the device demonstrated a good ability to separate biological particles (Red Blood Cells) at different concentrations (20%, 30%, 40%, 50%) in a PBS buffer.

Pancreatic ductal adenocarcinoma (PDAC) is a prevalent form of pancreatic cancer, contributing significantly to cancer-related mortality worldwide. Early lesions manifest within the exocrine pancreatic gland. Thus, in vitro fully human models of the exocrine pancreatic unit are needed to foster the development of more effective diagnosis and treatments. However, it is challenging to make these models anatomically and functionally relevant. Here, tomographic volumetric bioprinting was used to biofabricate human fibroblast-laden gelatin methacrylate-based pancreatic models, mimicking glandular structure. Indeed, this technique is an optically based method that uses reverse optical tomography to construct 3D objects in a layerless fashion.

Pancreatic epithelial cells, healthy or cancerous, were then seeded, and the development of a thin epithelium inside the lumen of the 3D model was demonstrated. Immunofluorescence and ELISA assays revealed higher activation of fibroblasts when they were co-cultured with cancer cells, replicating the realistic situation in vivo. To our knowledge, this is the first demonstration of a 3D bioprinted portion of pancreas that recapitulates its physiological 3-dimensional microanatomy, and which shows tumor triggered inflammation. It opens new avenues to the application of light-based additive manufacturing in tissue engineering, overcoming the difficulties associated with light scattering from cells in hydrogels.

Non-linear excitation microscopy offers superior in-vivo imaging but faces challenges in deep tissue. High numerical aperture beams suffer spherical aberrations, while tissue scattering impacts image quality. To address this, we propose implantable microlenses for precise focusing below the skin in lab animals. By using low numerical aperture lasers, we avoid spherical aberrations induced by high NA objectives. Our study presents various microlens designs differing in size, shape, and fabrication methods, all on glass or organo-hybrid ceramic substrates. This approach shows promise for enhancing deep tissue imaging, facilitating better understanding of biological processes in vivo.

Bioluminescent systems possess remarkable features, such as high quantum yield and no need for an external light source, that render them highly valuable bioanalytical tools for developing portable biosensors for monitoring molecules, cells, and bioactivities with applications spanning from agro-food to clinical fields. Therefore, bioluminescent biosensors have a great potential to support the “One Health” approach, to guarantee health to humans, pets, wildlife and our environment.

Here we report the development of novel bioluminescent biosensors implementing living cells and cell-free systems immobilized on paper and cotton threads and interfaced to a smartphone as light detector. The implementation of artificial intelligence algorithms to smartphone-based bioluminescence detection is also reported. A paper-based toxicity smartphone biosensor was developed providing, thanks to an Android AI mobile app, quantitative and user-friendly information.

The combination of bioluminescence biosensing with microfluidic thread-based analytical devices (μTADs) provided a sustainable low-cost alternative to paper based biosensing, especially to handle very low volumes of samples (less than 5 µl). We report a proof-of-principle application of bio-chemiluminescence biosensing on cotton threads and, to prompt future applications in point-of-care and point-of need settings, we exploited smartphone detection enabling easy detection of the bio-chemiluminescent signal directly on the thread.

This study deploys the application of Fiber Bragg Gratings (FBGs) in physiological pressure monitoring by integrating an elastomeric, biocompatible coating ranging from 300-500μm, designed to improve sensor functionality for in-vivo pressure monitoring applications. FBGs are favored for their sensitivity, immunity to electromagnetic interference, and compact size, making them ideal for embedding within medical devices such as catheters and guidewires. However, their use has been limited by low inherent pressure sensitivity (3.14 pm/MPa) and the impracticality of thicker coatings described in previous studies. Our approach demonstrates that this unique coating not only boosts the pressure sensitivity significantly—surpassing two orders of magnitude (43.10 times)—but also enhances the signal-to-noise ratio of the optical signal. These advancements enable potential applications in high-resolution manometry, gastrointestinal pressure monitoring, intracranial and intracoronary blood pressure measurements, marking a significant step forward in medical diagnostics and monitoring.

We discuss the implementation of Whispering Gallery Modes Microbubble resonators (MBRs) as unique platforms for photoacoustic (PA) detection and photothermal (PT) spectroscopy. In a first experiment, the MBR transducer allowed to detect the PA signal generated by a suspension of gold nanorods (GNRs) within its core, leveraging on the MBR sharp optical spectrum and high sensitivity towards mechanical perturbations. Both static and flow-cytometry configuration were tested, finding that the MBR mechanical modes help detection by decoupling the environmental noise from the PA oscillation. In a second experiment, the MBR transducer allowed to reconstruct the GNRs absorption spectrum through the photothermal (PT) conversion, leveraging on high sensitivity towards temperature variations. We verified the scattering-free nature of the detection by using milk-stained GNRs suspension. We also found that the active locking of the MBR resonance increases the system sensitivity by an order-of-magnitude. These results make MBRs interesting candidates for combined PA and PT characterisation of extremely small samples for medical diagnosis, quality controls in food safety and chemical production processes.

A novel technique for the realization of superimposed long-period gratings with different grating periods, based on the discretization of a continuous sinusoidal refractive index pattern with a step function, is described. The RI variation is induced step-by-step on a photosensitive optical fiber fiber with a 248nm UV laser beam. Two superimposed long-period grating (LPGs) with different grating periods, so coupling the light to two different cladding modes, were realized, and different sensitivity to refractive index and temperature were exploited for the simultaneous detection of both parameters. The proposed device can be used for the compensation of temperature fluctuations in biosensing.

Whispering Gallery Modes (WGM) are optical resonators with promising applications in chemical sensing. Nonetheless, only few examples regarding the detection of small molecules through WGM resonators have been reported in the literature to date. Here, we report on the detection of the anticancer drug Doxorubicin (DXR) using Layer-by-Layer (LbL)-functionalized WGM fluorescent microparticles. In particular, polystyrene sulfonate (PSS) is assembled in combination with the polycation polyallylamine hydrochloride (PAH) on the WGM surface and exploited as the DXR receptor. The PSS-functionalized WGM particles feature a linear change of the WGM resonance wavelength and linewidth when exposed to DXR at concentrations down to 1 μg mL-1, with good selectivity against other anticancer drugs. Remarkably, detection of DXR in interstitial fluid is also successfully demonstrated.

Optical Coherence Tomography (OCT) stands out for its ability to combine the high resolution of

microscopy with the penetration-depth of clinical imaging. However, in practice this is still limited to a few

millimetres. Interestingly, the imaging-depth of the latest swept-source systems is not limited by their spectral

width but by the analog-to-digital sampling rate. In lieu of slow reference arm length adjustments, we leverage

opto-electronic frequency shifting. This allows for depth adjustments on the microsecond timescale and a modest

detector bandwidth of 200 MHz . The opto-electronic scheme immediately gives us access to an 8 mm range,

a fourfold increase over the nominal 2 mm range of the source. Moreover, by circumventing the need for a

mechanical reference arm, changes in the axial displacement of the sample can be compensated in real-time.

This makes it attractive for imaging arbitrarily-curved surfaces. We showcase this with wide-field OCT imaging

of the curved retina.

Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues. In particular, Fluorescence Lifetime Imaging Microscopy (FLIM) has shown detailed profiles of tissue autofluorescence, enabling more informed and rapid tissue characterization, with the potential for translation from the research labs to bedside. We report here the test of our autofluorescence lifetime imaging probe device on four different clinical cases. More in detail, we examined four biopsies, one from a hepatocellular carcinoma (HCC), another from an intrahepatic cholangiocarcinoma (ICC), one from a gastrointestinal stromal tumor (GIST) and the last one from pancreatic ductal adenocarcinoma (PDCA). The results suggest that our autofluorescence lifetime imaging probe, together with phasor analysis, can offer a real-time tool to observe spectral and lifetime contrast on fresh tissues and, thus, is a suitable candidate for improving in situ tissue diagnostics during surgery.

Current surgical resections for Head and Neck Cancers aim for clear margins to prevent local recurrence. However, up to 20% of cases result in positive margins, with secondary surgery increasing the chances of death after 5 years. We envision a MMF endoscope that collects high resolution images using wavefront shaping to scan a 405 nm beam at the fiber tip and collecting fluorescence intensity and lifetime to map tumor margins and detect residual malignant cells. To address the question whether the information contained in the fluorescence and morphology can be used to classify cancer and normal tissues, we used images acquired with a microscope and artificial neural network. Initial findings show promise to separate cancer from normal tissue when training neural networks on FLIM data. Spatial and temporal resolution and required field of view for effective margin assessment are determined.

In this study, we introduce a novel optical setup tailored for measuring scattering spectra of small biological aggregates with minimal sample volumes. Calibration was achieved using Polystyrene beads (PS beads) based on Mie scattering principles, enabling accurate measurements of scattering intensities. Bovine serum albumin (BSA) served as a model for studying protein aggregation due to its predictable aggregation behaviour at elevated temperatures. Analysis of non-aggregated and aggregated BSA solutions revealed significant differences in particle size, underscoring the setup's capability to detect variations in aggregation states. Key findings demonstrate the system's efficacy in monitoring protein aggregation processes, which is critical for biochemical and pharmaceutical research. The precise calibration method and the use of BSA as a validation tool highlight the setup's sensitivity and accuracy in quantifying changes in particle concentration and size due to aggregation. This study provides a framework for analysing protein aggregation and offers insights into the aggregation's impact on scattering properties.

In this study, we report the results of two non-invasive optical methods, Raman microscopy (RM) and polarization-sensitive digital holographic imaging (PSDHI), for distinguishing prostate cancer cells from healthy ones. RM reveals cancer cells metabolize glucose faster, storing it as fatty acids and cholesteryl esters in lipid droplets (LDs). On the other hand, PSDHI shows significant morphological changes in LDs in glucose-incubated cancer cells, including number, volume, and refractive index. High birefringence in cancer LDs under perpendicular polarizations was observed, enabling fast discrimination with over 90% accuracy. PSDHI results align closely with Raman microscopy, suggesting its potential as a promising, high-speed technique for cancer screening purposes.

Venous thromboembolism (VTE) is a common and serious disease which encompasses deep vein thrombosis (DVT) and pulmonary embolism (PE). DVT is created when a blood clot forms in the deep veins of the leg and when the clot migrates through the bloodstream, to lung arteries, it creates a PE. VTE is the third cardiovascular cause of death overall and is responsible for 30000 annual deaths in Europe. After biological and clinical investigation, nearly half of VTE cases have no known origin (idiopathic VTE). Among the patients developing idiopathic VTE, about 30% of them would have a recurrent thromboembolic event, 70% would not be subjected to any recurrence. A balance must be struck between the risks of recurrent thrombosis if anticoagulant treatment is stopped versus the risks of bleeding associated with continued anticoagulation therapy that can go up to the course of decades. The search for new biomarkers allowing to best stear the treatment of patients is thus of major interest. Recent studies seem to link clot’s structure to a risk of recurrence. The aim of our work is to develop an innovative optical method, measuring the evolution of the scattering coefficient of a plasma during ex vivo clot formation.

Graphene constitutes a broadband energy acceptor, avoiding labeling, photobleaching and complicated photophysics. Graphene quenches fluorescence of fluorophores in a range of 0-40 nm, following a d-4 distance dependence. Due to Graphane Energy Transfer (GET) a single dye molecule shows a reduced fluorescence intensity and a shortened fluorescence lifetime as a function of its distance to graphene. This information can be used to determine the position of the dye molecule to graphene and to sensitively report on distance changes in real-time. In our first realization, we used DNA origami nanopositioners to place a fluorophore and other molecular components at a defined distance from graphene. With this approach, using single-molecule fluorescence microscopy techniques and several different assays we demonstrated among others: switching dynamics of a DNA pointer between two binding sites with high time resolution, dynamics of a flexible DNA tether influenced by viscosity or target binding, 3D superresolution imaging with isotropic nanoscale resolution, a biosensing assay with single DNA molecule detection in a novel unquenching assay format. Our recently developed tools to connect DNA and graphene enable single base-pair resolution. We use this approach to visualize structural properties of DNA which precede direct interactions with biomolecules and DNA-protein interactions.

Molecular beacons (MBs) represent a powered tool for the detection of RNAs, such as micro-RNA (miRNA) and messenger RNA (mRNA), which play an important role as indicators of the progress of different pathologies in the human body, from the chronic ones to cancer. Two examples are provided here of how the combination of molecular beacons with detection platforms based on surface enhanced Raman scattering (SERS) can lead to the realization of more performing and reliable biosensors. The first case concerns the use of a MB, engineered for specific detection of a miRNA associated with chronic obstructive pulmonary disease. Silver nanowires were used as SERS substrate on which the MBs are immobilized. A femtomolar detection limit has been reached. The second approach is based on soda-lime glass microrods on which silver nanoparticles were grown using the ion-exchange technique followed by an appropriate thermal annealing post-process. Production parameters were optimized aiming at exposing the embedded silver nanoparticles on the surface of the microrods. By functionalizing these nanoparticles with a MB specific for the mRNA for survivin, the microrods were successfully tested for SERS and fluorescence effects, allowing the detection of the complementary sequence.

In this study, we present an innovative optical biosensor designed for the precise detection of

Interleukin-6 (IL-6), a crucial cytokine associated with various pathological conditions. Our biosensor is

based on silicon porous material meticulously modified with a molecularly imprinted polymer (MIP),

ensuring specific and sensitive recognition of IL-6 molecules. Fabrication process involves the

electrochemical etching of silicon porous chips followed by the electrodeposition of MIP, tailored to

selectively bind IL-6 targets. Through rigorous testing across a range of IL-6 concentrations, our sensor

exhibits remarkable sensitivity, showcasing discernible optical responses proportional to the varying analyte

concentrations. Furthermore, we assessed the sensor's performance using bovine serum, a complex biological

matrix, to simulate real-world sample conditions. Encouragingly, the sensor maintains its selectivity and

optical response in the presence of serum components, affirming its robustness and applicability in practical

diagnostic settings.

A set of protein biomarkers are largely recognized as responsible of neurodegeneration mechanisms and hence as potential targets to be detected in low abundant concentrations in body fluids for performing early diagnosis. As an example, the Tau protein experiences a transition phase from a native disorder conformation into a preaggregation state, which leads to fibrillization processes. Here we show the possibility to detect Tau in urine samples at sub-picogram level, through the concentration effect of the pyro-electrohydrodynamic (p-jet) technique. An immunofluorescence protocol is applied to concentrated p-jet spots able to reduce drastically the diffusion effects in the antibody-antigen reaction. A set of diluted samples were prepared, and the fluorescence signal was detected by a confocal scanner. We achieved an excellent linear response with a significant signal-to-noise ratio down to 0.25 pg/mL. In perspective, the technique could be integrated into a compact device to be used for monitoring the early stage associated to neurodegenerative syndromes in different scenarios such as for example in long-term human space exploration missions.

We describe a novel one-dimensional photonic crystal design allowing for the concurrent excitation of transverse-electric and transverse-magnetic Bloch surface waves, thus paving the way for polarization-resolved sensing experiments. We discuss its application for the surface-enhanced sensing of oriented DNA molecules through nanoscale birefringence measurements.

Optical affinity biosensors with single molecule sensitivity are reported based on a combination of optical and molecular interaction - based amplification. Plasmonically-enhanced fluorescence detection that is suitable for readout of sufficiently large sensor surface areas is implemented by the use of tailored metallic nanostructures. These metallic nanostructures are chemically modified with antifouling polymer-based biointerfaces for specific capture of target molecular species. The response to specific affinity binding events is further enhanced by rolling circle amplification, through enzyme-free catalytic hairpin assembly method, and affinity mediated transfer, in order to associate the individual affinity capture target molecules with bright fluorescence spots enabling counting of affinity captured target molecules (‘digital assay format’). The presented methods will be put to context with liquid biopsy – based lung and melanoma cancer diagnostics through ultrasensitive detection of trace amounts of specific biomarkers circulating in blood.

Support from Gesellschaft für Forschungsförderung Niederösterreich m.b.H. project LS20-014 ASPIS, Austrian Science Fund via the project DIPLAB (I 5119-B and 21-16729K), Czech Science Fund through the project APLOMA (22- 30456J), European Union’s Horizon Europe program project VerSiLiB (No 101046217) and Operational Programme Johannes Amos Comenius financed by European Structural and Investment Funds and the MEYS (Project No. SENDISO -CZ.02.01.01/00/22_008/0004596) is acknowledged.

Over recent decades, metallic nanostructures have emerged as pivotal components in biosensing applications due to their exceptional optical transduction properties. In this study, we present a novel approach by integrating hybrid plasmonic/dielectric materials to enhance the sensitivity of large-scale plasmonic arrays. Specifically, we propose refining the fabrication process for Gold Nanomushrooms by incorporating dielectric Silicon Nitride instead of traditional pillars. This strategic modification aims to yield sensors with a heightened responsiveness to refractive index variation, tailored to address the needs of biomedical applications.

A Metal-Enhanced Fluorescence (MEF) based immunosensor has been developed for the detection of prostate-specific antigen (PSA), a crucial biomarker for prostate cancer. The biosensor consists of gold nanoparticles (AuNPs) randomly immobilized onto a glass substrate via an electrostatic self-assembly technique. A sandwich scheme has been employed for PSA recognition, with antibodies as capture bioreceptors covalently immobilized onto the AuNPs surface and a top fluorescently labeled bioreceptors layer. The biosensor has been tested on real samples, successfully detecting PSA in human serum within a rapid 40-minute assay time, achieving a limit of detection (LOD) of 150 pg·mL-1.

We recently developed a peculiar disaggregation‒based colorimetric sensing scheme where stable and reversible clusters of functionalized Core@satellite magnetic nanoparticles (CSMPs) are first created by surfactant‒induced depletion‒related forces and then fragmented in response to the detection events. The fragmentation of CSMPs clusters entails an unexpected increase of the extinction intensity of the colloids. Here, we provide a theoretical foundation for such optical behaviour by first modelling both the CSMPs and CSMPs clusters, and then investigating their optical response by finite-difference time-domain simulations.

This paper presents the design and implementation of a tailored optical system for rapid characterization of transmission-based localized surface plasmon resonance (LSPR) biosensors. The system incorporates a motorized monochromator, offering extreme versatility in experimenting with wavelength resolution and bandwidth of the illumination. This setup streamlines the process of determining optimal chip design parameters such as the density of nanoparticles and the number of detection wavelengths required, as measurements are fully automated and software-driven. Furthermore, the system facilitates straightforward detection of manufacturing errors, including misaligned spots or inhomogeneous particle distribution, enhancing overall efficiency and reliability in biosensor evaluation.