Non-invasive Optical Method Research Articles

Non-Invasive Functional Optical Brain Imaging Methods: A Review

Non-Invasive Functional Optical Brain Imaging Methods: A Review

Therapeutic Assessment of High-Intensity Focused Ultrasound for Vulvar Lichen Sclerosus by Active Dynamic Thermal Imaging and Hyperspectral Imaging—A Preliminary Study

Vulvar lichen sclerosus (VLS) is a common inflammatory condition associated with an increased risk of developing vulvar carcinoma. Recently, high-intensity focused ultrasound (HIFU) has been identified as a promising treatment modality for VLS in several clinical trials. However, in HIFU therapy, therapeutic outcome is routinely assessed visually by an expert using standardized grading criteria. Furthermore, such a therapeutic assessment cannot be made until at least three months after treatment. Therefore, an objective and timely method capable of quantitatively evaluating HIFU treatment effectiveness is desired, which may help identify patients whose treatment are insufficient in time and prevent the delay of re-treatment. The purpose of this study is to investigate the feasibility of using active dynamic thermal imaging (ADT) and hyperspectral imaging method (HSI) as two individual objective and non-invasive optical methods for quantitative assessment of the therapeutic response of HIFU treatment for VLS, promptly. From December 2018 to March 2019, 20 female patients of VLS, who underwent HIFU treatment, were evaluated using both ADT and HSI methods. Effective damage rate from ADT method and entropy of feature index from HSI method were used to develop a multivariate linear discriminant classification model for grading the effectiveness of HIFU treatment in comparison with clinical evaluation gold standard. Finally, ADT was able to correctly differentiate ineffective treatments from effective ones with a sensitivity of 100% and specificity of 100% while the sensitivity and specificity of HSI were 75% and 87.5%, respectively. The classification results demonstrate the clinical potential of ADT and HSI methods for non-invasive and quantitative assessment of HIFU treatment for VLS in time.

Blood flow rate estimation in optic disc capillaries and vessels using Doppler optical coherence tomography with 3D fast phase unwrapping.

The retinal volumetric flow rate contains useful information not only for ophthalmology but also for the diagnosis of common civilization diseases such as diabetes, Alzheimer's disease, or cerebrovascular diseases. Non-invasive optical methods for quantitative flow assessment, such as Doppler optical coherence tomography (OCT), have certain limitations. One is the phase wrapping that makes simultaneous calculations of the flow in all human retinal vessels impossible due to a very large span of flow velocities. We demonstrate that three-dimensional Doppler OCT combined with three-dimensional four Fourier transform fast phase unwrapping (3D 4FT FPU) allows for the calculation of the volumetric blood flow rate in real-time by the implementation of the algorithms in a graphics processing unit (GPU). The additive character of the flow at the furcations is proven using a microfluidic device with controlled flow rates as well as in the retinal veins bifurcations imaged in the optic disc area of five healthy volunteers. We show values of blood flow rates calculated for retinal capillaries and vessels with diameters in the range of 12-150 µm. The potential of quantitative measurement of retinal blood flow volume includes noninvasive detection of carotid artery stenosis or occlusion, measuring vascular reactivity and evaluation of vessel wall stiffness.

Race-specific differences in the phase coherence between blood flow and oxygenation: A simultaneous NIRS, white light spectroscopy and LDF study.

Race-specific differences in the level of glycated hemoglobin are well known. However, these differences were detected by invasive measurement of mean oxygenation, and their understanding remains far from complete. Given that oxygen is delivered to the cells by hemoglobin through the cardiovascular system, a possible approach is to investigate the phase coherence between blood flow and oxygen transportation. Here we introduce a noninvasive optical method based on simultaneous recordings using NIRS, white light spectroscopy and LDF, combined with wavelet-based phase coherence analysis. Signals were recorded simultaneously for individuals in two groups of healthy subjects, 16 from Sub-Saharan Africa (BA group) and 16 Europeans (CA group). It was found that the power of myogenic oscillations in oxygenated and de-oxygenated hemoglobin is higher in the BA group, but that the phase coherence between blood flow and oxygen saturation, or blood flow and hemoglobin concentrations is higher in the CA group.

Monitoring Charge Carrier Diffusion across a Perovskite Film with Transient Absorption Spectroscopy.

We have developed a new noninvasive optical method for monitoring charge carrier diffusion and mobility in semiconductor thin films in the direction perpendicular to the surface which is most relevant for devices. The method is based on standard transient absorption measurements carried out in reflectance and transmittance modes at wavelengths below the band gap where the transient response is mainly determined by the change in refractive index, which in turn depends on the distribution of photogenerated carriers across the film. This distribution is initially inhomogeneous because of absorption at the excitation wavelength and becomes uniform over time via diffusion. By modeling these phenomena we can determine the diffusion constant and respective mobility. Applying the method to a 500 nm thick triple cation FAMACs perovskite film revealed that homogeneous carrier distribution is established in few hundred picoseconds, which is consistent with mobility of 66 cm2 (V s)−1.

The fluorescent dye 3,3′-diethylthiatricarbocyanine iodide is unsuitable for in vivo imaging of myelination in the mouse

There is a growing interest to use non-invasive optical imaging methods to study central nervous system diseases. The application of a myelin-binding fluorescent dye, 3,3-diethylthiatricarbocyanine iodide (DBT) was recently described for in vivo optical imaging of demyelination in the mouse. In the present study we aimed at adapting the method to our optical imaging systems, the IVIS Lumina II to measure epifluorescence and the fluorescent molecular tomograph (FMT) for 3-dimensional quantification of the fluorophore.Epifluorescent imaging was performed 5−30 min after DBT injection which was followed by FMT imaging at 40 min. Two mice also underwent micro-CT imaging in the FMT cassette for the purpose of FMT-CT co-registration. Ex vivo imaging of the brain and other tissues of the head and neck was carried out 1 h after injection.Both the FMT-CT co-registration and the ex vivo imaging of organs proved that DBT poorly crossed the blood-brain barrier. The dye did not accumulate in the myelin sheath of the sciatic nerve. In contrast, there was an intense accumulation in the pituitary and salivary glands. The FMT-CT co-registration unequivocally demonstrated that the signal localized to the head did not originate from beyond the blood-brain barrier. No myelin binding was demonstrated by the ex vivo imaging either.In conclusion, DBT is unsuitable for in vivo imaging of myelination due to its poor BBB penetration, accumulation in other structures of the head and neck region and lack of selective binding towards myelin in vivo.

Application of hyperspectral imaging for analysis of embryonic development of lower vertebrates

The task of analyzing the characteristics of the embryonic development of lower vertebrates using non-invasive optical methods is considered. We propose to apply hyperspectral imaging for segmentation of embryo zones with different physical and chemical properties. This approach allows one to obtain information on the spatial distribution of optical spectral properties of the observed object. An experimental setup based on a microscope coupled with a spectral imaging add-on module utilizing a tunable acousto-optic filter has been created to implement this approach. Hyperspectral data arrays are formed as a series of spectral narrowband images obtained by successive tuning of the acousto-optic filter within the working spectral range. A series of experiments was conducted in which the development of embryos of the Misgurnus fossilis loach during their transition from the 32nd to the 33rd developmental stage was observed using the described experimental setup. Characteristic spatial regions corresponding to certain parts of the loach embryo were identified. The time dependences characterizing the change in the spectral properties of the selected regions were obtained. It is shown that joint processing of a series of spectral images obtained in different spectral bands within the working spectral range of an acousto-optical spectral imaging module allows one to effectively identify differences in the kinetics of the optical transmission spectral density for various regions of the axial mesoderm.

Технологии неинвазивных оптических методов определения уровня глюкозы в крови

The article studies the development of non-invasive optical methods for determining the concentration of glucose in the blood of patients with diabetes. Early diagnosis and day-to-day monitoring is essential to ensure a healthy life for patients with diabetes. Determining the concentration of glucose in the blood using conventional devices includes a chemical analysis of blood samples that are obtained by pricking a finger or extracting blood from the forearm. The pain, discomfort and inconvenience associated with modern invasive methods necessitated the study of non-invasive measurement methods. Non-invasive monitoring of blood glucose offers several advantages, including the absence of pain and biohazardous materials, the absence of sharp objects, an increase in the frequency of testing and, therefore, tighter control of glucose concentration. Given these potential benefits, the commercialization of non-invasive glucose monitoring devices has become a subject of increased interest. Some optical technologies may provide viable, non-invasive measuring devices. The purpose of this review study was to describe the main optical technologies for non-invasive monitoring of glucose and to compare their advantages and disadvantages.

(Invited) Optics-Based Wearable Device for Real-Time Monitoring of Newborn Jaundice

NoveltyA wearable optical device for real-time monitoring of newborn jaundice was developed (Fig. 1). Jaundice is caused by the accumulation of bilirubin in an infant's blood. Bilirubin levels measured using the newly developed sensor were comparable to those measured using a commercial jaundice meter(R = 0.86). Real-time monitoring of the bilirubin level in infants is required because the level can change rapidly and affect their growth. The real-time monitor evaluates jaundice by calculating the ratio of the reflected light intensity between blue (470 nm) and green (570 nm) wavelengths. The data can be transferred conveniently to a smartphone. The ability of the newly developed sensor to measure bilirubin levels was comparable to that of a commercial jaundice device (R = 0.86), indicating the sensor could successfully evaluate jaundice. This wearable sensor is an important advance in the healthcare of infants, as it provides real-time monitoring of jaundice, one of the most important medical parameters in preliminary medical screening tests for newborns. BackgroundThe liver of an infant is immature, and its function must be monitored periodically. An important parameter indicating liver function is bilirubin, which is generated during the breakdown of hemoglobin. The immature liver of an infant cannot breakdown the generated bilirubin quickly enough. As a result, bilirubin accumulates in the skin tissue, resulting in jaundice. Without appropriate treatment, bilirubin is deposited into the brain, which can lead to brain damage. Moreover, permissible bilirubin levels are dependent on the baby’s age. Therefore, it is crucial to measure bilirubin levels continuously in newborns in order to discover symptoms of jaundice as early as possible. Conventionally, bilirubin levels are measured using a blood sampling method[1] or an optical method[2]. The blood sampling method can measure bilirubin levels with high accuracy; however, this method is invasive. In light of this, the non-invasive optical method is more commonly used. However, even with the optical method, it is difficult to measure bilirubin levels continuously because conventional optical devices are large and burdensome to medical staff and infants. Furthermore, it is impossible for these two methods to detect a sudden change in the bilirubin level. The present study proposes the development of a wearable jaundice monitor that can be worn on the forehead of an infant and measure bilirubin levels in real time. Device Fabrication and Experimental ResultsAs shown figure 1, the wearable optical jaundice sensor was miniaturized by using two light-emitting diodes (LEDs) of different wavelengths (blue and green) and four photodiodes (PDs)(Fig. 1b). The flexible polyimide substrate of the device fabricated by soft lithography enables conformal coverage of the electronic components on soft, curved surfaces of the skin(Fig. 1c). Transmitted light from the LEDs irradiates the skin surface, and reflected light is detected by the PDs. The sensor detects the difference in the intensity of the reflected blue and green light. In addition, a wireless function, using a Bluetooth wireless network and original smartphone/Tablet applications, permits real-time measurements. The blue light is highly associated with the concentration of bilirubin, compared to the green light. For measurements of the reflected blue and green light, the LEDs are alternately illuminated in 40 ms. Calculation of the bilirubin concentration from the differences in the intensities is based on the Beer-Lambert Law. In a preliminary experiment, the calculated ratio of reflected light intensities was associated with the value measured using a commercial jaundice meter (R = 0.86). The present work represents an important step towards safer, more advanced technologies for infant healthcare made possible by the Internet of Things. Figure 1: Photograph of an actual wearable jaundice device. a. The package of the device is made of silicone rubber. b, c. Electrical components are mounted on flexible polyimide substrate. Optical sensing part is composed of four PDs, blue and green LEDs. Figure 1

Multimodal Optical Diagnostics of the Microhaemodynamics in Upper and Lower Limbs.

The introduction of optical non-invasive diagnostic methods into clinical practice can substantially advance in the detection of early microcirculatory disorders in patients with different diseases. This paper is devoted to the development and application of the optical non-invasive diagnostic approach for the detection and evaluation of the severity of microcirculatory and metabolic disorders in rheumatic diseases and diabetes mellitus. The proposed methods include the joint use of laser Doppler flowmetry, absorption spectroscopy and fluorescence spectroscopy in combination with functional tests. This technique showed the high diagnostic importance for the detection of disturbances in peripheral microhaemodynamics. These methods have been successfully tested as additional diagnostic techniques in the field of rheumatology and endocrinology. The sensitivity and specificity of the proposed diagnostic procedures have been evaluated.

Animal species identification in parchments by light

Recently, historical and conservation studies have attached an increasing importance to investigating the materials used in historic documents. In particular, the identification of the animal species from which parchments are made is of high importance and is currently performed by either genetic or proteomic methods. Here, we introduce an innovative, non-invasive optical method for identifying animal species based on light-parchment interaction. The method relies on conservation of light energy through reflection, transmission and absorption from the sample, as well as on statistical processing of the collected optical data. Measurements are performed from ultraviolet (UV) to near-infrared (NIR) spectral ranges by a standard spectrophotometer and data are processed by Principal Component Analysis (PCA). PCA data from modern parchments, made of sheep, calf and goat skins, are used as a database for PCA analysis of historical parchments. Using only the first two principal components (PCs), the method confirmed visual diagnostics about parchment appearance and aging, and was able to recognise the origin species of historical parchment of among database clusters. Furthermore, taking into account the whole set of PCs, species identification was achieved, with all results matching perfectly their proteomic counterparts used for method assessment. The validated method compares favourably with genetic and proteomic methods used for the same purpose. In addition to animals’ proteomic and genetic signatures, a unique “optical fingerprint” of the parchments’ origin species is revealed here. This new method is non-invasive, straightforward to implement, potentially cheap and accessible to scholars and conservators, with minimal training. In the context of cultural heritage, the method could help solving questions related to parchment production and, more generally, medieval writing production.

Estimating the transverse dimensions of cellulose fibres in wood and paper using 2D and 3D microscopy techniques

We describe a duo of experimental methods for determining the transverse dimensions of individual wood fibres using 2D optical microscopy and 3D X-ray tomography. These non-invasive optical sectioning methods, in conjunction with segmentation through image analysis, enables in situ identification of individual fibres in a paper sheet. The relationship between local fibre dimensions, i.e. diameter and fibre wall thickness, are established for kraft (NBSK) pulp fibres and compared to values obtained from wood. For the first time, we propose a criterion for transverse fibre collapse during the papermaking process that is based on both fibre diameter and wall thickness.

QTL Mapping of Diffuse Reflectance Indices of Leaves in Hexaploid Bread Wheat (Triticum aestivum L.)

Quantitative trait loci (QTL) mapping of diffuse reflectance indices of laminas in bread wheat (Triticum aestivum L.) has been first performed under controlled conditions of a regulated agroecobiological testing ground in the presence or absence of nitrogen fertilizers. Indices chosen for the study determine a range of important characteristics, such as the content of chlorophylls and anthocyanins, carotenoid/chlorophyll ratio, photochemical activity of the photosynthetic apparatus, light scattering on a lamina, assimilating leaf surface area, and grain productivity. In total, 31 QTLs have been mapped. A significant correlation has been revealed between the introduction of a nitrogen fertilizer and the five of six optical characteristics of the photosynthetic apparatus activity in bread wheat. The only exception is the reflectance index for near-infrared radiation (800 nm), which depends on the structural features of leaf tissues. No statistically significant correlation has been revealed between the thousand-grain weight and spectral characteristics of the diffuse reflectance of the lamina measured at the booting stage. However, a significant correlation between the number of grains formed in the spike of the main stalk and the traits characterizing activity of the photosynthetic apparatus (reflectance indices, leaf area) has been observed. Results of the performed variance, correlation, and QTL analyses confirm each other indicating reliability of the revealed effect of nitrogen nutrition level on the manifestation of the studied reflectance indices in bread wheat under strictly controlled conditions of an agroecobiological testing ground. Application of noninvasive optical methods provides a high-throughput assessment of photosynthetic intensity in plants and, therefore, can be used for efficient selection of promising wheat genotypes with high grain productivity under both controlled and field conditions.

Morphine Attenuates fNIRS Signal Associated With Painful Stimuli in the Medial Frontopolar Cortex (medial BA 10).

Functional near infrared spectroscopy (fNIRS) is a non-invasive optical imaging method that provides continuous measure of cortical brain functions. One application has been its use in the evaluation of pain. Previous studies have delineated a deoxygenation process associated with pain in the medial anterior prefrontal region, more specifically, the medial Brodmann Area 10 (BA 10). Such response to painful stimuli has been consistently observed in awake, sedated and anesthetized patients. In this study, we administered oral morphine (15 mg) or placebo to 14 healthy male volunteers with no history of pain or opioid abuse in a crossover double blind design, and performed fNIRS scans prior to and after the administration to assess the effect of morphine on the medial BA 10 pain signal. Morphine is the gold standard for inhibiting nociceptive processing, most well described for brain effects on sensory and emotional regions including the insula, the somatosensory cortex (the primary somatosensory cortex, S1, and the secondary somatosensory cortex, S2), and the anterior cingulate cortex (ACC). Our results showed an attenuation effect of morphine on the fNIRS-measured pain signal in the medial BA 10, as well as in the contralateral S1 (although observed in a smaller number of subjects). Notably, the extent of signal attenuation corresponded with the temporal profile of the reported plasma concentration for the drug. No clear attenuation by morphine on the medial BA 10 response to innocuous stimuli was observed. These results provide further evidence for the role of medial BA 10 in the processing of pain, and also suggest that fNIRS may be used as an objective measure of drug-brain profiles independent of subjective reports.

Visualization of flow conditions inside spiral jet mills with different nozzle numbers– Analysis of unloaded and loaded mills and correlation with grinding performance

Plenty of operational and geometric parameters have effects on spiral jet milling. Changes in the parameters cause distinctions in the flow conditions inside the mill and thereby also lead to consequences regarding the milled product. A new type of experimental spiral jet mill apparatus with almost entire optical accessibility enables a very convenient variation of the operational and geometric parameters as well as the determination of the flow conditions inside the spiral jet mill via non-invasive optical methods. Particle Image Velocimetry (PIV) measurements with diethylhexyl sebacat (DEHS) tracer droplets as well as solid barium sulphate micro particles were carried out to investigate the flow conditions inside the spiral jet mill. The differences between the net milling gas velocity fields and the velocity fields of loaded spiral jet mills were exposed. With the mill being loaded, the comminution zone decreases in the horizontal direction as well as decreases and deflects in the vertical direction. Besides, the analysis of the velocity fields inside the mill apparatus showed that decreasing milling nozzle numbers lead to increasing velocities inside the spiral jet mill, even if the mass flow rate of the gas supply is kept constant and the nozzle jet velocity is limited to sonic velocity at choked flow. With decreasing milling nozzle numbers, broader comminution zones and increasing lengths of the milling jets were investigated. As a consequence, a better grinding efficiency was expected and also confirmed with decreasing milling nozzle number by grinding experiments, as the new type of experimental apparatus was constructed in a fully operative way concerning the grinding ability.

Measuring the velocity fields of granular flows – Employment of a multi-pass two-dimensional particle image velocimetry (2D-PIV) approach

Measuring velocity fields plays a crucial role in investigating the dynamics of granular flows, which can improve the modeling of hazardous geophysical flows (e.g. avalanches and debris flows) and the control of powder flows in industrial applications. Non-invasive optical methods are invaluable tools for estimating this physical quantity at the laboratory scale. Despite the recent improvements of particle image velocimetry (PIV) algorithms, the employment of PIV to granular flows is still a non-trivial application, where there are several specific aspects to be carefully addressed. Here, we address the main challenges of granular PIV applications and systematically test the open-source window deformation multi-pass code, PIVlab [Thielicke and Stamhuis, J. Open Res. Soft., 2014], for dry granular flows in rotating drum and chute flow experiments. Three granular media (glass spheres, Ottawa sand and acetalic resin beads) with different optical properties are used as a broad test bench for validating the PIV approach. As well, comparisons between the estimations by PIVlab and those obtained by the commercial code, IDT ProVision-XS, are reported, where the advantages of the multi-pass approach are highlighted. This extensive experimental investigation allowed the evaluation of the accuracy of PIVlab in granular flow applications and also helped to assess the reliability of measurements of second-order statistics, such as the granular temperature. Finally, a guideline for setting a reliable PIV arrangement is suggested.

Blood pressure estimation from appropriate and inappropriate PPG signals using A whole-based method

Abstract Background and objective Blood pressure (BP) is one of the four vital signals that provides valuable medical information about the cardiovascular activity. In recent years, extensive studies have been conducted on non-invasive and cuff-less BP estimation using photoplethysmography (PPG) signals. PPG is a non-invasive optical method for measuring blood volume changes per pulse. In other words, the PPG waveform represents the mechanical activity of the heart. Methods In this paper, a new method for estimating the Mean Arterial Pressure (MAP), Diastolic Blood Pressure (DBP) and Systolic Blood Pressure (SBP) is proposed using only the PPG signal regardless of its shape (appropriate or inappropriate). Our proposed algorithm called whole-based, uses raw values of the PPG signal at a given time interval for estimating the BP. In other algorithms called parameter-based, use features which are extracted from PPG signals in time or frequency domain. These features related to precise spotting in the form of the PPG signal. In fact, compared to parameter-based methods, our algorithm is independent of the form of the PPG signal. Results Using the proposed algorithm, our results are completely met by the Association for the Advancement of Medical Instrumentation (AAMI) standard for both MAP and DBP estimations. The results are also very close to the standard boundary with an average error close to zero for SBP estimation. Also, according to the British Hypertension Society (BHS) standard, the proposed algorithm for DBP estimation got grade A, whereas it got grade B for estimation of MAP and got approximately grade C for SBP estimation. Conclusion The results demonstrate the applicability of the proposed algorithm in estimating BP noninvasively, cuff-less, calibration-free, and only by using the appropriate or inappropriate PPG signal.

Plasmonic Optical Imaging of Gold Nanorods Localization in Small Animals

Gold nanoparticles (GNP) have been intensively investigated for applications in cancer imaging and therapy. Most imaging studies focused on microscopic imaging. Their potential as optical imaging probes for whole body small animal imaging has rarely been explored. Taking advantage of their surface plasmon resonance (SPR) properties, we aim to develop a noninvasive diffuse optical imaging method to map the distribution of a special type of GNP, gold nanorods (GNR), in small animals. We developed an integrated dual-modality imaging system capable of both x-ray computed tomography (XCT) and diffuse optical tomography (DOT). XCT provides the animal anatomy and contour required for DOT; DOT maps the distribution of GNR in the animal. This SPR enhanced optical imaging (SPROI) technique was investigated using simulation, phantom and mouse experiments. The distribution of GNR at various concentrations (0.1–100 nM, or 3.5 ug/g–3.5 mg/g) was successfully reconstructed from centimeter-scaled volumes. SPROI detected GNR at 18 μg/g concentration in the mouse breast tumor, and is 3 orders more sensitive than x-ray imaging. This study demonstrated the high sensitivity of SPROI in mapping GNR distributions in small animals. It does not require additional imaging tags other than GNR themselves. SPROI can be used to detect tumors targeted by GNR via passive targeting based on enhanced permeability and retention or via active targeting using biologically conjugated ligands.

Non-invasive monitoring of hydraulic surge propagation in a wounded tobacco plant

BackgroundWhen a plant is wounded, a rapid hydraulic surge, acting probably as a systemic signal, spreads from the site of injury throughout the plant and leads to small transient deformation of tissues. So far, the propagation of hydraulic surge has been monitored by contact and thus potentially invasive methods.ResultsHere we present a non-invasive optical method, which allows simultaneous monitoring of micrometric shift of two opposite stem margins. The usefulness of this method was demonstrated by the measurement of the hydraulic surge propagation in a tobacco (Nicotiana tabacum (L.) cv. Samsun) after burning of its upper leaf. We have observed transient narrowing the stem below the burned leaf, which started within a few minutes after local burning. The comparison of the shift of the stem margin following vascular trace of the burned leaf and the margin on the opposite side of the stem has revealed that the stem deformation is highly asymmetric.ConclusionsThis optical method represents a novel tool to investigate the mechanism of systemic response of plants to local damage. Our results points out the complexity of the relationship between hydraulic surge propagation and stem deformation.

Fluid-Imbibition Coupled Interferometry Study of Surface Modifications in Nanoporous Anodic Alumina for Biosensing

Nanoporous anodic alumina (NAA) is a material with great perspectives in nanotechnology due to its cost-effective fabrication, its chemical stability and its versatility in applications such as biosensing or drug delivery[1]. Optical sensing with NAA has gained much attention in recent years[2,3]. Nano-optofluidic methods[4] based on a solid carrier matrix, a liquid carrying analytes and on the physico-chemical characteristics at their interfaces have been proven sensitive for substances as small as nanoparticles, viruses[5] and even ions[6]. Fluid imbibition-coupled laser interferometry (FICLI)[7] is an optofluidic technique that permits the investigation of the surface and geometrical properties of nanoporous structures by non-invasive optical methods. It consists of the measurement of interferometric light intensity fluctuations in a beam reflected in a nanoporous thin film membrane as it is being filled by a liquid. In FICLI, time-resolved interferograms are measured and then analyzed on the basis of mathematical models of the filling kinetics. With this the surface properties and the pore internal geometries can be estimated. Recently, we established for FICLI an accuracy better than 2 nm in determining changes in NAA pore radius after different modifications involving radius increase (by chemical wet etching) or decrease (by the deposition of layer-by-layer coatings). Such accuracy leads to consider NAA and FICLI as a platform to develop biosensing systems. Such application depends critically on the functionalization of the pore surface, as it is necessary to enable the selective immobilization of the desired biomolecules. In this work, we investigate the influence of three different NAA functionalization methods on the FICLI pore radius estimate: i) electrostatic binding of bovine serum albumin (BSA), ii) covalent binding for the attachment of streptavidin and biotin and iii) specific immune complexation of antibody-antigen pairs. The study was carried on NAA membranes produced in both oxalic and sulfuric acid electrolytes, and together a chemical wet etching step for a given amount of time (between 0 min. and 24 min.) permits to use a wide range of pore dimensions (between 15 nm and 44 nm). NAA inner pore surface was modified in different steps and the FICLI technique was applied to estimate the changes in filling dynamics and the consequent pore radius change after each step. For the electrostatic attachment of BSA, NAA membranes with different pore radius were used as-produced and measured before and after incubation in a BSA solution. For the covalent attachment of streptavidin, NAA samples with 40 nm initial pore radius were investigated after two steps: i) silanization with (3-aminopropyl)-triethoxysilane (APTES) with cross-linking activation with glutaraldehyde (GTA) and ii) incubation with streptavidin. Finally, immune complexation within NAA pores was tested on NAA samples with 40 nm initial pore radius attaching different kinds of immunoglobulin (IgG) to the inner pore surface. Several experiments were conducted, in all of them the first step was to attach electrostatically protein A, which has the ability to link with antibody or antigen portions. Then, samples were incubated following one of three possibilities: i) human IgG, ii) anti-human IgG and iii) anti-goat IgG from rabbit followed by incubation with goat IgG. Results show that for the case of the electrostatic attachment, FICLI is able to detect the formation of a BSA layer on the NAA pores, independently of the initial pore radius with slight differences observed for different electrolyte used in the preparation. For covalent immobilization of streptavidin, FICLI estimates a much bigger radius reduction than expected from the molecule size, what can be explained by a multi-layer deposition of streptavidin. Finally, FICLI is able to demonstrate that different IgG attach differently to the NAA pores with a determinant influence of the pore size. ACKNOWLEDGMENT This work was supported by the Spanish MINECO (TEC2015-71324-R), the Catalan authority AGAUR (2014SGR1344), and ICREA under the ICREA Academia Award. REFERENCES [1] J. Ferré-Borrull, E. Xifré-Pérez, J. Pallarès and L.F. Marsal in “Nanoporous Alumina: Fabrication, Structure, Properties and Applications”; Santos, A., Losic, D., Eds.; Springer-Verlag Berlin, 2015, p. 185. [2] G. Macias, J. Ferré-Borrull, J. Pallarès and L.F. Marsal, Analyst 140, 2015, p. 4848. [3] T. Kumeria, A. Santos, M.M. Rahman, J. Ferré-Borrull, L.F. Marsal, D. Losic, ACS Photonics 1, 2014, p. 1298. [4] D. Psaltis, S.R. Quake, C.H. Yang, Nature 442, 2006, p. 381. [5] A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, L. Novotny, ACS Nano 23, 2010, p. 1. [6] T. Kumeria, M.M. Rahman, A. Santos, J. Ferré-Borrull, L.F. Marsal, D. Losic, ACS applied materials & interfaces 6, 2014, p. 12971. [7] C. Eckstein, E. Xifré-Pérez, M. Porta-i-Batalla, J. Ferré-Borrull, L.F. Marsal, Langmuir 32, 2016, p. 10467.