This PDF file contains the front matter associated with SPIE Proceedings Volume 9529, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

The minimally-invasive quantitative observation of different cell types in a single culture is of particular interest for the analysis of the impact of pharmaceuticals, pathogens or toxins on different cellular phenotypes under identical measurement conditions and to analyze interactions between different cellular specimens. Quantitative phase microscopy (QPM), provides high resolution detection of optical path length changes that is suitable for quantitative tomographic imaging and stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and thus is in particular suitable for long term time-lapse investigations on cells in which for example morphology alterations due to toxins, drugs or genetic modifications are studied. We analyzed the feasibility of QPM, for the analysis of mixed cell cultures and explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with digital holographic microscopy (DHM) was utilized. Mixed cell cultures with different cell types were continuously observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted image segmentation algorithms. From the segmented quantitative phase images the area covered by the cells, the cellular dry mass as well as the mean cell thickness and volume were determined and used as parameters to quantify the reliability of data acquisition. The obtained results demonstrate that it is possible to characterize the growth of cell types with different morphology features separately in a single cell culture. This prospects new application fields of quantitative phase imaging in drug and toxicity testing in vitro.

Wide field, lensless microscopes have been developed for telemedicine and for resource limited setting [1]. They are based on in-line digital holography which is capable to provide amplitude and phase information resulting from numerical reconstruction. The phase information enables achieving axial resolution in the nanometer range. Hence, such microscopes provide a powerful tool to determine three-dimensional topologies of microstructures. In this contribution, a compact, low-cost, wide field, lensless microscope is presented, which is capable of providing topological profiles of microstructures in transparent material. Our setup consist only of two main components: a CMOSsensor chip and a laser diode without any need of a pinhole. We use this very simple setup to record holograms of microobjects. A wide field of view of ~24 mm², and a lateral resolution of ~2 μm are achieved. Moreover, amplitude and phase information are obtained from the numerical reconstruction of the holograms using a phase retrieval algorithm together with the angular spectrum propagation method. Topographic information of highly transparent micro-objects is obtained from the phase data. We evaluate our system by recording holograms of lines with different depths written by a focused laser beam. A reliable characterization of laser written microstructures is crucial for their functionality. Our results show that this system is valuable for determination of topological profiles of microstructures in transparent material.

Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn’s disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

Cellular morphology changes and volume alterations play significant roles in many biological processes and they are mirrors of cell functions. In this paper, we propose the Digital Holographic microscope (DH) as a non-invasive imaging technique for a rapid and accurate extraction of morphological information related to cell death. In particular, we investigate the morphological variations that occur during necrosis and apoptosis. The study of necrosis is extremely important because it is often associated with unwarranted loss of cells in human pathologies such as ischemia, trauma, and some forms of neurodegeneration; therefore, a better elucidation in terms of cell morphological changes could pave the way for new treatments. Also, apoptosis is extremely important because it’s involved in cancer, both in its formation and in medical treatments. Because the inability to initiate apoptosis enhances tumour formation, current cancer treatments target this pathway. Within this framework, we have developed a transmission off-axis DH apparatus integrated with a micro incubator for investigation of living cells in a temperature and CO2 controlled environment. We employ DH to analyse the necrosis cell death induced by laser light (wavelength 473 nm, light power 4 mW). We have chosen as cellular model NIH 3T3 mouse embryonic fibroblasts because their adhesive features such as morphological changes, and the time needed to adhere and spread have been well characterized in the literature. We have monitored cell volume changes and morphological alterations in real time in order to study the necrosis process accurately and quantitatively. Cell volume changes were evaluated from the measured phase changes of light transmitted through cells. Our digital holographic experiments showed that after exposure of cells to laser light for 90-120 min., they swell and then take on a balloon-like shape until the plasma membrane ruptures and finally the cell volume decreases. Furthermore, we present a preliminary study on the variation of morphological parameters in case of cell apoptosis induced by exposure to 10 μM cadmium chloride. We employ the same cell line, monitoring the process for 18 hours. In the vast group of environmental pollutants, the toxic heavy metal cadmium is considered a likely candidate as a causative agent of several types of cancers. Widely distributed and used in industry, and with a broad range of target organs and a long half-life (10-30 years) in the human body, this element has been long known for its multiple adverse effects on human health, through occupational or environmental exposure. In apoptosis, we measure cell volume decrease and cell shrinking. Both data of apoptosis and necrosis were analysed by means of a Sigmoidal Statistical Distribution function, which allows several quantitative data to be established, such as swelling and cell death time, flux of intracellular material from inside to outside the cell, initial and final volume versus time. In addition, we can quantitatively study the cytoplasmatic granularity that occurs during necrosis. As a future application, DH could be employed as a non-invasive and label-free method to distinguish between apoptosis and necrosis in terms of morphological parameters.

In this contribution we present a low cost, extremely simple, and highly stable scheme to update a standard microscope into a holographic microscope. The proposed architecture is named as SMIM (incoming from the initials of spatially-multiplexed interferometric microscopy) and it is based on a common-path interferometric configuration which is adapted into a conventional microscope with some specific constraints to allow holographic recording. The main layout modifications are three: i) the use of a coherent light source instead of the broadband one included in the microscope, ii) the insertion of a properly placed one-dimensional diffraction grating needed for the holographic recording, and iii) the use of spatial multiplexing at the input plane to allow reference beam transmission in a common light-path with the imaging branch. As consequence of the input plane spatial multiplexing, the field of view provided by the used microscope objective is reduced in comparison with the field of view reported by the same objective lens in conventional white light illumination mode. However, complex amplitude distribution of the inspected sample is retrieved after off-axis holographic recording and conventional digital image processing of the recorded hologram. The proposed update is experimentally validated in a standard upright microscope by using coherent illumination incoming from a commercial grade laser diode, by inserting a one-dimensional precision Ronchi ruling grating in the microscope embodiment, and by dividing the input plane into the spatially multiplexed areas. Experimental results are provided for the different microscope objectives included in the Olympus microscope showing calibration (USAF resolution test) as well as biological (red blood cells and sperm cells) images containing complex (amplitude and phase) information.

In this contribution we introduce MISHELF microscopy, a new concept and design of a lensless holographic microscope based on wavelength multiplexing, single hologram acquisition and digital image processing. The technique which name comes from Multi-Illumination Single-Holographic-Exposure Lensless Fresnel microscopy, is based on the simultaneous illumination and recording of three diffraction patterns in the Fresnel domain. In combination with a novel and fast iterative phase retrieval algorithm, MISHELF microscopy is capable of high-resolution (micron range) phase-retrieved (twin image elimination) biological imaging of dynamic events (video rate recording speed) since it avoids the time multiplexing needed for the in-line hologram sequence recording when using conventional phase-shifting or phase retrieval algorithms. MISHELF microscopy is validated using two different experimental layouts: one using RGB illumination and detection schemes and another using IRRB as illumination while keeping the RGB color camera as detection device. Preliminary experimental results are provided for both experimental layouts using a synthetic object (USAF resolution test target).

Digital Holography (DH) in microscopy allows to retrieve in an accurate way the spatial coordinates of multiple moving particles, performing 3D tracking of the sample in the entire field of view. In particular, a posteriori quantitative multifocus phase-contrast imaging, suitable for 3D tracking of micro-objects, is one of the main features of the holographic approach. However, classical methods need to decouple amplitude and phase contributions of the reconstructed complex wavefronts to calculate target positions in 3D, due to the fact that the lateral displacements can be calculated only after refocusing step. In order to overcome this limitation, recently, a novel method of the simultaneous calculation of both axial and lateral coordinates of moving particles has been proposed. This is based on the novel concept of wavefronts matching, i.e. the 3D positions of micro-object, moving in 3D volume, are obtained by aligning wo subsequent holographic complex reconstructions, calculated at the same distance. We test this approach in different experimental conditions in order to highlight its effectiveness in bio-microfluidic applications.

The Light Scattering Profile (LSP) of an individual cell provides a fast and accurate characterization of its morphological properties. By combining a camera-based small angle light scattering apparatus with a microfluidic-induced particle migration technique, it is possible to characterize cells in microfluidic flows. The scattering profile of an individual cell can be fully characterized by our optimized optical light collection system. Viscoelastic-induced particle migration by polyethylene oxide implemented in a low-cost microfluidic device composed of an alignment section and a measuring section opens the possibility of precise, label-free, individual cell analysis. We have studied living cells in microfluidic flows by our light scattering apparatus and by a Digital Holographic Microscope (DHM) system. Our DHM measurements provided an accurate 3D position tracking even in multiple cell conditions.

The in vitro cytotoxicity assessment of engineered nanoparticles commonly involves the measurement of different endpoints like the formation of reactive oxygen species, cell viability or cell death. Usually these parameters are determined by optical readouts of enzymatically converted substrates that often interfere with the tested nanomaterials. Using cell viability (WST-8) and cell death (LDH) as parameter we have initially investigated the toxic effects of spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with a matrix of four cell lines representing different functions: lung and kidney epithelial cells, macrophages and fibroblasts. In addition, we have used a label-free flow cytometer configuration to investigate interactions of particles and macrophages by side scatter signal analysis. Finally, we explored digital holographic microscopy (DHM) for multimodal label-free analysis of nanomaterial toxicity. Quantitative DHM phase images were analyzed for cell thickness, volume, density, dry mass and refractive index. We could demonstrate that silver spheres lead to more cytotoxic effects than rods in all four examined cell lines and both assay. Exemplarily a dose dependent interaction increase of cells with NM 300 and NM 302 analyzed by flow cytometry is shown. Furthermore, we found that the refractive index of cells is influenced by incubation with NM 300 in a decreasing manner. A 24 hours time-lapse measurement revealed a dose dependent decrease of dry mass and surface area development indicating reduced cell viability and cell death. Our results demonstrate digital holographic microscopy and flow cytometry as valuable label-free tools for nanomaterial toxicity and cell interaction studies.

Microcirculation plays a key role in the maintenance and hemodynamics of tissues and organs also due to its extensive interaction with the immune system. A critical limitation of state-of-the-art clinical techniques to characterize the blood flow is their lack of the spatial resolution required to scale down to individual capillaries. On the other hand the study of the blood flow through auto- or cross-correlation methods fail to correlate the flow speed values with the morphological details required to describe an intricate network of capillaries. Here we propose to use a newly developed technique (FLICS, FLow Image Correlation Spectroscopy) that, by employing a single raster-scanned xy-image acquired in vivo by confocal or multi-photon excitation fluorescence microscopy, allows the quantitative measurement of the blood flow velocity in the whole vessel pattern within the field of view, while simultaneously maintaining the morphological information on the immobile structures of the explored circulatory system. Fluorescent flowing objects produce diagonal lines in the raster-scanned image superimposed to static morphological details. The flow velocity is obtained by computing the Cross Correlation Function (CCF) of the intensity fluctuations detected in pairs of columns of the image. The whole analytical dependence of the CCFs on the flow speed amplitude and the flow direction has been reported recently. We report here the derivation of approximated analytical relations that allows to use the CCF peak lag time and the corresponding CCF value, to directly estimate the flow speed amplitude and the flow direction. The validation has been performed on Zebrafish embryos for which the flow direction was changed systematically by rotating the embryos on the microscope stage. The results indicate that also from the CCF peak lag time it is possible to recover the flow speed amplitude within 13% of uncertainty (overestimation) in a wide range of angles between the flow and the image scanning direction.

Digital holography is widely used nowadays for interferometric studies of various objects and processes. However, peculiarities of objects under study often imply difficulties in holograms recording, reconstruction and processing. One of the major factors is a typically large number of singular points at phase distributions caused by either low signal to noise ratio at the recorded holograms or sample inhomogeneities. The basic operations applied for absolute phase extracting from digital holograms are noise filtration, phase unwrapping and subtraction of phase distributions. In this paper we demonstrate that the sequence of these operations may drastically affect the resulting image quality and the data obtained. An optimized algorithm suitable for studies of dynamic processes in biological media on microscopic level has been developed. The algorithm was applied for monitoring of nonradiative deactivation processes occurring in onion cell specimens at photosensitized generation of singlet oxygen.

We propose a new recording modality, named here Space-Time Scanning Interferometry (STSI), which allows to capture interferograms using a linear array detector instead of a common 2D sensor. Object scanning is exploited to perform a different mapping of the holograms in the space-time domain. Three sensor rows are sufficient to yield the whole complex object field from a time sequence of interferograms. This approach is particularly useful in microfluidic microscopy, where the sample motion is intrinsically provided. We then introduce the Space-Time Digital Hologram (STDH), still possessing all the capabilities of a common DH, namely quantitative phase-contrast mapping of the samples and flexible refocusing starting from blind out-of focus recordings, but obtainable using a compact single line detector easily embeddable onboard a Lab-on-a-Chip platform. Above all, the proposed optofluidic approach is able to provide STDHs with unlimited FoV along the flow direction, independently of the set magnification factor and without the need for further processing such as hologram stitching. Hence, thanks to the possibility to refocus multiple flowing objects displaced in a liquid volume, STDH assures drastically enhanced throughput, quantitative and label-free, on-chip microscopy.

In humans, healthy mature erythrocytes or Red Blood Cells (RBCs) have globule structure and mostly important they lack a cell nucleus and most organelles, thus RBC is an envelope filled of uniform and transparent liquid. Abnormal RBCs may be fragmented or shaped like teardrops, crescents, needles, or a variety of other forms deviating from their regular ordinary shape. Here we show that seeing an erythrocyte-ensemble as nanolens-array, detection of abnormal cells can be made rapidly and efficiently without recurring to subjective shape analysis of image by the doctor or by sophisticated image processing tools, but rather by exploiting their abnormal shape alterations affecting the lens-focusing properties. Demonstration of how aberrations affect the focusing properties of the RBC is given by Hartmann- Shack approach and Zernike polynomial-fitting, as occurs for wavefront aberration correction in adaptive modern astronomic telescopes. The results show how the concept of biological lens could be addressed for revolutionary integration between photonics and biology and that a fast blood pre-screening can be performed by the proposed approach.

Azopolymer materials belong to family special materials, which are subject to photo-isomerization when illuminated by appropriate light wavelength. Optical characterization of azopolymer materials is interesting because they can be patterned when illuminated by coherent polarized light with potentially interesting applications in the biotechnology, photonic elements, molding templates, etch masks and micro-nanochannels. The interference lithography is an excellent tool to trigger the isomerization reaction on the material. During this work, switchable patterns were fabricated by means of a well established holographic set-up: surface relief gratings (SRGs) were realized with Lloyd’s mirror system. Moreover, optical characterization of the material was performed, starting from a commercial one and using a new way to analyse SRGs by means of Digital Holography Microscopy, to determine relevant parameters for the realization of the patterns with different shape and size. Some preliminary results of the influence of such patterns on the cell behavior were shown.

In the present paper, Holographic Optical Tweezers (HOT) is employed to trap and manage functionalized micrometric latex beads with the aim at probing cellular forces in no-adherent state. For the first time at best of our knowledge, a suspended cell, subjected to mechanical stress, structures its cytoskeleton when anchored to point-like bonds. We exploit the HOT arrangement to induce mechanical deformation in suspended NIH 3T3 fibroblast. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence. In our experiment, cell adhesion is prevented and the stimulus is applied through latex beads trapped by HOT and positioned externally to the cell membrane. Our aims are devoted to analyze cell response during the transition from an homogeneous and isotropic structure (as it’s in suspension) to a mechanically stressed state. To analyze the cell material interaction we combine the HOT arrangement with two imaging systems: a Digital Holography (DH) setup in microscope configuration that is an investigation method useful for quantitative, label-free and full-field analysis of low contrast object and a fluorescence modulus. HOT are exploited to induce cellular response to specific stimuli while DH allows to measure such responses in no-invasive way. Finally, fluorescence imaging is added to discriminate the inner cell structures.

An overview of the work recently conducted by our group on the development and applications of photovoltaic tweezers is presented. It includes the analysis of the physical basis of the method and the main achievements in its experimental implementation. Particular attention will be paid to the main potential applications and first demonstrations of its use in nano- and bio-technology. Specifically: i) fabrication of metallic nanoestructures for plasmonic applications, ii) development of diffractive components, iii) manipulation and patterning (1D and 2D) of various types of bio-objects (spores or pollen…) and iv) effects of PV fields of LiNbO₃ in tumour cells.

Digital Holography (DH) numerical methods have been developed to allow imaging through turbid media. DH is a wide used imaging technique as it provides non-invasive quantitative phase-contrast mapping as well as flexible numerical refocusing of samples acquired in lens-based or lensless conditions. However, a challenging issue has to be faced when the samples are immersed inside a dynamic turbid medium, as biological occluding objects out of interest provoke severe light scattering or unpredictable time-variable phase delays which scramble the object information, so that in many cases the sample is not visible at all. Here we show a simple technique, named Multi-Look Digital Holography (MLDH), able to fully recover the useful signal of the specimen of interest dipped inside the turbid liquid phase. Multiple hologram recordings are incoherently combined to synthesize the whole complex field carrying the useful information, thus revealing the hidden objects. In particular, it will be shown that both amplitude imaging and phase-contrast mapping of cells hidden behind a flow of Red Blood Cells can be obtained. Besides, qualitative comparison and quantitative evaluation show a remarkable improvement with respect to the image captured when the cells were immersed in a transparent medium. In other words, the RBCs have been demonstrated to accomplish an optical task, acting as a speckle noise de-correlation device.

Optical methods for study biological tissue and cell at micro- and nanoscale level step now over diffraction limit. Really it is single molecule localization techniques that achieve the highest spatial resolution. One of those techniques, called bleaching/blinking assisted localization microscopy (BaLM) relies on the intrinsic bleaching and blinking behavior characteristic of commonly used fluorescent probes. This feature is the base of BaLM image series acquisition and data analysis. In our work blinking of single fluorescent spot against a background of others comes to light by subtraction of time series successive frames. Then digital estimation gives the center of the spot as a point of fluorescent molecule presence, which transfers to other image with higher resolution according to accuracy of the center localization. It is a part of image with improved resolution. This approach allows overlapping fluorophores and not requires single photon sensitivity, so we use 8,8 megapixel CMOS camera with smallest (1.55 um) pixel size. This instrumentation on the base of Zeiss Axioscope 2 FS MOT allows image transmission from object plane to matrix on a scale less than 100 nm/pixel using 20x-objective, thereafter the same resolution and 5 times more field of view as compared to EMCCD camera with 6 um pixel size. To optimize excitation light power, frame rate and gain of camera we have made appropriate estimations taking into account fluorophores behaviors features and equipment characteristics. Finely we have clearly distinguishable details of the sample in the processed field of view.

A semi-cylindrical lens in Kretschmann geometry combined with a flow cell was designed for a commercial rotating compensator ellipsometer to perform internal reflection spectroscopic ellipsometry measurements, while allowing the use of multiple angles of incidence. A thin glass slide covered with a gold film was mounted between the half-cylindrical lens and a small-volume flow cell ensuring an improved sensitivity for protein adsorption experiments. The performance of the system was investigated depending on the angle of incidence, wavelength range and thickness of the gold films for surface plasmon resonance enhanced ellipsometric measurements, and a sensitivity increase was revealed compared to ellipsometric measurements with standard flow cells, depending on the measurement parameters and configuration. The sensitivity increase was demonstrated for fibrinogen adsorption.

Nanoparticles exhibit many unique and interesting optical properties which make them very useful in biomedical applications. In order to employ NPs for disease treatment, comprehensive knowledge of their important properties is crucial. One of these parameters is absorption coefficient. In this work, absorption coefficient of a nanofluid (Au nanoparticles in water) is measured by using Moiré deflectometry technique. Two laser beams are used: a comparatively high intensity laser beam as interacting beam and a low intensity as a probe beam. This method is fast, easy and nonscanning, also insensitive to vibrations.

The purpose of the work was the link establishment between the parameters characterizing the change phase difference of the wave pairs, passing through the cell and the parameters of the dynamics of speckle. It was assumed that the change in wave phases is caused by microscopic and macroscopic processes in the cell. Optical system is considered in theoretical part. It is consist of the source of coherent radiation, thin transparent diffuser, thin transparent biological object and thin lens which forms an image of the object. It is assumed that the diffuser consists of randomly located scattering point centers. Also, it is assumed that optical path length of single wave: a) changes with time t randomly due to the microscopic processes on the structure level, and b) are determined for the shape change (deformation) of the thin object. An expression for the time autocorrelation function η(t) of the radiation intensity I~ at some point in the observation plane is given. Function η(t) type for stationary and non-stationary change of the path difference of the waves is discussed. Typical experimental dependences I~(t) and η(t) are shown. It is shown that a focused selection of averaging time T allows creating the conditions for obtaining reproducible results, highlighting and analyzing processes occurring in cells at different velocities.

Time-resolved diffuse optical spectroscopy provides non-invasively the optical characterization of highly diffusive media, such as biological tissues. Light pulses are injected into the tissue and the effects of light propagation on re-emitted pulses are interpreted with the diffusion theory to assess simultaneously tissue absorption and reduced scattering coefficients. Performing spectral measurements, information on tissue composition and structure is derived applying the Beer law to the measured absorption and an empiric approximation to Mie theory to the reduced scattering. The absorption properties of collagen powder were preliminarily measured in the range of 600-1100 nm using a laboratory set-up for broadband time-resolved diffuse optical spectroscopy. Optical projection images were subsequently acquired in compressed breast geometry on 218 subjects, either healthy or bearing breast lesions, using a portable instrument for optical mammography that operates at 7 wavelengths selected in the range 635-1060 nm. For all subjects, tissue composition was estimated in terms of oxy- and deoxy-hemoglobin, water, lipids, and collagen. Information on tissue microscopic structure was also derived. Good correlation was obtained between mammographic breast density (a strong risk factor for breast cancer) and an optical index based on collagen content and scattering power (that accounts mostly for tissue collagen). Logistic regression applied to all optically derived parameters showed that subjects at high risk for developing breast cancer for their high breast density can effectively be identified based on collagen content and scattering parameters. Tissue composition assessed in breast lesions with a perturbative approach indicated that collagen and hemoglobin content are significantly higher in malignant lesions than in benign ones.

The EU-funded project VIAMOS1 proposes an optical coherence tomography system (OCT) for skin cancer detection, which combines full-field and full-range swept-source OCT in a multi-channel sensor for parallel detection. One of the project objectives is the development of new fabrication technologies for micro-optics, which makes it compatible to Micro-Opto-Electromechanical System technology (MOEMS). The basic system concept is a wafer-based Mirau interferometer array with an actuated reference mirror, which enables phase shifted interferogram detection and therefore reconstruction of the complex phase information, resulting in a higher measurement range with reduced image artifacts. This paper presents an experimental one-channel on-bench OCT system with bulk optics, which serves as a proof-of-concept setup for the final VIAMOS micro-system. It is based on a Linnik interferometer with a wavelength tuning light source and a camera for parallel A-Scan detection. Phase shifting interferometry techniques (PSI) are used for the suppression of the complex conjugate artifact, whose suppression reaches 36 dB. The sensitivity of the system is constant over the full-field with a mean value of 97 dB. OCT images are presented of a thin membrane microlens and a biological tissue (onion) as a preliminary demonstration.

Semen analysis is widely used as diagnostic tool for assessing male fertility, controlling and managing the animal reproduction. The most important parameters measured in a semen analysis are the morphology and biochemical alterations. For obtaining such information, non-invasive, label-free and non-destructive techniques have to be used. Digital Holography (DH) combined with Raman Spectroscopy (RS) could represent the perfect candidate for a rapid, non-destructive and high-sensitive morphological and biochemical sperm cell analysis. In this study, DH-RS combined approach is used for a complete analysis of single bovine spermatozoa. High-resolution images of bovine sperm have been obtained by DH microscopy from the reconstruction of a single acquired hologram, highlighting in some cases morphological alterations. Quantitative 3D reconstructions of sperm head, both normal and anomalous, have been studied and an unexpected structure of the post-acrosomal region of the head has been detected. Such anomalies have been also confirmed by Raman imaging analysis, suggesting the protein vibrations as associated Raman marker of the defect.

Confocal Raman microscopy is a powerful tool to measure small sample volumes or solids. Since commercial Raman microscopes are expensive and a change of the laser wavelength or the excitation path is hardly possible after the installation, we constructed a multimodal low-budget Raman microscope. Thus, it was possible to significantly increase the flexibility in terms of excitation wavelengths, paths, and planes. Furthermore, the asset costs were reduced by a factor of 1.7. By using commercial as well as home-built objectives to adapt the working distance and the magnification to the system under investigation, the self-constructed Raman microscope offers the possibility to measure big sample volumes, too. The obtained Raman spectra were validated by Raman spectra from a commercial Raman microscope. With a comparable measurement setting it was possible to increase the signal intensities, but with a slightly lower SNR. However, based on the great flexibility of the set-up, e.g., the laser power or the excitation wavelength can be adapted to increase the SNR. Furthermore, measurement times can be decreased. With this low-budget self-constructed Raman microscope high quality Raman microscopy and micro spectroscopy can be performed with a high flexibility to fast adapt the set-up to the sample under investigation which is not offered by commercial microscopes.

Blood borne oligonucleotides fragments contain useful clinical information whose detection and monitoring represent the new frontier in liquid biopsy as they can transform the current diagnosis procedure. For instance, recent studies have identified a new class of circulating biomarkers such as s miRNAs, and demonstrated that changes in their concentration are closely associated with the development of cancer and other pathologies. However, direct detection of miRNAs in body fluids is particularly challenging and demands high sensitivity -concentration range between atto to femtomolarspecificity, and multiplexing Here we report on engineered multifunctional microgels and innovative probe design for a direct and multiplex detection of relevant clinical miRNAs in fluorescence by single particle assay. Polyethyleneglycol-based microgels have a coreshell architecture with two spectrally encoded fluorescent dyes for multiplex analyses and are endowed with fluorescent probes for miRNA detection. Encoding and detection fluorescence signals are distinguishable by not overlapping emission spectra. Tuneable fluorescence probe conjugation and corresponding emission confinement on single microgel allows for enhanced target detection. Such suspension array has indeed high selectivity and sensitivity with a detection limit of 10^-15 M and a dynamic range from 10^-9 to 10^-15 M. We believe that sensitivity in the fM concentration range, signal background minimization, multiplexed capability and direct measurement of such microgels will translate into diagnostic benefits opening up new roots toward liquid biopsy in the context of point-of-care testing through an easy and fast detection of sensitive diagnostic biomarkers directly in serum.

A novel kind of miniaturized, all optical probe concept to measure the elasticity of biological tissues is here presented. The probe is based on fibre Bragg grating sensors (FBG) inscribed in optical fibres. The measurement procedure exploits the high strain sensitivity of Bragg gratings. A study on the reproducibility, reliability, and resolution of the sensor is presented and a first measurement on bovine cartilage tissue is reported. A linear elastic model of the cartilage has been used to analyse the data. The results indicate a good agreement with previous values given in the literature for micro-indentation.

Force sensing is a common practice used for the characterization of matter properties and in particular of bio-materials. Different optical methods have been used in the past to allow high resolution force measurements while avoiding uncertainties induced by external loading of contact sensors. In this paper, we propose the use of differential self-mixing interferometry, a self-aligned, cost effective and compact technique that allows the measurement of displacements with a theoretical resolution in the order of λ/2000 and a practical resolution in the order of λ/200 in practical applications. The DSMI sensor is used to detect the motion of a rectangular cross section cantilever placed on a piezoelectric stage. The measurements were compared with the signal received from the internal piezo stage capacitive sensor, which has a nominal resolution of 2nm. Results show that the DSMI sensor is able to follow accurately the cantilever displacement. A discussion of the potentials, limitations and required further developments of the method will also be presented.

Purpose Line sensors are cheap, fast and have high quantum effciencies. Here, we investigate whether these sensors can replace an area image sensor for the purpose of tissue thickness measurements. Material and Methods As part of a subject study high dynamic range (HDR) images of three subjects were acquired with an area image sensor. To simulate a line sensor as realistic as possible single or multiple lines were extracted from these HDR images. Thereby, horizontally extracted lines correspond to a parallel orientation of the line sensor relative to the incident angle of a laser beam. Vertically extracted lines correspond to an orthogonal orientation. Then, optical features were determined and converted into a tissue thickness using a machine learning algorithm. Results For the tested subjects the worst root mean square error (RMSE) of the learning process was 0:385 mm. The best RMSE was 0:222 mm. For all subjects, the mean RMSE and the standard deviation of RMSE values decreases with a larger number of extracted lines. The orientation of the line sensor turned out to be important for the RMSE. Vertically oriented line sensors achieve lower RMSEs than horizontally oriented sensors because of the influence of the incident angle. Furthermore, the head-pose of the subject seems to be important for the accuracy. Conclusion Line sensors deliver comparable results to previously analysed area image sensors. Nevertheless, the scattering of the values is higher and the size and orientation of the sensor and the head-pose have an influence on the RMSE of the learning process. Therefore, line sensors are feasible for tissue thickness estimation but they are a trade-off between accuracy and speed.

Nowadays is developed a lot of techniques for the refractive index measurements. One of them describe in this article. We are used method of optical microscopy, there is diffraction phase microscopy with transmissionreflection illumination for measurements of red blood cells (RBCs) refractive index. Theory of measurements refractive index and results of experiments also are presented in the article.

In this paper, dispersion equation of optical waveguide using metamaterial as buffer layer with non-linear cladding and substrate is pointed. The sensitivity of TE in metamaterial optical waveguide sensor is computed mathematically. The impacts of buffer layer with non-linear cladding and substrate on metamaterial optical waveguide sensor are also tried out. The effects of various parameters on sensitivity of sensor are obtained through MATLAB. It is expected that metamaterial as buffer layer with non-linear cladding and substrate profile has a huge application in leaky fibre sensor, gas sensor and chemical sensor for oil and under grounds mining industries.

Previously, a speckle interferometry technique and a device that allows quantitative evaluation of the metabolic activity of cultured cells were theoretically grounded and successfully tested. A speckle time-averaging technique was proposed to separate and study the processes occurring in cells at various velocities. The objective of the present research was comparing the parameters of speckle dynamics used to evaluate cell metabolism and averaged in areas of various size. Areas inside the speckle image of a cell as well as areas of the image plane with various numbers of cells were averaging areas. The target of the research was cells from L-41 culture placed onto a special optical tray with a nutrient solution immediately after defrosting. Time-average value T of digital radiation intensity in the TV camera pixels I (1) and the correlation coefficient of two digital images η (2) were used as speckle field change parameters. The digital images corresponded to a single frame area at two time moments. It is shown that in the area containing hundreds of cells dependence η(t) levels off, which indicate stationarity of random value I~ . Features of η(t) dependences obtained by averaging over large and small cell numbers image are discussed. Using the data obtained, we formulated recommendations on selecting area sizes for averaging physical values to study various processes occurring in cells.

At present work dynamic of biospeckles is used for studying processes occurring in cells which arranged in the one layer. The basis of many diseases is changes in the structural and functional properties of the molecular cells components as caused by the influence of external factors and internal functional disorders. Purpose of work is approbation of speckle-interferometer designed for the analysis of cellular metabolism in individual cells. As a parameter, characterizing the metabolic activity of cells used the value of the correlation coefficient (η) of optical signals proportional to the radiation intensity I, recorded at two points in time t. At 320x magnification for the cell diameter of 20 microns value η can be determined in the area size of 6 microns.

Spatial light interference microscopy (SLIM) is the optical method for visualization cell structure and measurement its dynamics. The distinctive feature of SLIM technique is introducing the additional spatial phase modulation of optical field in system of phase contrast microscope. In the currently known optical schemes of phase microscope the additional phase modulation is created by the liquid crystal spatial phase modulator (LCPM). It produces the phase shifts to the π/2, introducing the phase contrast between undiffracted and diffracted light from the sample. In our work we use the Michelson interferometer type optical system for introducing the phase delay between two waves. The phase shift is produced by micro displacement of one of the mirrors of the interferometer with the help of the piezoelectric element. Such modification allows setting the path difference of fraction of the wavelength between the undiffracted and diffracted components of the optical field, so it’s possible to get quantitative phase image of the object. This modernization of standard scheme of SLIM improves its performance due to higher speed of piezoelectric element comparing with speed of LCPM. The quality of result images of the investigated object depend on technical parameters of the optical scheme of microscope, including the size of illumination system aperture diaphragm. The purpose of this work is to investigate the influence of the aperture diaphragm on the phase image quality of RBC, which will be used further for the measurement RBC parameters and its dynamics.

Manual wavefront holoscopy, or simply manual w-holoscopy, is presented as a simplification of our recently reported concept of wavefront holoscope [B. Perucho and V. Micó, J Biomed Opt. 19(1):16017 (2014)]. The w-holoscope introduces an extremely simple visualization and inspection system for permanent marks in progressive addition lenses (PALs) as an application of digital in-line holography, the digital version allowing visualization (recognition and identification) and characterization (quantitative measurement) of the engravings in PALs. Here, we present a further simplification of such concept by avoiding digital elements in the setup. The layout is extremely simple and defines a new concept of marking reader for visualization and identification of engraved marks in PALs. Experimental results are reported using different PALs.

The possibilities of using gradient index (GRIN) lenses as the objective and relay lens in rigid endoscopic systems working in visible spectrum are investigated. The final aim of the project is to obtain a system providing diffraction limited image quality, which could be used for examining the gastrointestinal tract. This paper describes steps of the design of the system with working distance equal to 125 mm and angular field of view 25°. The diameter of the GRIN lenses is equal to 2,2 mm. Influence of the gradient of refractive index on the image quality is shown. Current results are analysed and future steps are described.