Multiple-pathways light modulation in Pleurosigma strigosum bi-raphid diatom.

Ordered, quasi-ordered, and even disordered nanostructures can be identified as constituent components of several protists, plants and animals, making possible an efficient manipulation of light for intra- and inter- species communication, camouflage, or for the enhancement of primary production. Diatoms are ubiquitous unicellular microalgae inhabiting all the aquatic environments on Earth. They developed, through tens of millions of years of evolution, ultrastructured silica cell walls, the frustules, able to handle optical radiation through multiple diffractive, refractive, and wave-guiding processes, possibly at the basis of their high photosynthetic efficiency. In this study, we employed a range of imaging, spectroscopic and numerical techniques (including transmission imaging, digital holography, photoluminescence spectroscopy, and numerical simulations based on wide-angle beam propagation method) to identify and describe different mechanisms by which Pleurosigma strigosum frustules can modulate optical radiation of different spectral content. Finally, we correlated the optical response of the frustule to the interaction with light in living, individual cells within their aquatic environment following various irradiation treatments. The obtained results demonstrate the favorable transmission of photosynthetic active radiation inside the cell compared to potentially detrimental ultraviolet radiation.

Application of Raman spectroscopy to the evaluation of F-actin changes in sea urchin eggs at fertilization.

The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca2+ signals and to guide sperm's entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca2+ release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg's subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.

Cancer metabolic features allow discrimination of tumor from white blood cells by label-free multimodal optical imaging.

Circulating tumor cells (CTCs) are tumor cells that have penetrated the circulatory system preserving tumor properties and heterogeneity. Detection and characterization of CTCs has high potential clinical values and many technologies have been developed for CTC identification. These approaches remain challenged by the extraordinary rarity of CTCs and the difficulty of efficiently distinguishing cancer from the much larger number of white blood cells in the bloodstream. Consequently, there is still a need for efficient and rapid methods to capture the broad spectrum of tumor cells circulating in the blood. Herein, we exploit the peculiarities of cancer metabolism for discriminating cancer from WBCs. Using deuterated glucose and Raman microscopy we show that a) the known ability of cancer cells to take up glucose at greatly increased rates compared to non-cancer cells results in the lipid generation and accumulation into lipid droplets and, b) by contrast, leukocytes do not appear to generate visible LDs. The difference in LD abundance is such that it provides a reliable parameter for distinguishing cancer from blood cells. For LD sensitive detections in a cell at rates suitable for screening purposes, we test a polarization-sensitive digital holographic imaging (PSDHI) technique that detects the birefringent properties of the LDs. By using polarization-sensitive digital holographic imaging, cancer cells (prostate cancer, PC3 and hepatocarcinoma cells, HepG2) can be rapidly discriminated from leukocytes with reliability close to 100%. The combined Raman and PSDHI microscopy platform lays the foundations for the future development of a new label-free, simple and universally applicable cancer cells' isolation method.

Noises investigations and image denoising in femtosecond stimulated Raman scattering microscopy.

In the literature of SRS microscopy, the hardware characterization usually remains separate from the image processing. In this article, we consider both these aspects and statistical properties analysis of image noise, which plays the vital role of joining links between them. Firstly, we perform hardware characterization by systematic measurements of noise sources, demonstrating that our in-house built microscope is shot noise limited. Secondly, we analyze the statistical properties of the overall image noise, and we prove that the noise distribution can be dependent on image direction, whose origin is the use of a lock-in time constant longer than pixel dwell time. Finally, we compare the performances of two widespread general algorithms, that is, singular value decomposition and discrete wavelet transform, with a method, that is, singular spectrum analysis (SSA), which has been adapted for stimulated Raman scattering images. In order to validate our algorithms, in our investigations lipids droplets have been used and we demonstrate that the adapted SSA method provides an improvement in image denoising.

Underwater Light Manipulation by the Benthic Diatom Ctenophora pulchella: From PAR Efficient Collection to UVR Screening.

Several species of diatoms, unicellular microalgae which constitute the main component of phytoplankton, are characterized by an impressive photosynthetic efficiency while presenting a noticeable tolerance versus exposure to detrimental UV radiation (UVR). In particular, the growth rate of the araphid diatom Ctenophora pulchella is not significantly affected by harsh treatments with UVR, even in absence of detectable, specific UV-absorbing pigments and even if it is not able to avoid high UV exposure by motility. In this work we applied a multi-disciplinary approach involving numerical computation, photonics, and biological parameters in order to investigate the possible role of the frustule, micro- and nano-patterned silica shell which encloses the cell, in the ability of C. pulchella to efficiently collect photosynthetic active radiation (PAR) and to simultaneously screen the protoplasm from UVR. The characterization of the photonic properties of the frustule has been accompanied by in vivo experiments conducted in water in order to investigate its function as optical coupler between light and plastids.

Integrated Photodetectors Based on Group IV and Colloidal Semiconductors: Current State of Affairs.

With the aim to take advantage from the existing technologies in microelectronics, photodetectors should be realized with materials compatible with them ensuring, at the same time, good performance. Although great efforts are made to search for new materials that can enhance performance, photodetector (PD) based on them results often expensive and difficult to integrate with standard technologies for microelectronics. For this reason, the group IV semiconductors, which are currently the main materials for electronic and optoelectronic devices fabrication, are here reviewed for their applications in light sensing. Moreover, as new materials compatible with existing manufacturing technologies, PD based on colloidal semiconductor are revised. This work is particularly focused on developments in this area over the past 5-10 years, thus drawing a line for future research.

Integrated Raman Laser: A Review of the Last Two Decades.

Important accomplishments concerning an integrated laser source based on stimulated Raman scattering (SRS) have been achieved in the last two decades in the fields of photonics, microphotonics and nanophotonics. In 2005, the first integrated silicon laser based upon SRS was realized in the nonlinear waveguide. This breakthrough promoted an intense research activity addressed to the realization of integrated Raman sources in photonics microstructures, like microcavities and photonics crystals. In 2012, a giant Raman gain in silicon nanocrystals was measured for the first time. Starting from this impressive result, some promising devices have recently been realized combining nanocrystals and microphotonics structures. Of course, the development of integrated Raman sources has been influenced by the trend of photonics towards the nano-world, which started from the nonlinear waveguide, going through microphotonics structures, and finally coming to nanophotonics. Therefore, in this review, the challenges, achievements and perspectives of an integrated laser source based on SRS in the last two decades are reviewed, side by side with the trend towards nanophotonics. The reported results point out promising perspectives for integrated micro- and/or nano-Raman lasers.

Fiber Amplifiers and Fiber Lasers Based on Stimulated Raman Scattering: A Review.

Nowadays, in fiber optic communications the growing demand in terms of transmission capacity has been fulfilling the entire spectral band of the erbium-doped fiber amplifiers (EDFAs). This dramatic increase in bandwidth rules out the use of EDFAs, leaving fiber Raman amplifiers (FRAs) as the key devices for future amplification requirements. On the other hand, in the field of high-power fiber lasers, a very attractive option is provided by fiber Raman lasers (FRLs), due to their high output power, high efficiency and broad gain bandwidth, covering almost the entire near-infrared region. This paper reviews the challenges, achievements and perspectives of both fiber Raman amplifier and fiber Raman laser. They are enabling technologies for implementation of high-capacity optical communication systems and for the realization of high power fiber lasers, respectively.

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering.

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 µm. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

Three-dimensional label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy.

Lipid droplets are lipid-storage organelles with a key role in lipid accumulation pathologies such as diabetes, obesity and atherosclerosis. Despite their important functions many aspects of lipid droplets biology are still unknown. This is partially due to the current use of exogenous labels to monitor their formation and remodelling by invasive imaging methods. Here, we apply stimulated Raman scattering microscopy to acquire images with high spatial resolution along with resolving capabilities of lipids and proteins and three-dimensional sectioning. Our images and data analysis demonstrate an increase in the number of large (>15µm2) lipid droplets in human adipocyte cells during differentiation process. In addition, spatially-resolved maps of lipids and proteins inside cells and three dimensional reconstructions of lipids at the initial and final steps of adipocyte differentiation are reported, too.

Combined Raman and polarization sensitive holographic imaging for a multimodal label-free assessment of human sperm function.

Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.

Integration of stimulated Raman gain and stimulated Raman losses detection modes in a single nonlinear microscope.

This work addresses some key challenges in the fields of bio and nanophotonics by stimulated Raman microscopy. We present the design and the implementation of a femtosecond stimulated Raman scattering microscope, equipped with three femtosecond laser sources, which allows the coexistence of stimulated Raman gain (SRG) and stimulate Raman losses (SRL) detection modes in a single microscopy setup and to generate images of the same region in succession, without adding or removing components. In order to demonstrate the switching between the two detection modes, SRL and SRG images of polystyrene beads are acquired and the images quality are evaluated and compared.

Diatom Valve Three-Dimensional Representation: A New Imaging Method Based on Combined Microscopies.

The frustule of diatoms, unicellular microalgae, shows very interesting photonic features, generally related to its complicated and quasi-periodic micro- and nano-structure. In order to simulate light propagation inside and through this natural structure, it is important to develop three-dimensional (3D) models for synthetic replica with high spatial resolution. In this paper, we present a new method that generates images of microscopic diatoms with high definition, by merging scanning electron microscopy and digital holography microscopy or atomic force microscopy data. Starting from two digital images, both acquired separately with standard characterization procedures, a high spatial resolution (Δz = λ/20, Δx = Δy ≅ 100 nm, at least) 3D model of the object has been generated. Then, the two sets of data have been processed by matrix formalism, using an original mathematical algorithm implemented on a commercially available software. The developed methodology could be also of broad interest in the design and fabrication of micro-opto-electro-mechanical systems.

Subcellular chemical and morphological analysis by stimulated Raman scattering microscopy and image analysis techniques.

The visualization of heterogeneous morphology, segmentation and quantification of image features is a crucial point for nonlinear optics microscopy applications, spanning from imaging of living cells or tissues to biomedical diagnostic. In this paper, a methodology combining stimulated Raman scattering microscopy and image analysis technique is presented. The basic idea is to join the potential of vibrational contrast of stimulated Raman scattering and the strength of imaging analysis technique in order to delineate subcellular morphology with chemical specificity. Validation tests on label free imaging of polystyrene-beads and of adipocyte cells are reported and discussed.

Label-free imaging and biochemical characterization of bovine sperm cells.

A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols.

Holographic imaging of unlabelled sperm cells for semen analysis: a review.

Male reproductive health in both humans and animals is an important research field in biological study. In order to characterize the morphology, the motility and the concentration of the sperm cells, which are the most important parameters to feature them, digital holography demonstrated to be an attractive technique. Indeed, it is a label-free, non-invasive and high-resolution method that enables the characterization of live specimen. The review is intended both for summarizing the state-of-art on the semen analysis and recent achievement obtained by means of digital holography and for exploring new possible applications of digital holography in this field. Quantitative phase maps of living swimming spermatozoa.

Optical properties of diatom nanostructured biosilica in Arachnoidiscus sp: micro-optics from mother nature.

Some natural structures show three-dimensional morphologies on the micro- and nano-scale, characterized by levels of symmetry and complexity well far beyond those fabricated by best technologies available. This is the case of diatoms, unicellular microalgae, whose protoplasm is enclosed in a nanoporous microshell, made of hydrogenated amorphous silica, called frustule. We have studied the optical properties of Arachnoidiscus sp. single valves both in visible and ultraviolet range. We found photonic effects due to diffraction by ordered pattern of pores and slits, accordingly to an elaborated theoretical model. For the first time, we experimentally revealed spatial separation of focused light in different spots, which could be the basis of a micro-bio-spectrometer. Characterization of such intricate structures can be of great inspiration for photonic devices of next generation.

Giant Raman gain in silicon nanocrystals.

Nanostructured silicon has generated a lot of interest in the past decades as a key material for silicon-based photonics. The low absorption coefficient makes silicon nanocrystals attractive as an active medium in waveguide structures, and their third-order nonlinear optical properties are crucial for the development of next generation nonlinear photonic devices. Here we report the first observation of stimulated Raman scattering in silicon nanocrystals embedded in a silica matrix under non-resonant excitation at infrared wavelengths (~1.5 µm). Raman gain is directly measured as a function of the silicon content. A giant Raman gain from the silicon nanocrystals is obtained that is up to four orders of magnitude greater than in crystalline silicon. These results demonstrate the first Raman amplifier based on silicon nanocrystals in a silica matrix, thus opening new perspectives for the realization of more efficient Raman lasers with ultra-small sizes, which would increase the synergy between electronic and photonic devices.