In this study, we utilized polarization-sensitive holotomography and Raman imaging to analyze prostate healthy (PNT2) and cancer (PC3) cells treated with glucose. After 48-hour incubation, distinct morphological differences were observed in cancer cells, including changes in volume, number, and refractive index of lipid droplets (LDs). Raman imaging confirmed the glucose uptake of these LDs. Cancer cells exhibited larger and more numerous LDs, with higher mean refractive-index and birefringence, compared to healthy cells. The study achieved over 90% accuracy in discriminating between cell types, highlighting its potential in cancer diagnostics. The research contributes to biomedical spectroscopy, offering a valuable tool for understanding cancer cell morphology and enabling early and precise cancer detection.
Simple photopolymer based on pentaerythritol tetrakis [3-mercaptopropionate] (PETMP) dispersed in Cellulose Acetate Butyrate (CAB) binder is developed to make surface and volume phase patterns transferred by means of a Direct Laser Writing (DLW) using 405nm laser. 1D and 2D gratings, as well as complex Computer Generated Holograms (CGHs) are printed and fully characterized exploiting different imaging techniques, including digital holography to highlight the contribution of surface phase shift and volume phase shift. Moreover, the diffraction efficiency of gratings is measured and correlated to the writing conditions.
Wounds that fail to heal impact the quality of life of 2.5 % of the total population. The costs of chronic wound care will reach $15–22 billion by 2024. These alarming statistics reveal the financial strain for both the medical industry and society. A solution can be found in compact and accessible sensors that offer real-time analysis of the wound site, facilitating continuous monitoring and immediate treatment, if required. Benefits of these sensors include reduction of cost and can extend the reach of healthcare to remote areas. The progression of a wound site can be closely monitored with holographic optical elements (HOEs) by real-time quantification of wound healing biomarkers, such as oxygen, temperature, pH and lactate. Fabrication of such wound monitoring sensors requires biocompatible, water-resistant photosensitive materials suitable for specific functionalisation with respect to wound analytes. Here, the design and fabrication of a HOE for delivering an excitation light beam to the sample chamber of a photoluminescence-based wound monitoring system is reported. We present a photopolymerisable hybrid sol gel (PHSG) material, capable of recording a 60 % diffraction efficiency holographic waveguide. A 1692 ± 5 lines/mm slanted transmission HOE has been theoretically designed and fabricated in PHSG films to in-couple a 633 nm beam into the oxygen sensing site. An identical grating has been used to out-couple the 633 nm beam out of the system. Stability of the PHSG grating post 476.5 nm recording was achieved by two techniques, 532 nm uniform illumination and UV-curing. The unform exposure to laser light was proved to be the more successful method since UV exposure was demonstrated to result in layer damage due to accumulated stress. The potential of waveguides as light filtering optical elements is also explored.
Here, we present a multimodal imaging approach based on Raman spectroscopy and PSDH imaging to detect circulating tumor cells. Particularly, the ability of tumor cells to internalize glucose faster than normal cells was investigated. Deuterated-glucose was used as Raman vibrational tag since its signal is present in the “silent” zone of Raman cell spectra. Raman information were correlated with the visualization of the cell birefringence by PSDH imaging. Each technique provides different information which combination gives a molecular and morphological signature of each cell, allowing to discriminate between normal and cancer cells with a sensitivity and specificity up to 80%.
Recent technological developments in ultrafast laser physics have permitted to make new kind of nonlinear microscopies, as microscopy based on Stimulated Raman scattering. These techniques are based on vibrational contrast mechanism for imaging with high sensitivity, high spatial and spectral resolution and 3D sectioning capability. The interest in the study of lipids and the possibility to image lipid droplets, thanks to their isolated Raman peaks associated with vibrational C-H bond, have encouraged investigation and identification of lipid structures inside cells, taking advantage of Stimulated Raman Scattering (SRS) imaging. In this work, we report and discuss label free images on biological environmental and structural analysis, to detect lipid microstructures inside adipocyte cells.
Stimulated Raman scattering microscopy allows vibrational contrast mechanism for imaging with high spectral and spatial resolution along with three-dimensional sectioning. In this paper, the implementation of a Stimulated Raman Scattering microscope (SRSM), obtained by the integration of a femtosecond SRS spectroscopy set-up with an optical microscope equipped with a scanning unit, is described. Femtosecond Stimulated Raman Scattering microscope is equipped with three femtosecond laser sources: a Ti:Sapphire (Ti:Sa), a synchronized optical parametric oscillator (SOPO) and a frequency converters for ultrafast lasers, i.e. a second harmonic generator optimized for the SOPO. The proposed implementation allows to cover all the regions of Raman spectra, taking advantage of two different laser combinations. The first, Ti:Sa and SHG laser combination can cover in SRL modality the fingerprint region (500 − 1700 cm−1 ) and the silent region. The second, Ti:Sa and OPO, can cover the C-H region or O−H region (2800 − 3200 cm−1 ) in SRG modality. In order to demonstrate its successful realization Stimulated Raman Gain (SRG) and Stimulated Raman Losses (SRL) images of polystyrene beads are reported and discussed.
In this work, we present successfully realization of a nonlinear microscope, not purchasable in commerce, based on stimulated Raman scattering. It is obtained by the integration of a femtosecond SRS spectroscopic setup with an inverted research microscope equipped with a scanning unit. Taking account of strength of vibrational contrast of SRS, it provides label-free imaging of single cell analysis. Validation tests on images of polystyrene beads are reported to demonstrate the feasibility of the approach. In order to test the microscope on biological structures, we report and discuss the label-free images of lipid droplets inside fixed adipocyte cells.
In the present work, a methodology for the analysis of subcellular morphology with chemical specificity for images from Stimulated Raman Scattering is suggested. In particular, a segmentation method based on a threshold algorithm and on a region growing process, to detect microstructures inside the cells, is proposed. Moreover, quantitative features for the segmented objects are extracted, in order to provide information about the possible morphological variations of microstructures in images acquired by means SRS technique.
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.
Volume Holographic Gratings is designed and fabricated to obtain a simple, lightweight and cheap light deflector. The entire process, starting from the chemical preparation of the photosensitive material, to the recording of Volume Holographic Gratings and their appropriate characterization, is reported. The recording material was a new photopolymer sensitive to light at 532nm. Results showed that the recorded Volume Holographic Grating presents a very high value of the diffraction efficiency, up to 94%. In addition, a flexible material is used to write Volume Holographic Gratings.
Sensor holograms utilize the diffraction principle of transmitting volume holographic grating (VHG) recorded within a photopolymer appositely functionalized to detect a specific stimulus or analyte. A change in the swelling or shrinking state or cross-linking density of the polymer can be caused by the hologram interaction with an analyte. This leads to a change in the recorded hologram sensor and thus, considering an incident monochromatic light and the VHG angular selectivity, to an angle shift of the diffracted maximum intensity. In this work, two new photopolymers based on a sol-gel matrix opportunely functionalized to be sensitive to transition metals or heavy metals were used as sensitive material to record VHGs. An interferometric set up with a laser source at 532nm was used to record VHGs and gratings of 1000 lines/mm were realized. When exposed to a solution of water and lead, an angle shift of about 3° of the first order diffraction of the grating was measured, demonstrating its capability to reveal the presence of heavy metal in water.
The current study relates to a Raman spectroscopy-based method for addressing the problem of sex assessment in
mammals. A direct method for sex predetermination in animals is based on the X- and Y-bearing sperm cells sorting
before insemination. Our Raman spectroscope allows distinguishing and characterizing the difference between X- and
Y-bearing sperm cells by detecting and analyzing their Raman spectra in a non-invasive and non-destructive way.
A digital holographic characterization technique is developed for studying changes in the refractive index when polymerization occurs. This all optical characterization technique enables real-time detection of the photopolymer refractive index changes during the recording process. In this paper, two different new photopolymers, sensitive to light at wavelength of 532 nm, were characterized by means of digital holography. We found a very high refractive index variation for both the new photopolymers, thus this materials could be suitable for holographic recordings with the great advantage of being inexpensive and easy to make.
Raman spectroscopy is a label-free and non-invasive method that measures the inelastic scattered light from a sample
giving insight into the vibration eigenmodes of the excited molecules. For these reasons, Raman spectroscopy has been
used as a powerful tool to investigate different biological tissues and living cells. In this paper, we present a Raman
spectroscopy-based method for sensitive biochemical characterization of bovine sperm cells. Importantly, by analysing
separate Raman spectra from the nucleus, acrosomale vesicle and tail of single sperm cells, we are able to identify
characteristic Raman features associated with DNA, protein and lipid molecular vibrations for discriminating among
different locations inside the cell with sub-micrometric resolution (∼0.3 μm). We demonstrate that our Raman
spectroscopy facilitates spectral assignment and increases detection sensitivity, opening the way for novel bio-imaging
platforms.
Diatoms are monocellular algae responsible of 20-25% of the global oxygen produced by photosynthetic processes. The protoplasm of every single cell is enclosed in an external wall made of porous hydrogenated silica, the frustule. In recent times, many effects related to photonic properties of diatom frustules have been discovered and exploited in applications: light confinement induced by multiple diffraction, frustule photoluminescence applied to chemical and biochemical sensing, photonic-crystal-like behavior of valves and girdles. In present work we show how several techniques (e.g. digital holography) allowed us to retrieve information on light manipulation by diatom single valves in terms of amplitude, phase and polarization, both in air and in a cytoplasmatic environment. Possible applications in optical microsystems of diatom frustules and frustule-inspired devices as active photonic elements are finally envisaged.
In this communication, we report some new results obtained in our laboratories in design, fabrication and
characterization of silicon-based optical structures and devices, including metamaterials, raman light amplifiers, and
biomatter-silicon interfaces for sensors and biochips.
In the last few years, silicon photonics has been characterized by a wide range of applications in several fields, from
communications to sensing, from biophotonics to the development of new artificial materials. In this communication,
we report a review of the main results obtained in our laboratories in design, fabrication and characterization of new
silicon-based optical structures and devices, including metamaterials, photodetectors, raman light amplifiers, and
porous silicon based bio-chemical sensors and biochips. Future perspectives in integration of silicon based MEMS
and MOEMS are also presented.
The stress in porous silicon during exposition to a liquid is investigated by an approach based on Raman scattering.
When the porous silicon structure is exposed to isopropanol or ethanol, a reversible blue shift of the Raman spectra is
observed. The blue shift of Raman scattering is ascribed to the contraction induced by the liquids that fill the pores.
The action of molecular interaction between a fluid and an adsorbent results in adsorption and wetting phenomena. However, the adsorbent is also submitted to the action of the molecular forces. In order to provide a large adsorption capacity, adsorbents with a large specific surface area are preferable. For this reason, for the study of adsorption phenomena, porous silicon is a material of great interest.
Wetting phenomena in porous silicon layers are experimentally investigated by Raman scattering. The experimental results prove a reversible blue-shift of Raman spectra of wetted porous silicon layers with isopropanol or ethanol with respect to unperturbed layers. We ascribe the shift to a compressive stress due to the increased lattice mismatch between the porous silicon layer and the bulk silicon substrate in wetting conditions. The use of two liquids having quite similar density and surface tension resulted, as expected, in quite comparable blue shift of the peak. This effect may be conveniently used in sensing applications of liquids on porous silicon layers.
In the last years, the possibility of light generation and/or amplification in silicon, based on Raman emission, has achieved great results. However, some significant limitations, inherent to the physics of silicon, have been pointed out, too. In order to overcome these limitations, a possible option is to consider low dimensional silicon. On this line of argument, an approach based on Raman scattering in porous silicon is presented. We prove two significant advantage with respect to silicon: the broadening of spontaneous Raman emission and the tuning of the Stokes shift. Finally, we discuss about the prospect of Raman amplifier in porous silicon.
Fluid-solid interfacial phenomena are a subject of much interest. In the adsorption phenomena, the adsorbent experiences the action of the molecular forces inducing strains.
In this paper, we experimentally investigate adsorption phenomena in porous silicon microcavities by spontaneous Raman scattering. Polarised Raman spectra are measured in a backscattering configuration using a diode laser at 404 nm. We observe a reversible blue shift of the Raman spectra exposing a porous silicon multilayer to air saturated with vapor of pentane or iso-propanol. We ascribe the shift of the Raman spectra to the strain in porous silicon due to the adsorption in the pore walls.
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