Maurizio Indolfi

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 of small lipid droplets by femtosecond-stimulated Raman scattering microscopy

In this paper, the implementation and main realization issues of a stimulated Raman scattering (SRS) microscope, obtained by the integration of a femtosecond SRS spectroscopy set-up with an optical microscope equipped with a scanning unit, are described. In order to characterize capabilities and limitations of SRS microscope, the optical resolution is measured and validation tests on polystyrene beads imaging are reported. In addition, taking account of strength of SRS vibrational contrast, label-free imaging of small biological structures, as such as lipid droplets (LDs), in adipocyte cells is carried out. The visualization of small LDs, with dimensions comparable to the optical lateral resolution of our system, is demonstrated.

Implementation of stimulated Raman losses and stimulated Raman gain microscopy using three femtosecond laser sources

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.

Implementation of stimulated Raman scattering microscopy for single cell analysis

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.

Automatic method for features extraction for images achieved by stimulated Raman scattering microscopy

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.

Stimulated Raman scattering in nanostructured materials

Nonlinear optics at nanoscale is an intriguing research field of great significance from both fundamental and applicative point of view. In this paper, the phenomena of stimulated Raman scattering in both nanostructured silica based materials and nanostructured silicon based materials are investigated and discussed.

Study of Raman amplification in nanostructured materials

In this paper, nanostructured materials based both on silica and silicon, are studied in order to obtain new materials with both large Raman gain coefficients and spectral bandwidth. In particular, silica-niobia based glasses have been characterized by linear Raman spectroscopy, while amorphous silicon nanoparticles and in silicon micro- and nano-crystals have been characterized by Stimulated Raman Scattering spectroscopy. Finally, results are discussed.

Tailoring nonlinear nanostructured materials for integrated sources based on stimulated Raman scattering

In this paper, the possibility to obtain new materials with both large Raman gain coefficients and spectral bandwidth is investigated. In particular, silica-niobia based glasses have been characterized by Raman spectroscopy. Our silica-based glass system (K2O-Nb2O5-SiO2 (KNS)) are obtained by a thermal treatment resulting in a nanocomposite material of easy and cheap production. Our glass composition have demonstrated both a strong Raman gain and a large bandwidth. These results can be related to nanostructured material domains.