Silvia Ghislanzoni

Scopus Publications

Estimation of biological variance in coherent Raman microscopy data of two cell lines using chemometrics
Rajendhar Junjuri, Matteo Calvarese, MohammadSadegh Vafaeinezhad, Federico Vernuccio, Marco Ventura, Tobias Meyer-Zedler, Benedetta Gavazzoni, Dario Polli, Renzo Vanna, Italia Bongarzone,et al.
Royal Society of Chemistry (RSC)
Broadband Coherent Anti-Stokes Raman Scattering (BCARS) is a valuable spectroscopic imaging tool forestimating the biological variance of cells in biomedical applications.

Optical Diffraction Tomography and Raman Confocal Microscopy for the Investigation of Vacuoles Associated with Cancer Senescent Engulfing Cells
Silvia Ghislanzoni, Jeon Woong Kang, Arianna Bresci, Andrea Masella, Koseki J. Kobayashi-Kirschvink, Dario Polli, Italia Bongarzone, and Peter T. C. So
MDPI AG
Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.

Full-Spectrum CARS Microscopy of Cells and Tissues with Ultrashort White-Light Continuum Pulses
Federico Vernuccio, Renzo Vanna, Chiara Ceconello, Arianna Bresci, Francesco Manetti, Salvatore Sorrentino, Silvia Ghislanzoni, Flavia Lambertucci, Omar Motiño, Isabelle Martins,et al.
American Chemical Society (ACS)
Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging nonlinear vibrational imaging technique that delivers label-free chemical maps of cells and tissues. In narrowband CARS, two spatiotemporally superimposed picosecond pulses, pump and Stokes, illuminate the sample to interrogate a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite recent technological advancements, BCARS microscopes still struggle to image biological samples over the entire Raman-active region (400–3100 cm–1). Here, we demonstrate a robust BCARS platform that answers this need. Our system is based on a femtosecond ytterbium laser at a 1035 nm wavelength and a 2 MHz repetition rate, which delivers high-energy pulses used to produce broadband Stokes pulses by white-light continuum generation in a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20 fs duration, with narrowband pump pulses, we generate a CARS signal with a high (<9 cm–1) spectral resolution in the whole Raman-active window, exploiting both the two-color and three-color excitation mechanisms. Aided by an innovative post-processing pipeline, our microscope allows us to perform high-speed (≈1 ms pixel dwell time) imaging over a large field of view, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, paving the way for applications in histopathological settings.

Reduced sulfatide content in deferoxamine-induced senescent HepG2 cells
Silvia Ghislanzoni, Gaia Martina Sarcinelli, Arianna Bresci, Francesco Manetti, Dario Polli, Antonella Tomassetti, Maria Teresa Radice, and Italia Bongarzone
Elsevier BV

Multimodal vibrational and multiphoton nonlinear optical microscopy as a non-invasive tool to prevent human tumor recurrence
Arianna Bresci, Francesco Manetti, Silvia Ghislanzoni, Federico Vernuccio, Salvatore Sorrentino, Chiara Ceconello, Renzo Vanna, Italia Bongarzone, Giulio Cerullo, and Dario Polli
SPIE

Plug-and-play stimulated Raman microscopy system for broadband coherent vibrational imaging
Francesco Crisafi, Benedetta Talone, Andrea Ragni, Gabriele Di Noia, Mujeeb Rahman, Jing He, Jeremiah Marcellino, Goutam Kar, Yarjan Samad, Boyang Mao,et al.
IEEE
Stimulated Raman scattering (SRS) microscopy is an emerging tool for biomedical imaging, with applications ranging from cell-drug interaction [1], cell sorting [2] to tissue analysis [3] and histopathology [4]. Current commercial systems [5] require users to deal with integration between laser, microscope and the detection system, resulting in trade-offs and, most importantly, lack of user-friendliness, thus hindering widespread application by non-specialists. This approach has pushed the laser market towards the development of high power (>100mW, >1nJ/pulse) narrowband (<1nm) tunable sources (Optical Parametric Oscillators (OPOs) and Fiber-OPOs) which can be coupled to off-the-shelf photodiodes and lock-in amplifiers, at the cost of detecting one or maximum two frequencies at a time [5].

Multimodal Coherent Raman and Multiphoton Nonlinear Optical Microscopy Reveals Early Risk of Tumour Recurrence after Anticancer Therapy in Human Cells
Arianna Bresci, Francesco Manetti, Silvia Ghislanzoni, Federico Vernuccio, Salvatore Sorrentino, Chiara Ceconello, Renzo Vanna, Italia Bongarzone, Giulio Cerullo, and Dario Polli
IEEE
Recent studies have shown that common anticancer treatments can induce cell senescence rather than death, which is a critical phenotype governing tumour recurrence, dormancy and treatment resistance [1]. This calls for the urgent development of safe, precise, and rapid tools to unveil early evidence of such therapy-induced senescence (TIS).

Deep Ensemble Learning and Transfer Learning Methods for Classification of Senescent Cells in Nonlinear Optical Microscopy Images
Francesco Manetti, Salvatore Sorrentino, Arianna Bresci, Federico Vernuccio, Chiara Ceconello, Silvia Ghislanzoni, Italia Bongarzone, Renzo Vanna, Giulio Cerullo, and Dario Polli
IEEE

Noninvasive morpho-molecular imaging reveals early therapy-induced senescence in human cancer cells
Arianna Bresci, Jeong Hee Kim, Silvia Ghislanzoni, Francesco Manetti, Lintong Wu, Federico Vernuccio, Chiara Ceconello, Salvatore Sorrentino, Ishan Barman, Italia Bongarzone,et al.
American Association for the Advancement of Science (AAAS)
Anticancer therapy screening in vitro identifies additional treatments and improves clinical outcomes. Systematically, although most tested cells respond to cues with apoptosis, an appreciable portion enters a senescent state, a critical condition potentially driving tumor resistance and relapse. Conventional screening protocols would strongly benefit from prompt identification and monitoring of therapy-induced senescent (TIS) cells in their native form. We combined complementary all-optical, label-free, and quantitative microscopy techniques, based on coherent Raman scattering, multiphoton absorption, and interferometry, to explore the early onset and progression of this phenotype, which has been understudied in unperturbed conditions. We identified TIS manifestations as early as 24 hours following treatment, consisting of substantial mitochondrial rearrangement and increase of volume and dry mass, followed by accumulation of lipid vesicles starting at 72 hours. This work holds the potential to affect anticancer treatment research, by offering a label-free, rapid, and accurate method to identify initial TIS in tumor cells.

Deep ensemble learning and transfer learning methods for classification of senescent cells from nonlinear optical microscopy images
Salvatore Sorrentino, Francesco Manetti, Arianna Bresci, Federico Vernuccio, Chiara Ceconello, Silvia Ghislanzoni, Italia Bongarzone, Renzo Vanna, Giulio Cerullo, and Dario Polli
Frontiers Media SA
The success of chemotherapy and radiotherapy anti-cancer treatments can result in tumor suppression or senescence induction. Senescence was previously considered a favorable therapeutic outcome, until recent advancements in oncology research evidenced senescence as one of the culprits of cancer recurrence. Its detection requires multiple assays, and nonlinear optical (NLO) microscopy provides a solution for fast, non-invasive, and label-free detection of therapy-induced senescent cells. Here, we develop several deep learning architectures to perform binary classification between senescent and proliferating human cancer cells using NLO microscopy images and we compare their performances. As a result of our work, we demonstrate that the most performing approach is the one based on an ensemble classifier, that uses seven different pre-trained classification networks, taken from literature, with the addition of fully connected layers on top of their architectures. This approach achieves a classification accuracy of over 90%, showing the possibility of building an automatic, unbiased senescent cells image classifier starting from multimodal NLO microscopy data. Our results open the way to a deeper investigation of senescence classification via deep learning techniques with a potential application in clinical diagnosis.

Multimodal multiphoton and vibrational microscopy reveals early therapy-induced senescence in human tumors: a non-invasive tool to prevent the risk of cancer relapse
Arianna Bresci, Francesco Manetti, Silvia Ghislanzoni, Federico Vernuccio, Salvatore Sorrentino, Chiara Ceconello, Renzo Vanna, Italia Bongarzone, Giulio Cerullo, and Dario Polli
SPIE
Recent studies have shown that common anticancer treatments can induce cell senescence rather than death, a critical phenotype governing tumor recurrence. This calls for the urgent development of safe, precise, and quick tools to unveil critical Therapy-Induced Senescence (TIS). Merging different coherent Raman and multiphoton techniques, we present label-free multimodal nonlinear optical (NLO) microscopy as a powerful tool to spot early TIS. We home-built a microscope including different NLO modalities: Stimulated Raman Scattering (SRS), forward and epi-detected Coherent Anti-Stokes Raman Scattering (CARS and E-CARS), and Two-Photon Excited Fluorescence (TPEF). The infrared laser source outputs synchronized narrowband 780 nm pump pulses and 950-1050 nm tunable Stokes pulses, so to match the CH-stretching region of the Raman spectrum. Thanks to the co-registration of these diverse techniques applied on label-free TIS cells and controls, we exposed quantitative hallmarks of early TIS, confirmed by comparing different optical signals monitored over 72 hours of treatment. TPEF from metabolic coenzymes combined with E-CARS from cardiolipin and cytochrome C indicated an early shrinking of mitochondria. CARS and SRS revealed lipid vesicles overproduction and accumulation. Nuclei enlarged irregularly, visualized via subtraction of SRS signals of proteins and lipids, and CARS from deoxyribose. We consider our results will strongly influence anticancer pre-clinical studies and translated clinical applications, helping to identify quickly, non-invasively, and quantitatively TIS in human tumors.

Multimodal vibrational and multi-photon nonlinear optical microscopy to assess chemotherapy-induced senescence in human cancer cells

Verbascoside protects pancreatic β-cells against er-stress
Alessandra Galli, Paola Marciani, Algerta Marku, Silvia Ghislanzoni, Federico Bertuzzi, Raffaella Rossi, Alessia Di Giancamillo, Michela Castagna, and Carla Perego
MDPI AG
Substantial epidemiological evidence indicates that a diet rich in polyphenols protects against developing type 2 diabetes. The phenylethanoid glycoside verbascoside/acteoside, a widespread polyphenolic plant compound, has several biological properties including strong antioxidant, anti-inflammatory and neuroprotective activities. The aim of this research was to test the possible effects of verbascoside on pancreatic β-cells, a target never tested before. Mouse and human β-cells were incubated with verbascoside (0.8–16 µM) for up to five days and a combination of biochemical and imaging techniques were used to assess the β-cell survival and function under normal or endoplasmic reticulum (ER)-stress inducing conditions. We found a dose-dependent protective effect of verbascoside against oxidative stress in clonal and human β-cells. Mechanistic studies revealed that the polyphenol protects β-cells against ER-stress mediated dysfunctions, modulating the activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response and promoting mitochondrial dynamics. As a result, increased viability, mitochondrial function and insulin content were detected in these cells. These studies provide the evidence that verbascoside boosts the ability of β-cells to cope with ER-stress, an important contributor of β-cell dysfunction and failure in diabetic conditions and support the therapeutic potential of verbascoside in diabetes.

Silvia Ghislanzoni

EDUCATION

Scopus Publications