NANOPHOTONICS GROUP
The Biophotonics and Biophysics group operates in the Department of Physics of the University of Milano-Bicocca. The research interests and application goals are in the field of the optical microscopy with non-linear excitation, in the use of gold and silver nanoparticles for biomedical applications and in the fluorescence image correlation microscopy. All these research fields are devoted to the study of the cellular dynamics and the interactions between nanoparticles and living cells. The group runs also a facility for optical nanoscopy with 70 nm optical resolution in the focal plane.
Permanent Staff: Giuseppe Chirico, Maddalena Collini, Laura D’Alfonso, Laura Sironi
Non-permanent: Staff Mykola Borzenkov (Post-Doc), Margaux Bouzin (Post-Doc), Amirbahador ZeynaLI (PhD), Mario Marini (PhD), Riccardo Scodellaro (PhD)
Research keywords: Biophysics, optical microscopy, Fluorescence Correlation Spectroscopy and Imaging, Nanoparticles, fluorescent proteins, numerical simulations of
polymeric and cellular systems.
• 192 publication [2000-2019]; 3820 citations.
RESEARCH TOPICS
I. Bioimaging
a) In-vivo non-linear excitation microscopy. Non-linear excitation can be exploited for biological imaging since most of the cellular and tissue components have intrinsic nonlinear excitation sensitivity. Moreover, most of the dyes used for conventional fluorescence microscopy can be excited by 2 or 3 photons with near infrared pulsed lasers. For tissue studies, second harmonic generation signals are also advantageously exploited to obtain the morphological information of the connective part of the tissues. The group applies these techniques to the study of the cellular dynamics in tissues (lymphocytes in lymph nodes) and to the intracellular dynamics (endocytosis, trafficking).
Relevant publications:
• F. Radaelli, L. D’Alfonso, M. Collini, F. Mingozzi, L. Marongiu, F. Granucci, I. Zanoni, G. Chirico & L. Sironi. μMAPPS: a novel phasor approach to second harmonic analysis for in vitro-in vivo investigation of collagen microstructure. Scientific Reports (2017) 7: 17468; IF:4.1
• F. Mingozzi, R. Spreafico, T. Gorletta, C. Cigni, M. Di Gioia, M. Caccia, L. Sironi, M. Collini, M. Soncini, M. Rusconi, Ulrich H von Andrian, Giuseppe Chirico, Ivan Zanoni, F. Granucci Prolonged contact with dendritic cells turns lymph node-resident NK cells into anti-tumor effectors. DOI 10.15252/emmm.201506164 | Published online 12.07.2016 EMBO Molecular Medicine (2016) 8: 1039- 1051. IF:10.6
b) Image correlation. Light fluctuations can be characterized by correlation and cross-correlation methods. Fluorescence correlation spectroscopy allows our group to study the interactions of biomolecules in vitro and in-vivo. The group has developed state of the art setups to investigate accurately the biomolecule binding and expression in cells. We are applying correlation methods on images to recover unique spatio-temporal information on the cellular and intracellular processes.
Relevant publications:
• Collini, M; Radaelli, F ; Sironi, L ; Ceffa, NG ; D’Alfonso, L ; Bouzin, M ; Chirico, G. Adaptive optics microspectrometer for cross-correlation measurement of micro fluidic flows. J. Biomed. Opt. (2019) 24: 025004; • C. A. Marquezin, N. G. Ceffa, F. Cotelli, M.Collini, L. Sironi, and G. Chirico. Image Cross-Correlation Analysis of Time Varying Flows. Anal. Chem. (2016) 88: 7115−7122.
• Sironi, Laura; Bouzin, Margaux; Inverso, Donato; D’Alfonso, Laura; Pozzi, Paolo; Cotelli, Franco; Guidotti, Luca G; Iannacone, Matteo; Collini, Maddalena; Chirico, Giuseppe, In vivo flow mapping in complex vessel networks by single image correlation. Scientific reports. (2014) 4: 7341.
c) Super-resolution optical microscopy. Nanoscopy is a branch of optical microscopy that overcomes the optical resolution set by diffraction of light. Nanoscopy overcomes this limitation by employing time and space structured illumination. Our group is implementing a particular methodology, called STED, to obtain microscopy images of cells at 50-70 nm spatial resolution. STED is applied to the field of nanotechnology for Medicine since it allows the detection of sub-micron sized particulate matter internalized in the cells or in the membrane domains (lipid rafts). These can be drug carriers in the form of metal nanoparticles decorated with stealth polymers and specific antibodies for cell receptors. Single molecule microscopy has been also demonstrated inhouse.
Relevant publications:
• Bouzin M, Chirico G., D’Alfonso L, Sironi L., Soavi G., Cerullo G., Campanini B. and Collini M. Stimulated Emission Properties Of Fluorophores by CW—STED Single Molecule Spectroscopy. J. Phys. Chem. B. (2013) 117(51):16405-15.
d) Photo-thermal imaging of nanostructures. The group has recently developed a new pump&probe technique to visualize thermal dissipation in light absorbing samples with a spatial resolution much lower than that set by the diffraction of the thermal radiation (wavelength ≅ 10 ??). This is applied currently to a variety of fields, from the physical characterization of polymeric blends to melanoma imaging for diagnosis.
Relevant publications:
• Marini, M.; Bouzin, M.; Zeynali, A.; M. Collini, G. Chirico. Super-Resolution Photo-Activated Thermography, Eur. Biophys. J (2019) 48: S203-S203
• Marini, M.; Bouzin, M.; M. Collini, Zeynali, A.; M. Borzenkov; G. Chirico. Super-Resolution Photo-Activated Thermography, Nature Comm. (2019) under review
II. Nano-biotechnology
a) Photo-thermal nanoparticles for counterfeiting. Since 2008 we are developing, in collaboration with the group of P. Pallavicini (Univ. Pavia), new anisotropic gold nanoparticles to induce thermal release by irradiation with near infrared laser (photothermal effect) or iron oxide nanoparticles designed for radiofrequency treatment of cancerous tissues (in collaboration with Cericol, Colorobbia, Empoli). Here we focus on the possibility to print inks composed of photothermal nanoparticles by means of ink-jet printing (collaboration with J. Peltonen, Abo Univ. Turku, FL). The possibility to tune the photothermal response of the nanoparticle inks can be exploited to code invisible thermal bar codes on a variety of surfaces. Applications in the field of counterfeiting is currently under development.
Relevant publications:
• Pallavicini, P.;Donà, A.; Casu A.,Chirico, G.;Collini, M.; Daccarro G.;Falqui, A.; Milanese C.; Sironi L.; Taglietti A. Triton X-100 for three-plasmon gold nanostars with two photothermally active NIR (near IR) and SWIR (short-wavelength IR) channels. Chem. Commun. (2013) 49: 6256-6267.
• Chirico, G., Dacarro, G., O’Regan, C., Peltonen, J., Sarfraz, J., Taglietti, A., et al. (2018). Photothermally Responsive Inks for Inkjet-Printing Secure Information. Part. Part. Syst. Charact. 2018 , 180009
b) Nano-thermal patches. Heating patches are widely applied medical devices able to relieve pain and foster healing. Current heating patches have a range of disadvantages: single use, slow and uncontrolled heating, side effects related to skin irritations or burns. Our NanoThermoPatch project (patented, Unimib) aims to the production of novel, “smart” patches whose thermal load can be remotely controlled by near infrared light irradiation. Our clear-cut, reachable objectives for the next 2 years are to improve patch properties (α prototypes have already been fabricated), to perform pre-clinical and clinical trials in order to reach TRL 7 and establish partnership with companies. The interdisciplinary team is composed of faculties of the University of Milano-Bicocca and Pavia.
Relevant publications:
• Patent:
c) Remotely activated nanoparticles based coatings for antimicrobial applications Antibacterial treatment is an essential issue in many diverse fields, from medical device treatments (for example prostheses coating) to food preservation. The use of photo-thermally active nanoparticles can lead to novel and re-usable materials that can be remotely activated on-demand to thermally eradicate bacteria and mitigate biofilm formation. We develop polyvinyl alcohol (PVA) hydrogel films containing non-toxic and highly photo-thermally active Prussian blue (PB) nanoparticles. The high and localized increase of temperature on the fabricated films resulted in an efficient antibacterial effect on Pseudomonas aeruginosa (P. aeruginosa) bacteria. In addition, the localized photo-thermal effect was also sufficient to substantially mitigate biofilms growth.
Relevant publications:
• Pallavicini P., Bassi B., Chirico G., Collini M., Dacarro G., Fratini E:, Grisoli P., Patrini M., Sironi L., Taglietti A., Moritz M., Sorzabal-Bellido I., Susarrey-Arce A., Latter E., Beckett A.J., Prior I.A., Raval R. & Diaz Fernandez Y.A. Modular approach for bimodal antibacterial surfaces combining photoswitchable activity and sustained biocidal release. Scientific Reports (2017) 7: 5259.
• Borzenkov, ; D’Alfonso, L.; Polissi, A.; Sperandeo, P.; Collini, M. ; Dacarro, G ; Taglietti, A ; Chirico, G ; Pallavicini, P. “Novel photo-thermally active polyvinyl alcohol-Prussian blue nanoparticles hydrogel films capable of eradicating bacteria and mitigating biofilms”. NANOTECHNOLOGY (2019) 30(29): 295702;
d) Proteinaceous 2-photon polymerized microstructures. The absorption of light from dyes leads often to the formation of free radicals that can then react with moieties of the protein aminoacids to enhance their reactivity. We exploit this possibility and mix proteins (BSA or lysozyme) with sensitizing dyes (Rose Bengal or Methylene Blue) and, by irradiating the mixture with a highly focused Near Infrared laser beam we induce two-photon absorption in the dye and induce cross-linking in the protein, thereby producing microstructures of well defined geometry. Furthermore, by mixing proteins with photothermally active nanoparticles (Prussian Blue NPs), we are currently producing microstructures that have a highly localized photothermal effect for applications in the tissue regeneration field.
Relevant publications:
• B. Zeynali, F. Ferrario, M. collini, M. Borzernkov, P. Pallavicini, L. Sironi, M. Bouzin; G. Chirico. Proteinaceous microstructures with photothermal efficiency by 2-photon polymerizataion of BSA. under preparation 2019.
• Marini, M.; Bouzin, M.; Zeynali, A.; M. Collini, G. Chirico. Super-Resolution Photo-Activated Thermography, Eur. Biophys. J (2019) 48: S203-S203
• Pallavicini P, Cabrini E, Cavallaro G, Chirico G, Collini M, D’Alfonso L, Dacarro G et al. Gold nanostars coated with neutral and charged polyethylene glycols:A comparative study of in–vitro biocompatibility and of their interaction with SH—SY5Y neuroblastoma cells. J. Inorg. Biochem. (2015) 151:123-31.
EQUIPMENT OF THE BIOPHOTONICS LAB.
The core facility is an optical microscopy laboratory that encompass two microscopes for multiphoton fluorescence and second harmonic generation imaging, Fluorescence Correlation Spectroscopy setup for in-vitro studies and a Leica CW STED super-resolution microscope. More in details:
- An Olympus BX51 microscope adapted for two-photon and second harmonic generation in-vivo microscopy (equipped with MaiTai HP Ti:Sapph laser, Spectra Physics). Thermostated chamber with atmosphere regulation (CO2+O2). Three acquisition channels.
- A Leica SP5 confocal microscope with STED extension for in-plane optical resolution of 70 nm. Extension for two-photon excitation by coupling with Tsunami/Millennia Ti:Sapph laser
(Spectra Physics). Spectral resolution of images in the 400-700 nm range. Fluorescence correlation extension for measurement of Fluorescence Correlation functions on ima - Homemade Fluorescence Correlation Spectrometer (FCS) equipped with Ti:Sapph laser (Tsunami, Spectra Physics) for single spot measurements and FCS imaging.
- Homemade all reflective raster scanning microscope equipped with EM-CCD for crosscorrelation imaging. The setup is optimized for infrared excitation used in two-photon spectroscopy and imaging. Source: Tsunami/Millennia Ti:Sapph laser (Spectra Physics).
GRANTED PATENTS
• International Patent, WO 2019/123227 A1, “Implantable Medical Device” (applicants: Polimi, CNR, Unimib; inventors: M. Raimondi, G. Cerullo, G. Chirico, R. Osellame, C.
Conci, T. Zandrini).
Submitted patents
• Italian Patent n. 102018000004053 was filed on 28.03.2018 (inventors: M. Borzenkov; G. Chirico; M. Collini, P. Pallavicini); an extended international patent (PCT/EP2019/057747 date of filing inventors)
RECENT PUBLICATIONS ( from 2017 )
• Dacarro, Giacomo, Pallavicini, Piersandro, Bertani, Serena Maria, Chirico, Giuseppe, D’Alfonso, Laura, Falqui, Andrea, Marchesi, Nicoletta, Pascale, Alessia, Sironi, Laura, Taglietti,
Angelo, Zuddas, Efisio. Synthesis of reduced-size gold nanostars and internalization in SHSY5Y cells. J. Coll. Interf. Sci. (2017) 505: 1055-1064
• Pallavicini P., Bassi B., Chirico G., Collini M., Dacarro G., Fratini E:, Grisoli P., Patrini M., Sironi L., Taglietti A., Moritz M., Sorzabal-Bellido I., Susarrey-Arce A., Latter E., Beckett A.J., Prior I.A., Raval R. & Diaz Fernandez Y.A. Modular approach for bimodal antibacterial surfaces combining photo-switchable activity and sustained biocidal release. Scientific Reports (2017) 7: 5259.
• Colombo, S; Broggi, S; Collini, M; et al. Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes. Biochem Biophys. Res. Commun. (2017) 487: 594-599
• Ceffa N., Cesana I., Collini M., D’Alfonso L., Carra S., Cotelli F., Sironi L., Chirico G. Spatiotemporal image correlation analysis of blood flow in branched vessel networks of zebrafish embryos. J. Biomed. Opt. (2017) 22(10), 106008.
• F. Radaelli, L. D’Alfonso, M. Collini, F. Mingozzi, L. Marongiu, F. Granucci, I. Zanoni, G. Chirico & L. Sironi. μMAPPS: a novel phasor approach to second harmonic analysis for in vitro-in vivo investigation of collagen microstructure. Scientific Reports (2017) 7: 17468.
• Ceffa N.G., Bouzin M., D’Alfonso L., Sironi L., Marquezin C.A., Auricchio F., Marconi S., Chirico G., Collini M. Spatio-temporal Image Correlation Analysis for 3D Flow Field Mapping in Microfluidic Devices. Anal. Chem. (2018) 90(3):2277-2284.
• Collini, Maddalena; Bouzin, Margaux; Chirico, Giuseppe. Out of the Randomness: Correlating Noise in Biological Systems. Biophys. J. (2018) 114(10): 2298-2307
• Taheri, F.; Isbilir, B.; Muller, G. ; Krieger, JW. ; Chirico, G. ; Langowski, J.; Toth, K. Random Motion of Chromatin Is Influenced by Lamin A Interconnections. Biophys. J. (2018) 114(10):
2465-2472
• Sarfraz, J.; Borzenkov, M.; Niemela, E.; Weinberger, C. ; Torngren, B.; Rosqvist, E.; Collini, M.; Pallavicini, P. ; Eriksson, J.; Peltonen, J.;Ihalainen, P.; Chirico, G. Photo-thermal and cytotoxic properties of inkjet-printed copper sulfide films on biocompatible latex coated substrates. Appl. Surf. Sci. (2018) 43: 1087-1095
• Borzenkov, M., Moros, M., Tortiglione, C., Bertoldi, S., Contessi, N., Faré, S., et al. Fabrication of photothermally active poly(vinyl alcohol) films with gold nanostars for antibacterial applications, Beilstein Journal of Nanotechnology. 9, 2040-2048. (2018).
• Chirico, G., Dacarro, G., O’Regan, C., Peltonen, J., Sarfraz, J., Taglietti, A., et al. (2018). Photothermally Responsive Inks for Inkjet-Printing Secure Information. Part. Part. Syst. Charact. 2018 , 180009
• Chirico, G, Gansen, A, Leuba, SH, Olins, AL, Olins, DE, Smith, JC, Tóth, K (2018). Jörg Langowski: His scientific legacy and the future it promises. BMC BIOPHYSICS, (2019) 11, 5
• Borzenkov, ; D’Alfonso, L.; Polissi, A.; Sperandeo, P.; Collini, M. ; Dacarro, G ; Taglietti, A ; Chirico, G ; Pallavicini, P. “Novel photo-thermally active polyvinyl alcohol-Prussian blue nanoparticles hydrogel films capable of eradicating bacteria and mitigating biofilms”. NANOTECHNOLOGY (2019) 30(29): 295702
• Collini, M; Radaelli, F ; Sironi, L ; Ceffa, NG ; D’Alfonso, L ; Bouzin, M ; Chirico, G. Adaptive optics microspectrometer for cross-correlation measurement of microfluidic flows. J. Biomed. Opt. (2019) 24: 025004
PROJECTS.
INFM projects.
2000-2004. Principal investigator at the two-photon facility of the INFM (University of Genoa). G.Chirico.
2001-2002. INFM funding for the project “Conformational dynamics of single molecules by spectroscopy and manipulation”, national PI (6 units), G.Chirico.
2000-2001. INFM funding for the project “Structural and Dynamic studies of proteins by Single Molecule Spectroscopies”, national PI (5 units) G.Chirico.
2001-2003 PAIS Project : ”FOLGIN, Stabilization of native intermediates in betalactoglobulin folding pathway: role of ligand interaction as detected by NMR and optical spectroscopies”.
2003 PAIS (INFM) “MEAB: multiphoton excitation of advanced bioprobes”
2003-2006. Fondo Integrativo Speciale per la Ricerca (FISR a sportello): “Nanoscreening automatico di singole cellule per lo studio di interazioni proteina-proteina con spettroscopia di fluorescenza a singolo o doppio fotone”. National PI (2 units) G.Chirico.
MIUR PRIN
2003-2005. MIUR PRIN: “Nanocapsules as a biomometic system for the study of molecular crowding by means of advanced fluorescence spectroscopy and microscopy methods”, national PI (2 units) G.Chirico.
2006-2009. MIUR PRIN:”Conformational substates and folding-unfolding pathways in green fluorescent protein: an experimental and theoretical study of discrete states in proteins” national PI, M.Collini
2008-2011., MIUR PRIN Studies of fluorescence correlation spectroscopy of photoactivable proteic constructs for dynamic application in optical microscopy by means of two color excitation”, local PI, M.Collini
Firb (RBNE01YSR8): “Molecular e biomolecular Nanostructures”. Local PI. Years 2002- 2005.
Cariplo Foundation projects.
-PI of the project “Construction and read out of 2D networks of fluorescent molecules by AFM: towards molecular optical memories” funded by Fondazione Cariplo (code: 2005-
1079; total cost: €180.000,00; final approved report on july 2008).
-Local PI for the 2010 Cariplo Foundation project “Gold nanorods (NR) and asymmetric nanoparticles (ANP) capped with a biocompatible polymer bearing binding groups for
molecules and metal cations: pharmacological and thermal antimicrobial action activated by near-IR irradiation” (code: ref. 2010-0454; total cost: €100000).
Fondazione Banca del Monte fundings.
Laboratory funding for the acquisition of an ANDOR spectral camera (value: 25.000,00 Euro) to be -coupled to Near Infrared microscopes. Year: 2005.
EU projects.
co-Principal Investigator (together with prof. F. Granucci, unimib) of an EU (FP7: Cooperation Health-2007-1.2-4) Health project (ENCITE: European Network for Cell Imaging and Tracking Expertise, Grant agreement No: 201842), on the study of the interactions between DC and NK cells in vivo by multiphoton intravital microscopy (PIs F. Granucci and G. Chirico; 2008-2012; gross funding: € 600.000,00).
Regione Lombardia projects.
PI in a two years project financed by Regione Lombardia nov-2010/nov 2012 (total cost € 300.000,00) on Raman Microscopy for Biomedical Applications, together with Politecnico di
Milano (Physics Dept).
Unimib Fundings.
-Invivo imaging Microscope, funded with €550000 in 2005-6 by the Università di Milano Bicocca, G.Chirico
– Microscopia di super-risoluzione, funded with €524000 in 2011 by Fondi per le Grandi Attrezzature dell’Universita’ di Milano-Bicocca, M.Collini.
– Fondi per quota competitiva 2014 Gold nanostars decorated with DNA G-quadruplexes for an unprecedented multimodal targeting of cancer, M.Collini
-2015 Innovation grant Unimib. Holoscope: an holographic microscope for medical imaging. PI: G. Chirico, P. Pozzi, E. D’Angelo, J. Mapelli
-Fondi quota competitiva 2017. I-Nano: A multidisciplinary network for smarter bioimaging, G. Chirico
Private Foundations: 2017 Zcube open accelerator program, Zambon, Milano, to G. Chirico, M. Borzenkov, M. Collini, P. Pallavicini.