OVERVIEW
ROUTINE LIGHT MICROSCOPY
ADVANCED LIGHT MICROSCOPY
ELECTRON MICROSCOPY
HIGH THROUGHPUT MICROSCOPY
MASS IMAGING
LABEL-FREE IMAGING OF CELLS AND TISSUES
SUPPORT AND TECHNOLOGY DEVELOPMENT

Euro-bioimaging ERIC infrastructure

Welcome to the Naples Euro-BioImaging ERIC facility located at the IBBC in CNR Research area NA-1. The facility is a continuation of the imaging facility developed by Alberto Luini and colleagues at the Mario Negri Sud Institute for Biomedical Research in the 1990s that pioneered the development of the modern Correlative video-Light Electron Microscopy (CLEM). It is the coordinating facility of the Advanced Light Microscopy Italian multi-modal multi-sited Node of Euro-BioImaging, a pan European research infrastructure project (for more information see: http://www.eurobioimaging.eu/).  It is managed and operated by the IBBC. The Naples Euro-BioImaging facility (and all other Euro-BioImaging facilities in the participating countries) started their interim operation since May 2016. The Naples facility can be accessed through the Euro-BioImaging portal or following the instruction reported here.

Brigthfield, Phase contrast, DIC and confocal microscopy

The facility is equipped with a phase contrast and DIC microscope, and two laser scanning confocal microscopes. All the systems have the temperature and CO2 control module, required for live imaging studies. The IBBC users will be provided batch training once in a year in the use of the routine ligth microscopes.

Services provided:

  • Bright-field, DIC and phase-contrast microscopy, for evaluation of general morphology and enzyme-based labelling of cells and tissues;
  • Wide-field fluorescence microscopy, for analysis of fluorescent marker distributions in cells and tissues;
  • Confocal microscopy, to investigate co-localisation of proteins and their three-dimensional (3D) space distribution
  • Image analysis station equipped with Zen off line analysis program, AxioVision off line analysis program, Leica image analysis suite, Image J, Cell profiler and Metamorph image analysis software. 

High speed confocal 4D imaging, spectral analysis, super-resolution imaging, imaging multiprotein complexes by multiplexing FRET/FLIM

The facility is equipped with two laser scanning confocal microscopes, one spinning disc confocal microscope and a structured-illumination based microscope. All the microscopes are equipped with temperature and CO2 control modules necessary for live imaging studies. The IBBC users will be provided batch training once in a year in the use of confocal microscopes and the apotome microscope and if sufficient numbers of people are present more additional courses will be considered. For the use of other microscopes individual training will be provided in case of need.

Services provided

  • Live-cell imaging (time-lapse microscopy), for monitoring intracellular organelles and protein dynamics in time and in 3D space;
  • Video Microscopy including Fluorescence recovery after photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) for quantitative evaluation of protein mobility within organelles, rates of inter-organelle exchange, and protein binding kinetics;
  • Fluorescence resonance energy transfer (FRET) and Fluorescence Lifetime Imaging (FLIM) , to evaluate the strength and intracellular topology of protein-protein interactions, and monitor intracellular dynamics of small signalling molecules and protein activities;
  • Lambda imaging;
  • Super resolution imaging (G-STED);
  • Cell microinjection, for acute modulation (activation or inhibition) of protein activities;
  • Image analysis station equipped with Zen off line analysis program, AxioVision off line analysis program, Leica image analysis suite, Image J, Cell profiler and Metamorph image analysis software.

Equipment

Zeiss LSM700

Confocal imaging, FRAP, FLIP, FRET, LAMBDA

Zeiss Axio zoom

Imaging of tissues at low resolutionat low resolution

INS

Apotome

Imaging with structured illumination, video imaging

g, FRAP, FLIP, FRET, LAMBDA

Leica SP5

Confocal imaging, FRAP, FLIP, Super resolution imaging (G-STED), Protein-protein interaction (FLIM, FRET)ues at low resolution

Nikon

Spinning disc confocal, Fast 3D live imaging, 3D deconvolution, high throughput confocal imaging

3D imaging (tomography and FIB-SEM), cryo-immunogold labelling 

The facility is equipped with a FEI Tecnai G2 Spirit BT electron microscope, a FIB-SEM microscope, microtomes for sectioning and a sample preparation lab. The Tecnai G2 BT Spirit microscope is equipped for automatic tilt series acquisition for tomographic studies. For regular users individual training on sample preparation and microscope use will be provided.
Others will have to utilize the services of the facility staff for experiments. Contact us for help with planning of the experiments

Services provided:

  • Routine Electron microscopy with epon embedding, for analysis of general cell and/or tissue ultrastructure;
  • Whole-mount electron microscopy preparation for investigation of ultrastructural changes in fractionated organelles or viral particles
  • Immuno-electron microscopy for ultrastructural evaluation of localisation (and co-localisation) of proteins of interest;
  • Correlative light-electron microscopy (CLEM), for electron microscopy analyses of structures observed in living cells before fixation, and of individual cells with various expression levels genes of interest, or particular intracellular distributions of a gene product.
  • Electron Tomography (ET) for 3D study of complex cellular structures, such as organelles, cytoskeleton, and chromosomes. It recovers the specimen’s 3D structure via computerized tomographic reconstruction from a set of 2D projections obtained with Transmission Electron Microscopy (TEM) at different tilt angles.
  • Cryo-immunogold labeling.
  • Electron Tomography and Focused ion beam – scanning electron microscopy (FIB – SEM)
  • Image analysis station equipped withFEI 3D reconstruction, : iTEM, Amira and IMOD.

Equipment

FEI Tecnai G2 Spirit BT

Equipped also for tomography acquisition

FEI Dual Beam SCIOS EM

Equipped for automated 3D imaging / reconstructionlow resolutionat low resolution

INS

Cryo ultra microtome

Preparation of frozen thin sections

Ultra microtome

Preparation of thin sections 

Imaging-based siRNA and drug screenings

The facility provides two microscopes that are equipped for high throughput image acquisition, a liquid handler station and sample preparation room equipped with a laminar flow hood and CO2 incubators. Users need to consult before planning the experiments.

Services provided
Contact us for details

Equipments

Olympus Scan^R

For high-throughput wide field imaging

Nikon spinning disk confocal

For high-throughput confocal imaging.

Image analysis:  Olympus image analysis suite, Cell Profiler.

Metabolomics analysis at cellular resolution 

The facility is equipped with one mass-spectrometer based imager and the necessary sample preparation equipment. The machine is capable for providing a mass-spectrometry based analysis of small molecules (lipids, metabolites) in tissue slices and cell culture samples. 

Equipment

AP-SMALDI10

Imaging mass spectometer

Label-free or deuterium-based imaging of intracellular metabolites 

The facility houses two Raman microscopes allowing high-resolution imaging  for the label-free characterization of materials and specimens in terms of their chemical composition. The Xplora INV Confocal Raman microscope (Jobin-Yvonne) is equipped with three laser sources (532nm, 630 nm and 780 nm), a DuoScan technology and it combines a sensitive confocal microscope with a high throughput spectroscopy system enabling sub-micron Raman imaging, adjustable laser spot sizes, and 100% coverage of a sample for macro-mapping. The home developed Raman microscope can be combined with other technologies (fibers, microfluidics, holography, optical tweezers) for high versatile experiments.

For regular users individual training on sample preparation and microscope use will be provided. Others will have to utilize the services of the facility staff for experiments. 

Contact us for help with planning of the experiments.

Equipment

Horiba Raman Imaging Microscope

 Olympus Raman Spectroscopy

Laboratory for sample preparation, laser micro dissection, micro injection and image analysis

Support

  • Laboratory for sample preparation, laser micro dissection, micro injection
  • Data analysis: Development of new image analysis software tools;
    Morphometric analysis of co-localisation, and intracellular protein distributions and dynamics;
    Electron microscopy-based morphometric analyses of the shape and size of cellular organelles and extracellular structures, and evaluation of local concentrations of gene products after their expression in cells of interest. 

Development

The Euro-BioImaging facility collaborates extensively with the imaging community in the city of Naples which includes the institutes of CNR, other research institutions like SDN and the Federico II university of Naples, to develop advanced microscopes and imaging techniques.

 The following technologies are in the process of development:

1) Raman spectroscopy-based imaging: We have developed a Raman microscopy/spectroscopy-based method for detection of lipids and other small molecules at sub-cellular resolution. 

2) Novel correlative methods: We are extending the reach of the correlative methods by integrating video microscopy with methods to image large multimolecular complexes by EM, or mass-spectrometry based imaging.

3) Image analysis: We are also developing methods based on cognitive image analysis systems for interpretation and classification of cellular images. This is carried out in collaboration with researchers at the ICAR, Napoli.  

Training

The Euro-BioImaging facility at IBBC organizes programs aimed at training young researchers in the use of basic as well as advanced microscopic techniques. We organize a yearly course on the theory and practice of microscopy for the prospective regular users of the facility. In addition, requests for special training sessions are also supported by appropriate training programs. 
We also organize workshops/meetings on imaging and microscopy.

Equipment


Microdissecter

Laboratory

Microinjector

Image analysis: image analysis station equipped with Zen off line analysis program, AxioVision off line analysis program, Leica image analysis suite, Image J, Cell profiler and Metamorph image analysis software. 


Access to Facility


For internal users:

Only certified independent users are allowed to use the microscopes.
Each certified user will be provided with a magnetic card to access the facility.
New users will have to participate in the annual training course on the use of microscopes before they will be allowed to access the facility. If you are not a certified user and you need to use the microscope, you can do so with the help of another experienced user from your group. Alternatively, you can contact the facility members for support.

Booking the microscopes:

Each research group is allotted timeslots to use the microscopes. The allocation chart can be found here.
These slots can be booked online.
The slots should be booked no later than 9:00 AM of that day; else the slot is considered free and other users may book the turn
The slots that have been booked should be occupied within 15min of the start of the booking time, else other users will be to free use the slot. Bookings need to be cancelled at least 24h in advance, else you will be charged for the use. In case you need more than the allotted time, you can book the free turns or time
The free slots should not be booked more than 48h in advance. 

Fee:

The charges for the use of the microscope and facility can be found here.
The costs of any damage to the instruments or the facility will be charged to the user.

For external users:

Being a part of the Euro-BioImaing network the requests from external users (other than IBBC) needs to be routed through the Euro-BioImaging portal. Details about this can be found here
We also encourage the potential users to contact the facility before so as to prepare the application together. Please write an email to this address: bioimaging@ibbc.cnr.it
You will be contacted by our staff as soon as possible to guide you through the procedures

For publications:

Depending on the nature of the request, an authorship in the eventual publication may be required. Please discuss about this with the facility head before starting the project. 
In any case (whether an authorship is provided or not) we request the users to please acknowledge the use of the BioImaging facility by inserting the following in the “Acknowledgement section” of their publications: “We thank the Euro-BioImaging facility at the Institute of Protein Biochemistry (CNR), Naples for help with microscopy experiments.”




Members & Contacts


Academic Advisor
Alberto Luini (Senior Associated Researcher)
Coordinator  
Seetharaman Parashuraman (Researcher)
Technology Advisors
Anna Chiara De Luca (Researcher)
Carmen Valente (Researcher)
Staff
Gabriele Turacchio (Technician)
Marinella Pirozzi (Researcher)
Valentina De Luca (Fellow)

Informatic support:  
Fabio Concilio


Selected Publications (2019-2020)


  1. Swartzwelter BJ, Barbero F, Verde A, Mangini M, Pirozzi M, De Luca AC, Puntes VF, Leite LCC, Italiani P, Boraschi D. Gold Nanoparticles Modulate BCG-Induced Innate Immune Memory in Human Monocytes by Shifting the Memory Response towards Tolerance. Cells. 2020 Jan 23;9(2). pii: E284.
  2. Loria AD, Dattilo V, Santoro D, Guccione J, De Luca A, Ciaramella P, Pirozzi M, Iaccino E. Expression of Serum Exosomal miRNA 122 and Lipoprotein Levels in Dogs Naturally Infected by Leishmania infantum: A Preliminary Study. Animals (Basel). 2020 Jan 8;10(1). pii: E100.
  3. Chiritoiu M, Brouwers N, Turacchio G, Pirozzi M, Malhotra V. GRASP55 and UPR Control Interleukin-1β Aggregation and Secretion. Dev Cell. 2019 Apr 8;49(1):145-155.e4.
  4. Subramanian A, Capalbo A, Iyengar NR, Rizzo R, di Campli A, Di Martino R, Lo Monte M, Beccari AR, Yerudkar A, Del Vecchio C, Glielmo L, Turacchio G, Pirozzi M, Kim SG, Henklein P, Cancino J, Parashuraman S, Diviani D, Fanelli F, Sallese M, Luini A. Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum. Cell. 2019 Mar 7;176(6):1461-1476.
  5. Sesorova IS, Karelina NR, Kazakova TE, Parashuraman S, Zdorikova MA, Dimov ID, Seliverstova EV, Beznoussenko GV, Mironov AA. Structure of the enterocyte transcytosis compartments during lipid absorption. Histochem Cell Biol. 2020 Mar 11.
  6. Parashuraman S, D’Angelo G. Visualizing sphingolipid biosynthesis in cells. Chem Phys Lipids. 2019 Jan;218:103-111.
  7. Pothukuchi P, Agliarulo I, Russo D, Rizzo R, Russo F, Parashuraman S. Translation of genome to glycome: role of the Golgi apparatus. FEBS Lett. 2019 Sep;593(17):2390-2411.
  8. Lania G, Nanayakkara M, Maglio M, Auricchio R, Porpora M, Conte M, De Matteis MA, Rizzo R, Luini A, Discepolo V, Troncone R, Auricchio S, Barone MV. Constitutive alterations in vesicular trafficking increase the sensitivity of cells from celiac disease patients to gliadin. Commun Biol. 2019 May 20;2:190.
  9. Varone A, Mariggiò S, Patheja M, Maione V, Varriale A, Vessichelli M, Spano D, Formiggini F, Lo Monte M, Brancati N, Frucci M, Del Vecchio P, D’Auria S, Flagiello A, Iannuzzi C, Luini A, Pucci P, Banci L, Valente C, Corda D. A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility. Cell Commun Signal. 2019 Mar 1;17(1):20.
  10. Tapia D, Jiménez T, Zamora C, Espinoza J, Rizzo R, González-Cárdenas A, Fuentes D, Hernández S, Cavieres VA, Soza A, Guzmán F, Arriagada G, Yuseff MI, Mardones GA, Burgos PV, Luini A, González A, Cancino J. KDEL receptor regulates secretion by lysosome relocation- and autophagy-dependent modulation of lipid-droplet turnover. Nat Commun. 2019 Feb 13;10(1):735.

show oldest publications >

  • Catara G, Grimaldi G, Schembri L, Spano D, Turacchio G, Lo Monte M, Beccari AR, Valente C, Corda D. PARP1-produced poly-ADP-ribose causes the PARP12 translocation to stress granules and impairment of Golgi complex functions. Sci Rep. 2017 Oct 25;7(1):14035.
  • Campelo F, van Galen J, Turacchio GParashuraman S, Kozlov MM, García-ParajoMF, Malhotra V.
    Sphingomyelin metabolism controls the shape and function of the Golgi cisternae. Elife. 2017 May 13;6. pii: e24603
  • Filadi R, Greotti E, Turacchio GLuini A, Pozzan T, Pizzo P.
    On the role of Mitofusin 2 in endoplasmic reticulum-mitochondria tethering. Proc Natl Acad Sci U S A. 2017 Mar 21;114(12):E2266-E2267
  • Brandolini L, d’Angelo M, Antonosante A, Villa S, Cristiano L, Castelli V, Benedetti E, Catanesi M, Aramini A, Luini A, Parashuraman S, Mayo E, Giordano A, Cimini A, Allegretti M.
    Differential protein modulation by ketoprofen and ibuprofen underlines different cellular response by gastric epithelium. J Cell Physiol. 2018 Mar;233(3):2304-2312.
  • Campelo F, van Galen J, Turacchio G,  Parashuraman S, Kozlov MM, García-Parajo MF, Malhotra V.
    Sphingomyelin metabolism controls the shape and function of the Golgi cisternae. Elife. 2017 May 13;6. pii: e24603
  • Hegde RN, Subramanian A, Pothukuchi P, Parashuraman S,  Luini A. Rare ER protein misfolding-mistrafficking disorders: Therapeutic developments. Tissue Cell. 2017 Apr;49(2 Pt A):175-185.
  • Rizzo R, Parashuraman S, D’Angelo G,  Luini A
    GOLPH3 and oncogenesis: What is the molecular link? Tissue Cell. 2017 Apr;49(2 Pt A):170-174.
  • Capasso S, Sticco L, Rizzo R, Pirozzi M, Russo D, Dathan NA, Campelo F, van Galen J, Hölttä‐Vuori M, Turacchio G, Hausser A, Malhotra V, Riezman I, Riezman H, Ikonen E, Luberto C, Parashuraman SLuini A and D’Angelo G.
    Sphingolipid metabolic flow controls phosphoinositide turnover at the trans‐Golgi network. EMBO J. May 2, 36 (9): 1117–1297 (2017)
  • Filadi R, Greotti E, Turacchio GLuini A, Pozzan T, Pizzo P. On the role of Mitofusin 2 in endoplasmic reticulum-mitochondria tethering. Proc Natl Acad Sci U S A. 2017 Mar 21;114(12):E2266-E2267.
  • Raote I, Ortega Bellido M, Pirozzi M, Zhang C, Melville D, Parashuraman S, Zimmermann T, Malhotra V.TANGO1 assembles into rings around COPII coats at ER exit sites.J Cell Biol. 2017 Mar 9; 216 (3).
  • Gaglio D, Valtorta S, Ripamonti M, Bonanomi M, Damiani C, Todde S, Negri AS, Sanvito F, Mastroianni F, Di Campli A, Turacchio G, Di Grigoli G, Belloli S, Luini A, Gilardi MC, Colangelo AM, Alberghina L, Moresco RM. Divergent in vitro/in vivo responses to drug treatments of highly aggressive NIH-Ras cancer cells: a PET imaging and metabolomics-mass-spectrometry study. Oncotarget. 2016 Aug 9;7(32):52017-52031.
  • Chesi G, Hegde RN, Iacobacci S, Concilli M, Parashuraman S, Festa BP, Polishchuk EV, Di Tullio G, Carissimo A, Montefusco S, Canetti D, Monti M, Amoresano A, Pucci P, van de Sluis B, Lutsenko S, Luini A, Polishchuk RS. Identification of p38 MAPK and JNK as new targets for correction of Wilson disease-causing ATP7B mutants. Hepatology. 2016 Jun;63(6):1842-59.
  • Schiattarella GG, Cattaneo F, Pironti G, Magliulo F, Carotenuto G, Pirozzi M,, Polishchuk R, Borzacchiello D, Paolillo R, Oliveti M, Boccella N, Avvedimento M, Sepe M, Esposito G, Trimarco B, Feliciello A, Perrino C. Akap1 deficiency promotes mitochondrial aberrations and exacerbates cardiac injury following permanent coronary ligation via enhanced mitophagy and apoptosis. PLoS One. May 2, 11 (5) (2016).
  • Curwin AJ, Brouwers N, Alonso Y Adell M, Teis D, Turacchio G,Parashuraman S, Ronchi P, Malhotra V. ESCRT-III drives the final stages of CUPS maturation for unconventional protein secretion. Elife. 2016 Apr 26;5  
  • Filadi R, Greotti E, Turacchio GLuini A, Pozzan T, Pizzo P. Mitofusin 2 ablation increases endoplasmic reticulum-mitochondria coupling. Proc Natl Acad Sci U S A. 2015 Apr 28;112(17):E2174-81.
  • Chrisam M, Pirozzi M, Castagnaro S, Blaauw B, Polishchuck R, Cecconi F, Grumati P, Bonaldo P. Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice. Autophagy. 2015;11(12):2142-52.
  • Hegde RN, Parashuraman S, Iorio F, Ciciriello F, Capuani F, Carissimo A, Carrella D, Belcastro V, Subramanian A, Bounti L, Persico M, Carlile G, Galietta L, Thomas DY, Di Bernardo D, Luini A. Unravelling druggable signalling networks that control F508del-CFTR proteostasis. Elife. 2015 Dec 23;4.