presentazione standard di powerpoint · nikon a1r inverted confocal microscope for fast live...
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![Page 1: Presentazione standard di PowerPoint · Nikon A1R inverted confocal microscope for fast live imaging of single cells Single cell FRET analysis: in vivo caclium imaging at plant vacuolar](https://reader030.vdocuments.mx/reader030/viewer/2022041111/5f1573c485157d57cb69b08b/html5/thumbnails/1.jpg)
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DIVISIONE SERVIZI PER LA RICERCA -
UFFICIO AUDIT
NOLIMITSi
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DIVISIONE SERVIZI PER LA RICERCA -
UFFICIO AUDIT
NOvel LIve bioImaging MIlano c tTà Studi
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DIVISIONE SERVIZI PER LA RICERCA -
UFFICIO AUDIT
NOvel LIve bioImaging MIlano citTà Studi
![Page 5: Presentazione standard di PowerPoint · Nikon A1R inverted confocal microscope for fast live imaging of single cells Single cell FRET analysis: in vivo caclium imaging at plant vacuolar](https://reader030.vdocuments.mx/reader030/viewer/2022041111/5f1573c485157d57cb69b08b/html5/thumbnails/5.jpg)
Subscribers departments @ NOLIMITS:
1. Dip. di Bioscienze (DBS) (capofila). Alex Costa
2. Dip. di Scienze Farmacologiche e Biomolecolari (DiSFeB). Fabrizio Gardoni
3. Dip. di Scienze Biomediche per la Salute (SCIBIS). Elena Donetti
4. Dip. di Scienze Agrarie e Ambientali (DISAA). Piero Attilio Bianco
5. Dip. di Biotecnologie Mediche e Medicina Traslazionale (BIOMETRA). Roberto Cerbino
6. Dip. di Scienze per gli Alimenti, la Nutrizione e l'Ambiente (DeFENS). Luisa Pellegrino
7. Dip. di Scienze della Salute (DISS). Daniele Bottai
8. Dip. di Chimica. Luca Chiarello
9. Dip. di Oncologia (DIPO). Niccolo’ Bolli
10. Dip. Dipartimento di Scienze e Politiche Ambientali (ESP): Michela Sugni
11. Dip. di Fisica: Cristina Lenardi
Facility manager and technicians @ NOLIMITS:
1. Nadia Santo; D2-Area Tecnica, Tecnico-Scientifica ed Elaborazione Dati. Facility manager
2. Miriam Ascagni; D1-Area Tecnica, Tecnico-Scientifica ed Elaborazione Dati.
3. Laura Madaschi; D3-Area Tecnica, Tecnico-Scientifica ed Elaborazione Dati
4. Norma Lattuada; C4- Area Tecnica, Tecnico-Scientifica ed Elaborazione Dati
DIVISIONE SERVIZI PER LA RICERCA -
UFFICIO AUDIT
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Single photon confocal microscopes
Inverted Nikon A1R+ with FLIM, FRET-FLIM and FCS
Inverted Nikon A1R with SIM (Super Resolution,115 nm X,Y;
270 nm Z)
Inverted Zeiss 510
Multi photon confocal microscope
Upright Nikon A1 with MP Dual Red pulsed laser
Digital slide scanner
Scanning Electron Microscope (SEM)
Transmission Electron Microscope (TEM)
Magnetic Resonance Imaging (MRI).
Workstation and software for imaging analysis
Equipment
DIVISIONE SERVIZI PER LA RICERCA -
UFFICIO AUDIT
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Locations
Other locations:
Anin, Via G. Celoria 26
Locali Ex-Cima, Via G. Celoria 26
BIOMETRA, Via Vanvitelli, 32
DISAA, DeFENS, Via G. Celoria 2
DiSFeB, Via Balzaretti, 9
Main location:
Ex aula G08, Via C. Golgi 19
DIVISIONE SERVIZI PER LA RICERCA -
UFFICIO AUDIT
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Which tools can we use for single cell analyses?
Confocal Microscopes @ the
UNITECH NOLIMITS (bio-imaging facility)i
Nikon A1R Nikon A1 3D-SIM
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Miriam
Ascagni
Confocal microscope high end for fast live imaging (cell cultures, animals, microorganisms, plants and food...)
Ex aula G08, Via C. Golgi 19
➢ Lasers: 405 nm, 445 nm, 488 nm, 561 nm,
633 nm
➢ Control of temperature and CO2
➢ Hybrid Scanner for ultrahigh speed imaging
and simultaneous photo-activation
➢ 3 PMTs, 2 GaAsP
➢ Perfect Focus
➢ Deconvolution software
Nikon A1R
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Ex aula G08, Via C. Golgi 19
➢ Lasers: 405 nm, 445 nm, 488 nm, 561
nm, 633 nm
➢ Control of temperature and CO2
➢ Hybrid Scanner for ultrahigh speed imaging
and simultaneous photo-activation
➢ 3 PMTs, 2 GaAsP
➢ Perfect Focus
➢ Deconvolution software
Miriam
Ascagni
Confocal microscope high end for fast live imaging (cell cultures, animals, microorganisms, plants and food...)
Nikon A1R
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Sensor for in vivo Calcium detection: Cameleon (YC3.6)
Features:
• Ratiometric (FRET based): correct for unequal dye loading,
illumination intensity, optical path
length, less sensitive to bleaching etc.
• Genetically encoded
• Strong signal to noise ratio
• High Ca2+ specificity
• Low pH sensitivity
• Targetable to different cellular
compartments
Ratio=cpVenusEm530/CFPEm480
Nagai et al., 2004
HIGH
[Ca2+]
CFP
cpVenus
440 nmCaM
M13
480 nm
LOW
[Ca2+]
Abs Em Abs Em
400 500 600
Wavelength (nm)
Ab
san
d E
mS
pe
ctr
a
FRET
530 nm440 nm
CFP cpVenus
CaM
M13
Ca2+Ca2+
Ca2+Ca2+
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Nikon A1R inverted confocal microscope
for fast live imaging of single cells
Single cell FRET analysis: in vivo caclium imaging at plant vacuolar membrane
➢ Lasers: 405 nm, 445 nm, 488 nm, 561 nm, 633 nm
➢ Control of temperature and CO2
➢ Hybrid Scanner for ultrahigh speed imaging and
simultaneous photo-activation
➢ 3 PMTs, 2 high sensitive GaAsP
➢ Perfect Focus
➢ Deconvolution software
CFP cpVenus FRET
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…ongoing generation of a tonoplast localized YC3.6 Cameleon…
Doccula and Costa, unpublished
Arabidopsis stable plants transformed with nTP-YC3.6
ECFP cpVenusYC3.6
Kd=250 nMN-tag-fusion
-0,2
-0,1
0
0,1
0,2
0,3
0,4
0,5
0 100 200 300 400 500 600
ROI 1ROI 2ROI 3ROI 4ROI 5
Time (sec)
DR
/R0
ATP
Doccula and Costa, unpublished
Ratio cpVenus/CFP
[Ca2+]
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ε-subunit of Bacillus subtilis ATP synthase
ATeam1.03-nD/nA
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The effect of CCCP on the ATeam response in different
Arabidopsis seedling tissues
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FLIM and FRET-FLIM for
in vivo protein-protein interaction analysis
Ex aula G08, Via C. Golgi 19
➢ Lasers: 405 nm, 445 nm, 488 nm, 561 nm,
633 nm
➢ Control of temperature and CO2
➢ Hybrid Scanner for ultrahigh speed imaging
and simultaneous photo-activation
➢ 3 PMTs, 2 GaAsP
➢ Perfect Focus➢ Pulsed lasers: 440 nm, 485 nm
➢ Hybird Sensors for photon counting
Miriam
Ascagni
Confocal microscope high end for fast live imaging (cell cultures, animals, microorganisms, plants and food...)
Nikon A1R
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9 months seasoned cheese
10 µm
Fat
Proteins
Ex aula G08, Via C. Golgi 19
➢ Lasers: 405 nm, 488 nm, 561 nm, 633 nm
➢ Spectral GaAsP detector for l scan
➢ 3 PMTs, 2 GaAsP
➢ Control of temperature and CO2
➢ Piezo stage
➢ Perfect Focus
➢ Deconvolution software
Miriam Ascagni
Confocal microscope high end for fast live imaging (cell cultures, animals, microorganisms, plants and food...)
Nikon A1
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Confocal microscope high end for fast live imaging (cell cultures, animals, microorganisms, plants and food...)
Nikon A1
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Deconvolution for high
resolution live cell
imaging
Confocal microscope high end for fast live imaging (cellcultures, animals, microorganisms, plants and food...)
Nikon A1
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Deconvolution for high
resolution live cell
imaging
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Courtesy of Dr. M. Paroni
Bacteria Zebrafish ear
Courtesy of Prof. A. Binelli
Microplastics in zebrafish
Courtesy of Prof. L. Del Giacco
Samples from users working at the Department of Biosciences
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Ex aula G08, Via C. Golgi 19
➢ Lasers: 405 nm, 488 nm, 561 nm,
633 nm
➢ Spectral GaAsP detector for l scan
➢ 3 PMTs, 2 GaAsP
➢ Control of temperature and CO2
➢ Piezo stage
➢ Perfect Focus
➢ Deconvolution software
3D-SIM in vivo Super
Resolution imaging
(115 nm x,y; 270 nm Z)
Laser per SR: 488 nm, 561 nm, 633 nm
Miriam Ascagni
Confocal microscope high end for fast live imaging (cell cultures, animals, microorganisms, plants and food...)
Nikon A1 with 3D SIM
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Nikon A1 inverted confocal microscope
with 3D-SIM for in vivo Super resolution
Courtesy of Prof. G. Cappelletti
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Arabidopsis stomata expressing PM-localised GFP
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Which tool can we use for single cell analyses in entire
organisms?
Nikon A1 MP
Multiphoton microscope @ the
UNITECH NOLIMITS (bio-imaging facility)i
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Red light penetrates tissue more deeply than light of shorter wavelengths
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Upright Multiphoton Confocalfor imaging in vivo on animal, plants and ticktissues, organoids, brain slices…
Multiphoron
Confocal Stops
Here
Nikon MP A1R
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Stabulario, Via G. Celoria 26
➢ Multiphoton Laser 820-1300 nm + 1040 nm
➢ Hybrid Scanner for ultrahigh speed imaging
and simultaneous photo-activation
➢ Four GaAsP detectors
➢ Deconvolution software
➢ CFI75 Apochromat 25XC W 1300 (N.A. 1.10)
Laura Madaschi
Upright Multiphoton Confocalfor imaging in vivo on animal, plants and ticktissues, organoids, brain slices…
Nikon MP A1R
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Fish from Prof. L. Del Giacco
Nikon MP A1RZebrafish tail vasculature
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Arabidopsis thaliana ovules
Nikon MP A1R
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Which tool can we use for single cell analyses for histopathology
analyses?
Digital Slide Scanner @ the
UNITECH NOLIMITS (bio-imaging facility)i
Hamamatsu NanoZoomer S60 Digital slide scanner
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Digital slide scanner
➢ Automatic slides acquisitions (auto focus, z-stack
for thick samples…)
➢ Bright field and fluorescence (DAPI/FITC/TRIC)
➢ Remote data acquisition
➢ 20 x and 40 x magnification
Laura Madaschi
Ex aula G08, Via C. Golgi 19
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Courtesy of Prof. L. Del GiaccoCourtesy of Prof. M. Muzi Falconi
Courtesy of Prof. K. Petroni Courtesy of Dr. A. Amadeo
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Cryo-Ultramicrotomes for electron microscopy specimens preparation
Norma Lattuada
Cryo CR-X system
Ex aula G08, Via C. Golgi 19
Nadia Santo
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SEM and TEM
electron microscopySEM-EDS TEM-EELS
Norma Lattuada
Zeolin accumulationTMV 30000
Locali Ex-Cima, G. Celoria 26; DISAA, DeFENS, G. Celoria 2; BIOMETRA, Vanvitelli 32
Nadia Santo
HeLa cell
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Alle macchine possono accedere indipendente: borsisti, dottorandi, assegnisti,
ricecatori etc..
L’uso indipendente delle macchine è possibile previo corso teorico/pratico.
Un sistema di gestione delle prenotazioni permette di controllare la disponibilità delle
macchine oppure richiedere prestazioni fornite dai tecnici. L’accesso sarà consentito a
utenti UNIMI utilizzando credenziali di Ateneo.
Si possono organizzare corsi per dottorati o altri workshop…
….some info….
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Per motivi di prevenzione, sicurezza e tutela della salute delle persone, nonché per preservare i beni
dell’Ateneo, devono essere osservate le seguenti disposizioni:
1) la frequenza al di fuori dell'orario ufficiale è riservata al personale docente e tecnico amministrativo;
2) nei laboratori o comunque nei locali ove sussistono rischi di varia natura non è consentito operare in
solitudine;
3) studenti, dottorandi, specializzandi, tirocinanti, borsisti, volontari frequentatori, così come individuati
dal "Regolamento per l’accesso dei laureati frequentatori alle strutture dell’Università", possono
accedere al di fuori dell'orario ufficiale solo previa autorizzazione del personale tecnico e se è
assicurata la concomitante presenza di personale universitario che professionalmente sia in grado di
svolgere una funzione attiva di sorveglianza e controllo sulle attività svolte e sul corretto impiego delle
apparecchiature;
4) il possesso delle chiavi della struttura deve essere autorizzato dal Coordinatore del CS o dal
Responsabile Tecnico che a tal fine predispone un apposito elenco delle persone consegnatarie. In
alcun modo le chiavi possono essere consegnate a personale non strutturato;
5) gli utenti autorizzati che accedono a NOLIMITS sono tenuti a verificare lo stato in cui lasciano il
laboratorio e a provvedere al ripristino delle condizioni in cui i locali e gli strumenti sono stati
consegnati.
Al fine di garantire la sicurezza e la tutela della salute di tutti i soggetti che debbano accedere alle
strutture universitarie al di fuori dell'orario ufficiale di apertura dell'Ateneo, si riportano di seguito l'orario
ufficiale di apertura dell'Ateneo:
dalle ore 7,30 alle ore 20,00 (dal lunedì al venerdì);dalle ore 8,00 alle ore 12,00 (il sabato).
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Tariffario
CODICE
PRESTAZIONE
DESCRIZIONE
PRESTAZIONE
DESCRIZIONE
AGGIUNTIVAUNITA' TARIFFA IMPORTO (Euro) IMPOSTE
TIPOLOGIA PIANO
DI UTILIZZO
1
Utilizzo
Microscopio
Confocale FRET-
FLIM senza
assistenza di
personale tecnico
Per gli utenti
delle strutture:
abbonate
1 ora 17.00 ESENTE
2
Per gli utenti
delle strutture:
dell'Università
degli Studi di
Milano
1 ora 25.00 ESENTE
3
Per le altre
strutture
universitarie,
enti e
istituzioni di
ricerca
pubblica
1 ora 37.50 22% IVA 25%
4
Per enti e
strutture:
private o
assimilabili
1 ora - 22% IVA 25%
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Contacts:
Scientific Coordinator
Alex Costa [email protected]
Facility Manager
Nadia Santo [email protected]
Confocal Microscopes
Miriam Ascagni [email protected]
Multiphoton and Slide Scanner
Laura Madaschi [email protected]
Electron Microscopes
Norma Lattuada [email protected]
Administration
Andrea Iula [email protected]
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More info about tariff and rules @
http://www.unimi.it/ricerca/finanziamenti_ateneo/90200.htm