School of Photonics Cortona March 30 – April 3 characteriza)on of func)onality and morphology of )ssue disease by non linear microscopy Francesco S. Pavone European Laboratory for Non-‐linear Spectroscopy (LENS), Sesto Fioren:no, Italy State-of-the-art methods Computed tomography Magne:c resonance imaging fMRI State-of-the-art methods Computed tomography Magne:c resonance imaging • • • • • CT Scan costs range from $1,200 to $3,200. Usually completed within 5 minutes. radiaAon dose from CT ranges from 2 to 10 mSv, the same as from background in 3 to 5 years. Able to image bone, soF Assue and blood vessels all at the same Ame. • • • MRI costs range from $1200 to $4000 (with contrast). Scanning typically run for about 30 minutes. MRI machine control/limit energy deposiAon in paAent. Suited for SoF Assue evaluaAon, e.g. ligament and tendon injury, spinal cord injury, brain tumors etc. Main Limitations • Cost • Size • Difficult to be implemented in a surgical scenario Optical alternatives Op:cal biopsy • To use light for noninvasively determine tissue conditions Imaging • Various techniques • Morphological examina:on • Endoscopic implementa:on • Direct visualiza:on Contrast mechanisms • Scattering • Fluorescence • Raman Spectroscopy • Auto or induced Fluorescence • Func:onal informa:on • Data analysis required Wide-field imaging White light & Narrowband imaging • Narrowband imaging uses laser light for highlighting absorbing structures in tissues Narrowband imaging White light Fluorescence imaging with photosensi:zers White light Fluo White light Fluo • Improved sensitivity • Low specificity NIR imaging • Use probes for specific labelling of cancer cells • High contrast imaging of cancer Direct comparison with WL and NBI • Better discrimination using fluo • It uses exogenous agents OCT and elastography WL vs OCT endoscopy Op:cal coherence tomography (OCT) of occult lung cancer during bronchoscopy. White arrows: superficial tumor. A,C White light bronchoscopy imaging. B, D Corresponding OCT in vivo imaging. Laser scanning confocal endoscopy Mauna Kea Techologies Cell-‐Vizio Op:scan Five-‐1 Confocal endomicroscopy already commercially available Pancreas Liver Cornea Non-linear endoscopy Non-‐linear endoscopy Radial resolu:on Axial resolu:on Rivera et al, PNAS 108, 17598 (2011) Non-linear endoscopy Tendon -‐ SHG • Unaveraged intrinsic fluorescence images of mouse lung at 50, 60, and 70 μm from the :ssue surface. a: lumen – w: alveolar wall • 4.1 fps, 800-‐nm, FOV 110 μm. • Power: 75 mW Colon -‐ TPF • Unaveraged SHG images of mouse tail tendon at 10, 20, and 30 μm from the surface. • 4.1 fps, 800-‐nm, FOV 110 μm. • Power: 30 mW Lung -‐ TPF • Five frames averaged intrinsic fluorescence images of mouse colon at 35, 45, and 55 μm from the surface. e: enterocytes – c: crypts – g: goblet cells • 4.1 fps, 800-‐nm, FOV 110 μm. • Power: 75 mW Fiber-based spectroscopy • Illumination of tissue through optical fibers • Collection of signal using fibers • Endoscopic capability • No imaging • Only spectroscopic data Fluorescence and reflectance spectroscopy “Teragnostic” Nanoparticles Molecular Imaging • Non-‐linear imaging in combina:on with molecular specific fluorescent nanopar:cles • Non-‐invasive diagnos:cs • Monitoring transdermal drug delivery Dijkhuizen et al, Transl Stroke Res 2 (2011) Gold nanopar:cle Boisselier et al, Chem Commun (2008) Nanoparticles (diagnostics) Cells with gold NP Cells w/o gold NP Yelin et al, Opt Express 11 (2003) Nanoparticles (therapeutics) Ex:nc:on spectra Thermal effect -‐ pulsed Thermal effect -‐ CW • Same laser for imaging and therapy Laser: CW – 4 W/cm2 Wavelength: 810 nm NP concentra:on: 5x109 cm-‐3 Terentyuk et al, J Biomed Opt 14 (2009) Morphology and chemical content in tumor detec)on Morphology Two photon and Second Harmonic Generation The diagram The diagram comparing the photo-physical pathways for two-photon excited fluorescence (left side) and resonance enhanced second harmonic generation (right side). Deep imaging Two-photon excitation advantages: • The fluorescence signal depends nonlinearly on the density of photon providing an absorption volume spatially confined to the focal region. • For commonly used fluorescence probes two-photon absorption occurs in the near-infrared wavelength range (700-1000 nm): less scattering than visible light (one photon), and deeper penetration imaging depth. Signal generation and fluorescence collection in clear and in scattering tissues TPF = Micron-scale resolution in deep tissue Helmchen F, Denk W. Deep tissue two-photon microscopy. Nat Methods. 2(12):932-40. (2005) Single Molecule Hyper Rayleigh Scattering A molecule can produce HyperRayleigh Scattering HRS when it comprises donor and acceptor moieties spanned by a uniaxial charge transfer path, known as a conjugation path. A polyenic stilbene derivative in its aromatic (top) and quinoid (bottom) configurations. There is a net change in dipole moment between both configurations. SHG Theory (Coherent Summation) A) The HRS from a population of N molecules: A) Randomly oriented molecules scatter incoherently and the total HRS power scales as N B) Aligned molecules scatter coherently, producing second-harmonic generation (SHG) whose power is well collimated and scales as N? B) Chemical content Fluorescence Life2me Imaging Microscopy (FLIM) FLIM TPE Fluorescence Decay τ = (Γ +k )−1 nucleus cytoplasm Fluorescence Light Proper:es • Intensity (excita:on light, cross sec:on) • Spectrum (energy band structure) • Life:me (relaxa:on phenomena, collisions, environment) LASER pulse Fluorescence light Two photons cross sections Absorp:on 700-‐1000 nm : Ti:Sapphire wavelength range • Deep imaging capability inside :ssues • Limited out-‐of-‐focus absorp:on • Reduced absorp:on and scagering with respect to UV light 600-‐1600 nm : Op:cal therapeu:c window of :ssues Can be microscopy chemically more selecAve? • • • IR vibraAonal spectroscopy can be more selecAve with respect to electronic transiAons (see one or two photons microscopy), because of sharper and more selecAve absorpAon lines. Imaging resoluAon is anyway reduced when moving to longer wavelength Spontaneous Raman Sca^ering avoids this problem, permi_ng so a direct chemical imaging of unstained samples (avoiding photobleaching and labelling perturbaAons), and keeping the high resoluAon. CARS/Raman Chemically-‐selec:ve vibra:onal technique 2ω pump − ωstokes = Ω • By properly tuning Pump & Stokes • Imaging of a par:cular vibra:onal transi:on LABEL-‐FREE VIBRATIONAL IMAGING!!! Morphological Measurements (TPEF - SHG) Bladder Tumor multiphoton vs histology Carcinoma in situ Healthy Mucosa Cells Connective tissue border Excitation wavelength: 740 nm (TPEF-green) + 840 nm (SHG-blue) Resolution: 1024 × 1024 pixels Pixel dwell time: 5 µs Scale bars: 10 µm Bladder Tumor Morphology (TPE autofluorescence) Healthy Mucosa Excitation wavelength: 740 nm (NADH) Resolution: 1024 × 1024 pixels Pixel dwell time: 5 µs Laser power: 10 mW Scale bars: 10 µm Carcinoma in situ Differences • Nucleus to Cytoplasm ratio • Cell shape • Cell density Bladder Tumor Morphology (nuclear dimension) Carcinoma in situ Healthy Mucosa Measuring the cell area and the nucleus area on thresholded images • 50 cells (HM) • 50 cells (CIS) • measured cellular area • measured nuclear area • Cell-to-nucleus area ratio Opt. Express 18, 3840-3849, 2010 Colon Tumor TPF vs Histology 20X 40X Excita:on wavelength: 740 nm TPEF detec:on: 430-‐490 nm (NADH) Pixel dwell :me: 5 µs Laser power: approx. 30 mW Healthy mucosa Polyp Carcinoma Histological images taken using a 20X objec:ve on H&E stained :ssue sec:ons of healthy colon mucosa (A), adenomatous polyp (C), and adenocarcinoma (E). Corresponding TPF images (taken with a 20X objec:ve -‐ 0.9 NA) acquired from ex vivo fresh biopsies of healthy colon mucosa (B), adenomatous polyp (D), and adenocarcinoma (F). Scale bars: 40 µm. Histological images taken using a 40X objec:ve on H&E stained :ssue sec:ons of healthy colon mucosa (G), adenomatous polyp (J), and adenocarcinoma (L). Corresponding TPF images (taken with a 40X objec:ve – 1.3 NA) acquired from ex vivo fresh biopsies of healthy colon mucosa (H), adenomatous polyp (K), and adenocarcinoma (M). Scale bars: 30 µm Colon Tumor Cellular morphology (high magn) Healthy mucosa Adenomatous Polyp Adenocarcinoma Excita:on wavelength: 740 nm TPEF detec:on: 430-‐490 nm (NADH) Pixel dwell :me: 5 µs Laser power: approx. 30 mW Cell-‐to-‐nucleus ra:o Asymmetry HM: 6.7 ± 1.8 P: 3.3 ± 0.8 CT: 2.1 ± 0.3 HM: 1.3 ± 0.2 P: 2.6 ± 0.6 CT: 1.7 ± 0.3 Biomed. Opt. Express 4, 1204-1213 (2013) Functional techniques (Red-Ox) Functional imaging (Red-Ox) Oxida:ve Phosphoryla:on Glycolysis • Low efficiency • Does not require Oxygen • Only NADH involved • High efficiency • Requires Oxygen • NADH & FAD involved Neoplas:c Healthy h^p://www.biology-‐innovaAon.co.uk Functional imaging (Red-Ox) TPEF cross sec:on Fluo emission Excita:on wavelengths: Huang et al., Biophys J (2002) NADH • 740 nm (NADH) • 890 nm (FAD) FAD Detec:on wavelengths: • 460 nm (NADH) • 510 nm (FAD) 740 nm 890 nm 460 nm 510 nm Red-‐Ox Ra:o maps ROxRatio = I FAD − I NADH I FAD + I NADH …with images 890, 510 nm -‐ I_FAD (890 nm, 510 nm) – I_NADH (740 nm, 460 nm) I_NADH(740 nm, 460 nm) + I_FAD (890 nm, 510 nm) 740, 460 nm 740, 460 nm 890, 510 nm + Red-‐Ox Ra:o Index map = Bladder Tumor Red-Ox Healthy Mucosa Carcinoma in situ Excita:on wavelength: 740 nm (NADH) / 890 nm (FAD) Image dimension: 20 µm × 20 µm Resolu:on: 32 × 32 pixels Detec:on range: 460 nm (NADH) / 515 nm (FAD) Lambda-‐resolu:on: 13 nm/channel Scale bars: 5 µm Opt. Express 18, 3840 (2010) Red-Ox (Colon) Healthy mucosa 30 µm depth 60 µm depth 90 µm depth Polyp Colon tumor Excita:on wavelength: 740 nm (NADH) / 890 nm (FAD) Image dimension: 100 µm × 100 µm Resolu:on: 512 × 512 pixels Detec:on range: 460 nm (NADH) / 510 nm (FAD) Brain tumor (preliminary results) imaging (TPF-SHG) Single image FOV: 230 µm • SHG from collagen • TPF from cells Magnified Detail FOV: 100 µm Brain tumor imaging (FLIM) TPF image Magnified Detail FOV: 100 µm FOV: 230 µm • Isolated cell • FLIM imaging • Spectral imaging FLIM image MulAmodal TPE, SHG, CARS Aorta (multimodal) CARS + TPEF + SHG RED: CARS from lipid deposi:on in atherosclero:c plaques GREEN: TPEF from elas:c fibers BLUE: SHG from collagen Field of View: 3.6 mm × 3.15 mm Resolu:on: 8192 × 7168 pixels Pixel dwell :me: 6.4 µs Objec:ve: 20X -‐ NA0.5 Atherosclerotic plaques Strong deposi:on Mild deposi:on CARS Pump wavelength: 671.5 nm Stokes wavelength: 830.4 nm Ship: 2850 cm-‐1 CH-‐stretch An:stokes wavelength: 576 nm Pixel dwell :me: 6.4 µs Laser power: 25 mW Pump-‐Stokes TPEF Light deposi:on No deposi:on Excita:on wavelength: 671.5 nm TPEF detec:on: up to 550 nm Pixel dwell :me: 6.4 µs Laser power: approx. 25 mW SHG Excita:on wavelength: 830.4 nm SHG detec:on: 412-‐418 nm Pixel dwell :me: 6.4 µs Laser power: approx. 25 mW Field of View: 0.45 mm × 0.45 mm SHG for cholesterol CARS Introducing Raman maps SHG Raman maps Cholesterol SHG Imaging Cholesterol crystal CARS SHG GREEN: TPEF from epithelial cells RED: CARS from lipid deposi:on BLUE: SHG from cholesterol J Biophoton, DOI 10.1002/jbio.201300055 (2013) SHG Analysis FFT analysis on plaque collagen Collagen -‐ SHG Control region FFT -‐ Control Control Deposi:on 0.87 ± 0.05 Deposi:on region FFT -‐ Deposi:on 0.72 ± 0.05 GLCM analysis on collagen Control region Deposi:on region Correla:on length analysis Tissue type Correlation length Mean fibre size Normal arterial wall 1.79 ± 0.14 µm 1.89 ± 0.37 µm Plaques deposition 1.35 ± 0.51 µm 1.43 ± 0.37 µm • Correla:on length compared with mean fibre diameter (measured from images) • Regular oscilla:ons in the control region – Noisy in the deposi:on region Fiber bundle spacing Normalized Power spectrum • Dis:nct peak @ spa:al frequency of 0.11 µm-‐1. • Higher values in the deposi:on at higher spa:al frequency. Clinical studies In vivo nonlinear microscope SHG, TPE, SLIM Laser Source • Ti:Sapphire Relaying Op:cs • 7-‐mirror ar:culated arm Microscope Head • Custom-‐made Applied Physics B: Lasers and Optics, pp. 359-365, 2008 Custom microscope head Detec:on plate Objec:ve • Olympus XLUM 20X objec:ve lens (NA 0.9-‐WD 2 mm) Excita:on plate Detectors • Hamamatsu H7422 PMT (propor:onal mode) • Becker&Hickl PML-‐16-‐SPEC (mul:spectral SPC detector) In vivo test Epidermis TPEF Dermis -‐ SHG Basal layer Granular layer Spinous layer -‐ 80 / -‐200 µm 5 µµm 0 m m -‐ 5-‐ -‐ 5 24µ FOV = 200 µm FOV = 400 µm Laser resurfacing Abla:ve lasers Abla:ve Er:YAG frac:onal Non-‐abla:ve Abla:ve CO2 frac:onal frac:onal Raham et al. Lasers Surg. Med. 41:78-‐86 (2009) • Abla:ve resurfacing • Non-‐abla:ve resurfacing • Minimally abla:ve (frac:onal) resurfacing Thermal effect causes fibroblasts s:mula:on and the produc:on of new collagen Laser resurfacing (Group I) Age < 35 years • Any significant modifica:on observed. • Collagen fibers are quite similar before and aper the treatment. • Amorphous component (hyaluronic acid, glicosaminoglicanes) is similar before and aper the treatment. Excita:on wavelength: 900 nm Image dimension: 400 µm × 400 µm Resolu:on: 512 × 512 pixels Pixel dwell :me: 20 µs Scale bars: 40 µm Laser resurfacing (Group II) 35 years < Age < 60 years • Slight modifica:ons observed. • Collagen fibers slightly increase in density. • Amorphous component (hyaluronic acid, glicosaminoglicanes) increases as demonstrated by a more cloudy appearance. Excita:on wavelength: 900 nm Image dimension: 400 µm × 400 µm Resolu:on: 512 × 512 pixels Pixel dwell :me: 20 µs Scale bars: 40 µm Laser resurfacing (Group III) Age > 60 years • Strong modifica:ons observed. • Collagen fibers increase in density and they exhibit a reduced thickness. • Amorphous component (hyaluronic acid, glicosaminoglicanes) undergoes a dras:c improvement. Excita:on wavelength: 900 nm Image dimension: 400 µm × 400 µm Resolu:on: 512 × 512 pixels Pixel dwell :me: 20 µs Scale bars: 40 µm Spectral SAAID mapping Acquired emission spectrum from skin dermis SHG − TPEF SAAID = SHG + TPEF In terms of images SHG SHG TPEF TPEF -‐ SAAID = SHG TPEF + Spectral unmixing of SHG and TPEF SAAID analysis Excita:on wavelength: 900 nm SHG detec:on: 437 nm – 463 nm TPEF detec:on: 476 nm – 618 nm Image dimension: 64 µm × 64 µm Resolu:on: 64 × 64 pixels Pixel dwell :me: 0.2 ms Scale bars: 10 µm Group I Group II Group III Psoriasis • Inflammatory skin disease • Chronic condi:on • Hereditary with environmental triggering factors • Abnormal differen:a:on of kera:nocytes • Autoimmune • No cure Treatments • Topical treatment (mild cases)-‐ creams and ointments • Phototherapy (moderate cases) -‐UVA,UVB exposure • Systemic agents (severe cases) – immuno-‐suppressives Histological appearance Healthy Psoria2c Lowes M. A. et al, "Pathogenesis and therapy of psoriasis”, Nature, 445 (2007). • Thickening of the epidermis • Elongated rete ridges • Elongated dermal papillae • Dilated blood vessels Microscopic invesAgaAon of Epidermis Healthy skin Autofluorescence ObjecAve : N.A. 1.3, 40× ExcitaAon: 740 nm Pixel dwell Ame: 20μs FOV: 50 μm Image size: 512×512 pixels Microscopic invesAgaAon of Epidermis Psoriasis Autofluorescence ObjecAve : N.A. 1.3, 40× ExcitaAon: 740 nm Pixel dwell Ame: 20μs FOV: 50 μm Image size: 512×512 pixels -‐ 10 μm -‐20 μm -‐40 μm -‐50 μm -‐70 μm -‐80 μm • Abnormal corneum • Smaller cytoplasm • InfiltraAng papillae Stratum Spinosum Healthy Psoria)c • Sparse cellular arrangement • Large nucleus • Smaller cytoplasm Healthy skin dermis - 85 µm -100 µm -115 µm -130 µm -150 µm -170 µm Excita:on wavelength: 900 nm Image dimension: 400 µm × 400 µm Resolu:on: 512 × 512 pixels Pixel dwell :me: 20 µs Psoriasis skin dermis - 85 µm -100 µm -115 µm -130 µm -150 µm -170 µm Excita:on wavelength: 900 nm Image dimension: 400 µm × 400 µm Resolu:on: 512 × 512 pixels Pixel dwell :me: 20 µs • Elongated papillae • High density of papillae 3D appearance of Dermis Healthy Psoriasis Papillae Diameter ~ 7 μm Length ~ 30 μm Papillae Diameter ~ 15 μm Length ~ 100 μm 3D rendering • 20 images • 5μm lateral distance • ImageJ 3D Viewer: Brightest point method • Volume size : 100μm × 100μm × 100μm Clinical study: Chemical content analysis by means of an opAcal pen Experimental setup Optical fiber probes • Small dimension = 2 mm • High temperature resistance (Plasma Steriliza:on 40-‐70°C). • Bio-‐compa:ble components. • One central fiber for light delivery. Diameter: 100 mm; NA 0.22 • 24 fibers for light collec:on. Diameter: 100 mm; NA 0.22 Central light delivering fibres • High VIS transmission @ 378 nm, 445 nm (high OH-‐); • High NIR transmission @ 785 nm (low OH-‐); Probe size Raman special-filtered tip Cleaning Laser Line Filter Long Pass collecting fibers filter Samples Samples Examined samples • Op:cal research Lab inside the dermatological clinic. • Fresh biopsies examined within 10 minutes from excision. • Cross-‐check with histo-‐pathology • • • • Melanocy2c lesions 10 malignant melanoma 10 melanocy:c nevi 20 healthy skin sample (skin surrounding the lesion) 2 seborreic keratosis Acquisi2on seRngs 378 nm • power 2 mW • integra:on :me: 0.1-‐4 s • 10 spectra per sample • Average spectrum 445 nm • power 20 mW • integra:on :me: 0.1-‐4 s • 10 spectra per sample • Average spectrum Raman – 785 nm • power 150 mW • integra:on :me: 10 s • 10 spectra per sample • PCA Data processing (Raman extraction) • Acquisi:on of Raw spectrum • Polynomial fivng (fiph degree) • Subtrac:on • Extrac:on of Raman spectrum Fluorescence @ 378 nm excitation • Red ship of Melanoma with respect to Nevus and Healthy Skin • Red ship of Nevus with respect to Healthy Skin Fluorescence @ 445 nm excitation • Red ship of Melanoma with respect to Nevus and Healthy Skin • Red ship of Nevus with respect to Healthy Skin Subtracted signal (785 nm excitation) • Fiph degree polynomial fivng func:on Raman @ 785 nm excitation Ammide III Ammide I Tissue classification Scoring algorithm (global) Scoring algorithm (NIR + Raman) Scoring algorithm (Fluo 378+445 nm) • Cross-‐valida:on • Cross-‐valida:on • Leave-‐one-‐out approach • Leave-‐one-‐out approach • ScoreNIR = PCA1 (NIR) -‐ PCA1 (Raman) • ScoreVIS = PCA1 (378nm) + PCA1 (445 nm) • Cross-‐valida:on • Sensi:vity: 56% • Sensi:vity: 78% • Leave-‐one-‐out approach pecificity: 89% • Specificity: 89% • Score = ScoreVIS + • S ScoreNIR • Sensi:vity: 89% • Specificity: 100% J Biophoton DOI 10.1002/jbio.201200230 (2013) Additional lesions (work in progress) Seborreic Keratosis Fluorescence @ 378 nm Fluorescence @ 445 nm • Good discrimina:on of Seborreic Keratosis against Melanoma Colon tumor Healthy Mucosa vs Adenomatous Polyp vs Adenocarcinoma Fluorescence @ 378 nm Fluorescence @ 445 nm Subtracted BG @ 785 nm Raman @ 785 nm Brain Tumor Dysplas2c 2ssue vs Tumor (glioma) Fluorescence @ 378 nm Fluorescence @ 445 nm Subtracted BG @ 785 nm Raman @ 785 nm One simple conclusion Morpho chemistry can be powerfully analyzed by the combinaAon of methodologies using non linear interacAon (TPE, SHG, SLIM) on imaging with mulAdimensional iperspectral analysis (using a combinaAon of Raman, one photon fluorescence, one photon lifeAmes) on whole volume or choosing detailed parAculars of the image Acknowledgement Biophotonics group @ LENS People R. Cicchi, Dr. D. Kapsokalyvas, LENS, University of Florence Dr. A. Sturiale, Prof. F. Tonelli Department of Clinical Physiopathology, Surgical Unit, University of Florence Dr. A. Crisci, Prof. M. Carini Division of Urology, Department of Surgical and Medical Cri:cal Area, University of Florence Dr. F. Giordano and Dr. R. Guerrini Neurosurgery unit, pediatric hospital Meyer, Florence Dr. V. De Giorgi, Dr. P. Campolmi, Dr. N. Pimpinelli Division of Dermatology, Department of Surgical and Medical Cri:cal Area, University of Florence Dr. V. Maio, Prof. D. Massi, Dr. G. Nesi Division of Human Pathology and Oncology, Department of Surgical and Medical Cri:cal Area, University of Florence Prof. J. Popp, Prof. B. Dietzek, Dr. C. Maahaeus, Dr. T. Meyer Ins:tute for Photonic Technology (IPHT), Jena Prof. B. Brehm Internal Medicine and Cardiology, Catholic Clinic, Koblenz, Germany Dr. A. Laaermann Ins:tute of Pathology, Department of Neuropathology, Frierich-‐ Schiller University, Jena, Germany ZEISS | ON YOUR CAMPUS E’ per noi un grande piacere poterLa invitare anche quest’anno all’evento“ZEISS on Your Campus” nella sua edizione 2014, che si svolgerà al Polo Scientifico e Tecnologico di Sesto Fiorentino c/o il Laboratorio Europeo per la Spettroscopia Non-lineare (LENS) e il Dipartimento di Fisica dell’Università di Firenze. E’ disponibile l’applicazione Light Lab App. per iPhone e iPad, che consente di ottimizzare il setup dell’imaging in fluorescenza; l’applicazione è scaricabile gratuitamente da iTunes Store. L’istante che conferma il successo del Vostro operato. Questo è il momento per cui lavoriamo www.zeiss.com/micro-apps ZEISS on Your Campus (ZOYC) è un’iniziativa globale di Carl Zeiss, gratuitamente finalizzata alla formazione scientifica di ricercatori e studenti nell’ambito della microscopia . Anche l’edizione di quest’anno si focalizzerà sulle basi dell’acquisizione di immagini destinate a pubblicazioni scientifiche e sulle tecnologie emergenti nell’ambito della microscopia ottica. I temi trattati saranno: • • • Le basi della microscopia Tecniche di microscopia avanzata Scegliere la tecnologia più idonea in base al tipo di applicazione Invito con la collaborazione di Incontriamoci al “ZEISS on Your Campus 2013” Le sopra elencate sessioni sono applicabili a qualsiasi tipo di configurazione o sistema di microscopia. E’ prevista la possibilità di analizzare eventuali campioni introdotti dai partecipanti. La invitiamo a prendere visione del programma riportato sul retro del presente invito; Le sarà possibile scegliere le argomentazioni più interessanti od appropriate per la Sua attività oppure scegliere di presenziare all’intero corso per un training generale e completo. con la collaborazione di Al fine di poter garantire l’adeguata organizzazione e la massima resa del corso, per partecipare è indispensabile effettuare la propria iscrizione on line visitando il nostro sito Carl Zeiss S.p.A. con socio unico V.le delle Industrie 20 20020 Arese (MI) Email: [email protected] www.zeiss.it Martedì 20 maggio 09.30 Benvenuto Fiorentino Mercoledì 21 maggio - Prof. F. Pavone, LENS, Sesto 09.45 Benvenuto - Giovanni Borsani, Carl Zeiss S.p.A. 10.30 Coffee Break 11.00 Lighsheet Fluorescence Microscopy Paysan, Carl Zeiss Microscopy GmbH - Dr. J. 11.45 Micron-scale neuroanatomy of the whole mouse brain by confocal light-shift microscopy Dr L. Silvestri, LENS, Sesto Fiorentino 09.30 Benvenuto 09.30 Benvenuto 09.45 Le nuove tecniche di imaging intravitale dal confocale al multifotone - Dr. A. Cometta Carl 09.45 Laser Microdissection: an introduction to LCM Dr. Ulrich Sauer, Carl Zeiss Microscopy GmbH Zeiss S.p.A. 10.30 Coffee Break 10.30 Coffee Break 11.00 Laser Microdissection: sample preparation and down-stream analysis - Dr. Ulrich Sauer, Carl Zeiss 11.00 In vivo morpho-functional brain imaging by two-photon fluorescence microscopy - Dr. A. L. A. Mascaro - LENS, Sesto Fiorentino Microscopy GmbH 11.45 Discussione, Question & Answer 11.45 A close-up view on superresolution - J. Paysan, Carl Zeiss Microscopy GmbH 12.30 Whole slide imaging: a virtual home page for fluorescence microscopy - Dr. O. Clark, Carl Zeiss Microscopy GmbH 12.30 Apotome e Digital Imaging: vantaggi dell’acquisizione con luce strutturata in rapporto alle altre tecniche di microscopia Fabio Villa, Carl Zeiss S.p.A 13.15 Lunch 13.15 Lunch 14.30 Hands on in gruppi, a rotazione sulle seguenti stazioni: 14.30 Hands on in gruppi, a rotazione sulle seguenti stazioni: • ZEISS Lightsheet Z.1 • ZEISS LSM 780 with Axio Observer Z.1 • ZEISS Axio Zoom.V16 • ZEISS ApoTome.2 with Axio Observer Z.1 • ZEISS PALM MicroBeam IV • ZEISS Axio Scan.Z1 Giovedì 22 maggio 12.45 Chiusura lavori e formazione gruppi per sessione Hands on 13.00 Lunch - • ZEISS Lightsheet Z.1 • ZEISS LSM 780 with Axio Observer Z.1 • ZEISS Axio Zoom.V16 • ZEISS ApoTome.2 with Axio Observer Z.1 • ZEISS PALM MicroBeam IV • ZEISS Axio Scan.Z1 E’ PREVISTA LA POSSIBILITÀ DI ANALIZZARE EVENTUALI CAMPIONI INTRODOTTI DAI PARTECIPANTI. 14.00 Hands on in gruppi, a rotazione sulle seguenti stazioni: • ZEISS Lightsheet Z.1 • ZEISS LSM 780 with Axio Observer Z.1 • ZEISS Axio Zoom.V16 • ZEISS ApoTome.2 with Axio Observer Z.1 • ZEISS PALM MicroBeam IV • ZEISS Axio Scan.Z1
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