Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes indicators and two-photon microscopy

Átírás

1 Institute of Experimental Medicine, Hungarian Academy of Sciences Pázmány Péter Catholic University Faculty of Information Technology and Bionics Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes indicators and two-photon microscopy Gergely Katona Femtonics - Two-photon Imaging Center BSS 2015

2 Institute of Experimental Medicine, Hungarian Academy of Sciences Pázmány Péter Catholic University Faculty of Information Technology and Bionics 1. About Femtonics 2. Two photon imaging 3. ROI scannning 4. The 3D acousto-optical microscope 5. 3D scanning of neurons and dendrites 6. Future plans Gergely Katona Femtonics - Two-photon Imaging Center

3 About Femtonics Spin-off : Institute of Experimental Medicine of HAS (MTA KOKI) History Femtonics Ltd. was founded in 2005, it started its operations in Its main focus is the manufacturing and selling of laser scanning microscopes primarily for the purposes of brain research and diagnostics Location The Hungarian company has its HQ in Budapest, but it has local sales centers in several countries. Laser-scanning microscopes: Two-photon microscopes and laboratories, confocal microscopes, FLIM and FRET microscopes Market Worldwide: research facilities, pharmaceutical companies, biologists, biophysicists, brain researchers

4 Combined 2P and confocal FEMTO 2D FEMTO 3D-AO FLIM and FRET microscope Epifluorescens + UV Unique scanning features!

5 R&D strategy Mix scientific and company results

6 Femtonics s developments BME Pál Maák (Nature Methods) Ádám Gali (OTKA) PPKE Prof. Tamás Roska (Infobionika, BIK) István Ulbert Electronics develop. Optical develop. OITI (OII) Loránd Erős (human diagnostic) Self-designed and manufactured motherboard acousto optical control card z = 0 Quantum chemistry Biological measur. Software develop. Chemical synthesis Microscope production Precision mechanics Brain research Imaging Laser develop. Electrophysiology Gene technology International Collaborations Botond Roska, Daniel Hillier (Basel) Christophe Bernard (Marseilles) James Poulet (Berlin) Ivo Vanzeta (Marseilles) Valentin Nagerl (Bordeaux) Veronica Egger (Munich) David Fitzpartick (Max Planck Florida) Ryohei Yasuda (USA) Optical engineering in ZEMAX Software in MATLAB, C++, FPGA codes Medical research Medical technology develop. Lab. equipment SZTE Károly Osvay (ELI, FP7 grant, laser development) Gábor Tamás (Cortical Mic.of HAS, PNAS, 3D imaging) MTA KOKI (IEM HAS), Tamás Freund Two-Photon Imaging Center Zoltán Nusser, (Nature Neuroscience 2012) Szabolcs Káli, Tamás Freund, (in progress) Sylvester E. Vizi (Nature Methods, PNAS) Norbert Hájos, (PNAS, 2011) Attila Gulyás (in progress) Ádám Dénes, Emília Madarász (OTKA)

7 Intellectual Property AKTASZÁMUNK 504-EP 504-US 504-CIP 505-US 505-EP 521-US 521-CA 521-CN 521-EP 527-EP 527-US 637-PCT 656-PCT 659-PCT CÍM Laser scanning microscope (utazó detektor) Laser scanning microscope (utazó detektor) Laser scanning microscope for scanning along a 3D trajectory (Rollercoaster) Focusing system comprising acoustooptic deflectors for focusing an electromagnetic beam (AOD) Focusing system comprising acoustooptic deflectors for focusing an electromagnetic beam (AOD) Focusing system comprising acoustooptic deflectors for focusing an electromagnetic beam (AOD) Method and measuring system for scanning multiple regions of interest (multiple free line scan) Compensator system and method for compensating angular dispersion Methon for scanning along a continuous scanning trajectory with a scanner system Methos for measuring a 3-dimensional sample via measuring device comprising a laser scanning microscope and such measuring device BEJELENTÉSI HIVATALI SZÁM NAP Közzététel száma /998,667 US VESZ, KG, RB FELTALÁLÓK RÖVID LEÍRÁS STÁTUSZ Közzététel Link A PCT/HU2009/ nemzeti szakasza érdemi vizsgálat folyamatban /081,035 US VESZ, KG, RB 12/998,667 folytatása érdemi vizsgálat folyamatban /737,426 US VESZ, KG, RB /138,059 US ,748,525 CA CN /998,668 US PCT Optical microscope system PCT Laser scanning microscope (utazó detektor) Laser scanning microscope for scanning along a 3D trajectory (Rollercoaster) Focusing system comprising acoustooptic deflectors for focusing an electromagnetic beam (AOD) Method and measuring system for scanning multiple regions of interest (multiple free line scan) Acousto-optic deflector comprising multiple electro-acoustic transducers E E E PCT/HU2012/ PCT/HU2012/ PCT/HU2012/ PCT/HU2013/ PCT/HU2013/ EP EP EP EP WO 2013/ még nincs közzétéve WO 2013/ még nincs közzétéve még nincs közzétéve RB, KG, VESZ VESZ, KG, RB MP, RB, KG, VESZ, VM, CSA, SZG MP, RB, KG, VESZ, VM, CSA, SZG MP, RB, KG, VESZ, VM, CSA, SZG MP, RB, KG, VESZ, VM, CSA, SZG RB, KG, VESZ, KA, TG RB, KG, VESZ, KA, TG KG, VM, MP, RB, SZG KG, VM, MP, RB, SZG, KA, CB, MP KG, CSF, MP, RB európai szbadalmi bejelentés 2 magyar (504, 505) elsőbbséggel PCT/HU2009/ nemzeti szakasza PCT/HU2009/ nemzeti szakasza a PCT/HU2009/ nemzeti szakasza a PCT/HU2009/ nemzeti szakasza a PCT/HU2009/ nemzeti szakasza a PCT/HU2009/ nemzeti szakasza európai szabadalmi bejelentés egy magyar (520) elsőbbséggel PCT/HU2009/ nemzeti szakasza magyar elsőbbségű PCT bejelentés közvetlen PCT bejelentés magyar elsőbbségű PCT bejelentés kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési, kiterjesztési díj, évi évdíj befizetve érdemi vizsgálat folyamatban kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési, kiterjesztési díj, évi évdíj befizetve megadott, köv évdíj április 15-ig nemzeti szakaszként elindítva, évi fenntartási díj befizetve, 2014.dec 30-ig kell kérni az érdemi vizsgálatot érdemi vizsgálat folyamatban, díj megfizetve kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési díj, évi évdíj befizetve kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési díj, évi évdíj befizetve közzétett, érdemi vizsgálat folyamatban Nemzeti szakaszok elindítva; USA, Kína, Kanada, India, Európa, Japán Nemzeti szakaszok elindítva; USA, Kína, Kanada, India, Európa, Japán Nemzeti szakaszok elindítva; USA, Kína, Kanada, India, Európa, Japán EPODOC&II=0&ND=3&adjacent=true&locale=en_EP&FT=D&d ate= &cc=ep&nr= a1&kc=a1 EPODOC&II=0&ND=3&adjacent=true&locale=en_EP&FT=D&d ate= &cc=us&nr= a1&kc=a1 US&NR= A1&KC=A1&FT=D&ND=&date= &DB=&locale=en_EP US&NR= A1&KC=A1&FT=D&ND=4&date= &DB=EPODOC&locale=en_EP EPODOC&II=0&ND=3&adjacent=true&locale=en_EP&FT=D&d ate= &cc=ep&nr= a2&kc=a2 US&NR= A1&KC=A1&FT=D&ND=4&date= &DB=EPODOC&locale=en_EP CA&NR= A1&KC=A1&FT=D&ND=6&date= & DB=EPODOC&locale=en_EP CN&NR= A&KC=A&FT=D&ND=5&date= & DB=EPODOC&locale=en_EP EPODOC&II=0&ND=3&adjacent=true&locale=en_EP&FT=D&d ate= &cc=ep&nr= a1&kc=a1# EPODOC&II=1&ND=3&adjacent=true&locale=en_EP&FT=D&d ate= &cc=ep&nr= a1&kc=a1 US&NR= A1&KC=A1&FT=D&ND=4&date= &DB=EPODOC&locale=en_EP WO&NR= A1&KC=A1&FT=D WO&NR= A1&KC=A1&FT=D WO&NR= A1&KC=A1&FT=D RB, KG, MP közvetlen PCT bejelentés kutatási jelentést várjuk várható közzétételt követően lesz elérhető RB, KG, MP közvetlen PCT bejelentés kutatási jelentést várjuk várható közzétételt követően lesz elérhető Aktaszámunk oltalom formája megjelölés áruosztályok ügyszám státusz Link 721-CTM Közösségi védjegy FEMTONICS 9,10,41,42 CTM lajstromozott More than 17 patents The National Intellectual Property Office's Innovation prize (2012) ails/trademarks/

8 Publications Wertz A, Trenholm S, Yonehara K, Hillier D, Raics Z, Leinweber M, Szalay G, Ghanem A, Keller G, Rózsa B, Conzelmann KK, Roska B (2015) Single-cell-initiated monosynaptic tracing reveals layer-specific cortical network modules Science Katona G, Szalay G, Maak P, Kaszas A, Veress M, Hillier D, Chiovini B, Vizi ES, Roska B, Rozsa B (2012) Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nature Methods. IF= Wolfgang G. Bywalez, Balázs Rózsa, et al. & Veronica Egger (2015) Electrical compartmentalization in olfactory bulb granule cell spines/local postsynaptic sodium channel activation in olfactory bulb granule cell spines Neuron (in press) IF=16.48 Jan Tønnesen, Gergely Katona, Balázs Rózsa, & Valentin Nägerl (2014) Nanoscale spine neck plasticity regulates compartmentalization of synapses Nature Neuroscience IF=15.25 Balázs Chiovini, Gergely F. Turi, Gergely Katona, Attila Kaszás, Dénes Pálfi, Pál Maák, Gergely Szalay, Mátyás Forián Szabó, Gábor Szabó, Zoltán Szadai, Szabolcs Káli, Balázs Rózsa (2014) Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves Neuron IF=16.48 Noemi Holderith, Andrea Lorincz, Gergely Katona, Balázs Rózsa, Akos Kulik, Masahiko Watanabe, Zoltan Nusser (2012) Release probability of hippocampal glutamatergic terminals scales with the size of the active zone Nature Neuroscience IF=15.25 G. Katona, A. Kaszás, GF. Turi, N. Hájos, G. Tamás, E. S. Vizi, B. Rózsa (2011) Roller Coaster Scanning Reveals Spontaneous Triggering of Dendritic Spikes in CA1 Interneurons PNAS IF=9.7 Lőrincz* A, Rózsa B, Katona G, Vizi ES, Tamás G (2007) Differential distribution of NCX1 contributes to spine-dendrite compartmentalization in CA1 pyramidal cells PNAS IF=9.7 Chiovini B, Turi GF, Katona G, Kaszás A, Erdélyi F, Szabó G, Monyer H, Csákányi A, Vizi ES, Rózsa B. (2010) Enhanced Dendritic Action Potential Backpropagation in Parvalbumin-positive Basket Cells During Sharp Wave Activity. Neurochem Res. 35(12):

9 Number of employees Turnover (1000 HUF) Growth Two-photon" word in the field of neuroscience /internet/ Canada 50 Guelph, 40 New York, 30 South Carolina, USA EU E x p o r t Employees Budapest, Hungary Ramat Gan, Israel Singapore Ausztralia Income (khuf) % YEAR Employees Research Scientists % 116 % YEAR

10 Two photon microscopy GCaMP6f injection

11 Why neuroscience? Neuroscience... If brain transplantation were possible, that would be the only organ transplantation procedure where it is better to be a donor. is very close Where is the soul? is of social impact Brain diseases represent third of all health expenses. is business Prosthetics, brain machine interface is the science of the future the IT revolution at the end of the 21th century is the challenge of ultimate complexity Is it possibble to understand our functioning?

12 Why two-photon microscopy? Anatomy Function

13 Technology comparison Technology Positives Negatives Electron microscopy Superb resolution Not compatible with living tissues Spinning disc confocal, multipoint two-photon Electrode arrays High frame rates Fine temporal resolution Penetration <100 um Invasive, no anatomy and localization, only suprathreshold signals Patch-clamp Fine temporal resolution, detailed signals Invasive, no anatomy and localization, cumbersome in vivo fmri Noninvasive, whole body No cellular resolution, speed on the order of seconds

14 Why two-photon microscopy Deep penetration ~700 µm Low phototoxicity Hours of measurements Subcellular resolution ~400 nm Speed up to khz Functional measurements

15 The two-photon effect One-photon absorption Nonlinear effect Small cross section Square proportional with intensity Two-photon absorption Mode-locked lasers Focusing 15

16 Pulsed laser increases 2p excitation kp 0 Pulesd laser P 0 CW laser 1 k T Same average power: P 0 T = kp 0 1 k T T Two-photon excitation: CW : ~ P 0 2 T pulsed: ~ (kp 0 ) 2 1 k T + 0 = k P 0 2 T T = 10 ns 1 T = 100 fs k = 105 k

17 distance Focusing increases 2p excitation A ~ d 2 Flux ~ d -2 2p Excitation ~ d -4 Focal plane

18 The two-photon effect One and two-photon excitation

19 The two-photon microscope Mode-locked Ti:S laser MHz repetition rate dichroic mirror Scanning mirrors Infrared laser excitation PMT Detector femtosecond impulse width Sample Visible fluorescent light excitation localized to the focal point

20 Comparison to confocal microscopy Penetration Less tissue scattering for infrared light Focal excitation Excitation happens only in the focal point causing clearer background and less pohototoxicity Detection efficiency Even scattered emitted photons reach the detectors with high probability allowing the use of low excitation intensities. Tunability Mode-locked lasers are often tunable allowing the tuning of excitation wavelength to the dye in use Price Wider spectrums Broader spectrums and the excitation wavelengths limit the number of dyes usable at the same time

21 Vm (mv) One in vitro measurement 20 µm Rat hippocampal brain slice DIC image Patch-clamp Oregon Green BAPTA 1 Ca 2+ concentration sensitive fluorescent dye Two-photon excitation measurements % df/f 500 ms 21

22 df/f 500 ms y(um) y(um) Line-scan Scanline composed of any number of lines and curves -140 spine 1 2 µm Arbitrary positions in the focal plane 1-30ms scan times, depending on the complexity of the scanline ( 3kHz bandwidth of the galvos) sp d sp sp d Simultaneous fluorescent measurement -145 dendrite 1,0 0,8 0,6 0,4 0,2 0,0-0,2 dendrite 0 5 spine time [ms] x(um) spine 1 Subsequent scanning over different curvatures 5 m Synchronized digital outputs (shutters, triggers, electric stimulation, drug application, etc...)

23 y(um) Resolution of single synaptic inputs to a neuron No spine activated cell Opt. A 100 μm One spine activated More spine activated Action potential activated μm control 0 benzamil in CA1 pyramidal cells. 400 Proc Natl Acad Sci U S A 104: A. Lorincz and B. Rozsa contributed equally to this work. Lorincz A, Rozsa B, -258 Katona 200 G, Vizi ES, Tamas G (2007) Differential distribution of NCX1 contributes to spine-dendrite compartmentalization

24 ROIs are sparsely located

25 Signal-to-noise ratio Low number of photons Poisson distribution: SNR = sqrt(n) Not possible to increase excitation (phototoxicity) Number of collected photons ~ Time spent on ROI Scan only the ROIs! ROI-scanning Line scan Small, multiple frame scan Point scan

26 Z-scannig is a challenge XY scanning with galvanometric mirrors Z scanning with objective positioning ~100 ms Piezoelectric objective positioner ~5 ms Liquid lenses ~ 5 ms Focal plane Denk W et al. Photon upmanship: Why multiphoton imaging is more than a gimmick, Neuron, 1997 But neurons are fast, in 3D!!!

27 There are many-many-many neurons to record in 3D We need a special scanning solution: Acousto-optical scanning

28 Acousto-optical deflectors and lenses Acousto-optical deflection Acousto- Optic Medium Diffracted light piezoelectric driver RF sound enter the AO cell d sin (α n ) = 2n λ/2 Piezoelectric driver Incident light Δ Δf Acousto-optic medium Acousto-optical focusing Acousto- Optic Medium F RF sound enters the AO cell piezoelectric driver K f RF sound enter the AO cell x Piezoelectric driver

29 3D AO microscope x 2D-AO scanning and drift compensation y Beam expander AO lens AO lens Tc 3 Angular dispersion compensation AO z-focusing q Tc 4 PMT PMT m m Beam stabilization q Dispersion compensation in vivo Or in vitro Faraday isolator Ti:S laser Mai Tai DeepSee Katona G., Szalay G., Maák P., Veress M., Kaszás A., Hillier D., Chiovini B., Vizi ES., Roska B., Rózsa B. (2012) Nature Methods

30 Custom developed electronics system Digital IO module Analog IO & PMT module AO drive module (v5) Motherboard

31 Driving functions and organization of the AO measurement Idő AO cycle X1 AOD frequency y1 AOD frequency x2 AOD frequency y2 AOD frequency Paramater upload Dead time I/O update to all AO cards Focusing OK PMT data download Data omitted Data stored

32 Driving functions where and

33 Control software

34 3D virtual reality Shutter glasses Position sensors b 3D mouse bird Virtual cursor LCD monitor

35 Product developed. Patents: WO , WO , WO , US , US , US , WO , US , EP Parameters Large scanning volumes ( up to 1100 x 1100 x 3000 µm 3 ) Preserved resolution ( ~400 nm in the center ) Max. scanning speed: ~ 53 khz/point Examples 2000 regions (cells, dendrites 25 Hz population imaging 600 regions (cells, 90 Hz 5-10 regions (cells, khz propagation speed measurements - Temporal superresolution microscopy Scanning modes Random access point scanning 3D trajectory scanning 3D stripe scanning Tilted frame scanning All conventional scanning modes Femto3D-AO Acousto-optic 3D-scanning two-photon microscope

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38 How to use the microscope? Multiple scanning approaches High speed scanning Movement artefact corrections

39 Different scanning methods Random access point scanning 2. Multiple 3D trajectory scanning 3. Multiple 3D frame scanning 4. Multiple 3D folded frame scanning 5. Multiple 3D line scanning s 1 ROI 4.

40 Random-access point scanning Use ROI scannig approach: 3D random acces point scanning for in vivo measurement! GCaMP6 responses measured with resonant scanning 3D Ca2+ response 1s Each column corresponds to a neuron 30Hz per a single z-plane 300Hz in the 3D volume 0 20µm Cell # 80

41 GCaMP6 measurements 500 cells selected for the fast, random-access measurement Ca2+ activity in df/f basis Cell #1 100% ΔF/F Advantages: Long term learning task can be investigated No artefact from the loading and clearing characteristic of OGB-AM Higher absolute fluorescene, deeper regions can be measured Simultaneous spine and neuronal network measurement Genetically targetable to specific cell types. Slow Z-stack for volume information 0% 50% df/f 1s Cell # t[ms]

42 Random-access point scanning

43 SPW-EPSP ΔF/F Multiple 3D trajectory scanning 2 ms 30 µm Point-by-point 2 ms Continuous trajectory scanning 10 µm 5a 6a 4a 7a 3a 2a 1a 9b 8b 7b 6b 5b 4b 3b 2b 1b measurement of dendritic spikes in PV neurons arrows indicate distance along dendrites 1a 2a 3a time, 200 ms 4a 5a 7a 50 µm SPW-EPSP = Sharp wave associated subthreshold EPSP Chiovini et. al (Neuron) 2014

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45 Multiple 3D frame scanning 3B. Multiple 3D frame scanning Simultaneous 3D imaging of apical and basal dendritic regions region #1 (apical dendrites) region #2 (basal dendrites)

46 Multiple 3D frame scanning Ca2+ transients from the cells marked on the lower video with corresponding colors 20% df/f 2000ms 289 cells following motion artefacts elimination in ΔF/F Imaging of 100 neurons raw data

47 Multiple 3D frame scanning

48 Visual stimulus evoked network activity in vivo

49 Multiple 3D folded frame scanning Raw data from a virtual 2D plane 20µm Simultaneously measured dendritic regions - Data showed in df/f

50 Multiple 3D folded frame scanning µm

51 Motion artefact correction time Multiple 3D line scanning Heartbeat frequency movement Responses from individual spines 1 s 1 ROI 20 nm 2s 50% ΔF/F spine #2 2s 50% ΔF/F spine #2 spine #6 spine #6 spine #9 Szalay et l. (manusript in prep.) spine #9 spine #11 spine #11 without motion correcting spine scans with motion correction

52 Future plans In vivo cellular activity measurements on animals performing learning tasks Algorithm to analyze network activity Movement artefact corrections (offline and online) Deeper, faster scanning technologies (adaptive optics, novel lasers, calcium dyes)

53 3x3D AO scanning methods New microscope able to measure multiple brain areas simultaneously Scan head #1 for imaging 2D-AO scanning and drift compensation Scan head #2 for photo stimulation Beam expander 2D-AO scanning and drift compensation x y Beam expander AO lens AO lens AO z-focusing Beam stabilization Tc 3 Angular dispersion compensation AO z-focusing q Tc 4 Beam stabilization Dispersion compensation 3D scan head #3 m m q Ti:S laser Mai Tai DeepSee Faraday isolator Detector unit PMT PMT Input port #1 Input port #2 Input port #3 Optical multiplexer Output port #1 Output port #2 & detectors Output port #3 & detectors Dispersion compensation Laser amplifier V1 LGN Optical adapters Faraday isolator Laser amplifier Ti:S laser Mai Tai DeepSee

54 copyright 2009 Femtonics Kft. CONFIDENTIAL Unauthorized use or disclosure of this information to any third party is strictly prohibited by

55 Summary Femtonics is a close collaboration with research institutes Introduced two-photon microscopy and the need for ROI scannig approaches I ve showed a 3D acousto-optic microscope able to record neuronal activity in vivo I showed novel two-photon scanning methods 3D random acces trajectory scennig 3D multiple frame scannig 3D folded-frame scannig I showed how to use it for motion compensation in vivo Neuroscience is fun!

56 Why neuroscience? Neuroscience... If brain transplantation were possible, that would be the only organ transplantation procedure where it is better to be a donor. is very close Where is the soul? is of social impact Brain diseases represent one third of the health expenses. is business Prosthetics, brain machine interface is the science of the future the IT revolution at the end of the 21th century is the challenge of ultimate complexity Is it possibble to understand our functioning?

57 Thank You for your attention We look for new colleagues and students for both research and the company!

58 Thank you. IEM HAS Two-photon Imaging Center Rózsa Balázs Szalay Gergely Bojdán Alexandra Chiovini Balázs Katona Gergely Pálfi Dénes Tompa Tamás Sulcz-Judák Linda Szadai Zoltan Juhász Gábor All employees of BME, Department of Atomic Physics Maák Pál Collaborating partners: Botond Roska (FMI, Basel) David Fitzpatrick (Max Plank, USA) Daniel Hillier (FMI, Basel) Valentin Nägerl (Bordeux) Ádám Kepecs (CSH) Pál Maák & Máté Veress (BME) Szabolcs Káli (IEM HAS) Christophe Bernard (Marseille), James Poulet (Berlin), Veronica Egger (Münich) Zoltán Nusser (IEMHAS, Budapest), Gábor Szabó Ferenc Erdélyi (IEM HAS) Ádám Dénes (IEM HAS) Ádám Gali (SZFKI, Budapest) Ibolya Molnár (ELTE, Budapest) Károly Osvay (SZTE, Szeged) Ivo Vanzeta (Marseille), István Ulbert (PPKE, Budapest) Scientific and R&D grants Hungarian-French (TÉT_0389), GOP-1.1.1, Swiss-Hungarian SH/7/2/8, KMR_0214, OTKA (K83251, K105997), FP7-ICT HBP, Magyary Zoltán ( ), TÁMOP B-11/2/KMR , KTIA_NAP_