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

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

Institute of Experimental Medicine, Hungarian Academy of Sciences Pázmány Péter Catholic University Faculty of Information Technology and Bionics

1. 2. 3. 4. 5. 6.

About Femtonics Two photon imaging ROI scannning The 3D acousto-optical microscope 3D scanning of neurons and dendrites Future plans Gergely Katona Femtonics - Two-photon Imaging Center

About Femtonics

”Spin-off”:

Institute of Experimental Medicine of HAS (MTA KOKI)

History

Location

Laser-scanning microscopes: Two-photon microscopes and laboratories, confocal microscopes, FLIM and FRET microscopes

Market

• Femtonics Ltd. was founded in 2005, it started its operations in 2007. Its main focus is the manufacturing and selling of laser scanning microscopes primarily for the purposes of brain research and diagnostics

• The Hungarian company has its HQ in Budapest, but it has local sales centers in several countries.

• Worldwide: research facilities, pharmaceutical companies, biologists, biophysicists, brain researchers

Combined 2P and confocal FEMTO 2D

FEMTO 3D-AO

FLIM and FRET microscope Epifluorescens + UV

Unique scanning features!

R&D strategy

Mix scientific and company results

Femtonics’s developments

BME

PPKE

Pál Maák (Nature Methods) Ádám Gali (OTKA)

Prof. Tamás Roska (Infobionika, BIK) István Ulbert

Electronics develop. Self-designed and manufactured motherboard

Software develop.

Biological measur.

Precision mechanics

Microscope production

OITI (OII)

Optical develop.

Loránd Erős (human diagnostic)

Laser develop.

Imaging

Gene technology

acousto optical control card z=0

Quantum chemistry

Chemical synthesis

Optical engineering in ZEMAX

Medical research

Brain research

Medical technology develop.

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)

Electrophysiology

Lab. equipment

SZTE Software in MATLAB, C++, FPGA codes

International Collaborations

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)

Intellectual Property BEJELENTÉSI HIVATALI SZÁM NAP

Közzététel száma

FELTALÁLÓK

RÖVID LEÍRÁS

STÁTUSZ

Közzététel

Link

8462010

EP2146234

RB, KG, VESZ

európai szbadalmi bejelentés 2 magyar (504, 505) elsőbbséggel

kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési, kiterjesztési díj, 3-4-5-6. évi évdíj befizetve

2010.01.20

http://worldwide.espacenet.com/publicationDetails/biblio?DB= EPODOC&II=0&ND=3&adjacent=true&locale=en_EP&FT=D&d ate=20100120&CC=EP&NR=2146234A1&KC=A1

2011.07.27

12/998,667

US2011279893

VESZ, KG, RB

A PCT/HU2009/000096 nemzeti szakasza

érdemi vizsgálat folyamatban

2011.11.17

Laser scanning microscope (utazó detektor)

2013.11.15

14/081,035

US20140055852

VESZ, KG, RB

12/998,667 folytatása


2014.02.27

505-US

Laser scanning microscope for scanning along a 3D trajectory (Rollercoaster)

2011.01.13

12/737,426

US2011211254

VESZ, KG, RB

PCT/HU2009/000057 nemzeti szakasza


2011.01.09

http://worldwide.espacenet.com/publicationDetails/biblio?CC= US&NR=2011211254A1&KC=A1&FT=D&ND=4&date=2011090 1&DB=EPODOC&locale=en_EP

505-EP

Laser scanning microscope for scanning along a 3D trajectory (Rollercoaster)

2009.07.14

E09785758

EP2307921

VESZ, KG, RB


kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési, kiterjesztési díj, 3-4-5. évi évdíj befizetve

2011.04.13


2009.12.30

13/138,059

US2012044569

a PCT/HU2009/000112 nemzeti szakasza

megadott, köv évdíj 2017. április 15-ig

2012.02.23


2009.12.30

2,748,525

CA2748525


nemzeti szakaszként elindítva, 2-3-4. évi fenntartási díj befizetve, 2014.dec 30-ig kell kérni az érdemi vizsgálatot

2010.07.08

http://worldwide.espacenet.com/publicationDetails/biblio?CC= CA&NR=2748525A1&KC=A1&FT=D&ND=6&date=20100708& DB=EPODOC&locale=en_EP


érdemi vizsgálat folyamatban, díj megfizetve

2012.01.25

http://worldwide.espacenet.com/publicationDetails/biblio?CC= CN&NR=102334065A&KC=A&FT=D&ND=5&date=20120125& DB=EPODOC&locale=en_EP

AKTASZÁMUNK

CÍM

504-EP


2008.12.31

504-US


504-CIP

521-US

521-CA

521-CN

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)

2009.12.30

200980157413.8 CN102334065

MP, RB, KG, VESZ, VM, CSA, SZG MP, RB, KG, VESZ, VM, CSA, SZG MP, RB, KG, VESZ, VM, CSA, SZG

http://worldwide.espacenet.com/publicationDetails/biblio?DB= EPODOC&II=0&ND=3&adjacent=true&locale=en_EP&FT=D&d ate=20111117&CC=US&NR=2011279893A1&KC=A1 http://worldwide.espacenet.com/publicationDetails/biblio?CC= US&NR=2014055852A1&KC=A1&FT=D&ND=&date=20140227 &DB=&locale=en_EP

521-EP

Focusing system comprising acoustooptic deflectors for focusing an electromagnetic beam (AOD)

2009.12.30

E09804142.9

EP2399162

MP, RB, KG, VESZ, VM, CSA, SZG


kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési díj, 3-4-5. évi évdíj befizetve

2011.12.28

http://worldwide.espacenet.com/publicationDetails/biblio?DB= EPODOC&II=0&ND=3&adjacent=true&locale=en_EP&FT=D&d ate=20111228&CC=EP&NR=2399162A1&KC=A1#

527-EP

Method and measuring system for scanning multiple regions of interest (multiple free line scan)

2008.12.31

E08462011

EP2187252

RB, KG, VESZ, KA, TG

európai szabadalmi bejelentés egy magyar (520) elsőbbséggel

kutatási jelentéssel közzétett bejelentés, érdemi vizsgálat folyamatban, megjelölési díj, 3-4-5-6. évi évdíj befizetve

2010.05.19


2009.11.17

12/998,668

US2011279667

RB, KG, VESZ, KA, TG


közzétett, érdemi vizsgálat folyamatban

2011.11.17


2012.01.05

PCT/HU2012/00 0003

WO 2013/098568

KG, VM, MP, RB, SZG

magyar elsőbbségű PCT bejelentés

Nemzeti szakaszok elindítva; USA, Kína, Kanada, India, Európa, Japán

2013.07.04

http://worldwide.espacenet.com/publicationDetails/biblio?CC= WO&NR=2013098568A1&KC=A1&FT=D

2012.01.05

PCT/HU2012/00 0001

még nincs közzétéve

KG, VM, MP, RB, SZG, KA, CB, MP

közvetlen PCT bejelentés


2013.07.11


2012.01.05

PCT/HU2012/00 0002

WO 2013/098567

KG, CSF, MP, RB

magyar elsőbbségű PCT bejelentés


2013.07.03


2013.11.28

PCT/HU2013/0001 14


RB, KG, MP


kutatási jelentést várjuk

várható 2015.05.28 közzétételt követően lesz elérhető

2013.11.28

PCT/HU2013/0001 15


RB, KG, MP


kutatási jelentést várjuk

várható 2015.05.28 közzétételt követően lesz elérhető

527-US 637-PCT 656-PCT

659-PCT

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

725-PCT

Optical microscope system

726-PCT

Acousto-optic deflector comprising multiple electro-acoustic transducers

Aktaszámunk

oltalom formája

megjelölés

áruosztályok

ügyszám

státusz

Link

721-CTM

Közösségi védjegy

FEMTONICS

9,10,41,42

CTM011630555

lajstromozott

https://oami.europa.eu/eSearch/#det ails/trademarks/011630555

More than 17 patents

The National Intellectual Property Office's Innovation prize (2012)

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= 25.95 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):2086-95

Growth “Two-photon" word in the field of neuroscience /internet/

120

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Employees

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40 Guelph,

Canada

30 New York,

80

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Ausztralia 2008 2009 2010 2011 2012 2013 2014

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Income (kHUF)

15 Employees

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Research Scientists

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Turnover (1000 HUF)

350000

20 1000000

5

Singapore

Export

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Ramat Gan, Israel

South Carolina, USA

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Two photon microscopy

GCaMP6f injection

Why neuroscience? „If brain transplantation were possible, that would be the only organ transplantation procedure where it is better to be a donor.”

Neuroscience... … 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?”

Why two-photon microscopy? Anatomy

Function

Technology comparison Technology

Positives

Negatives

Electron microscopy

Superb resolution

Not compatible with living tissues

Spinning disc confocal, multipoint two-photon

High frame rates

Penetration <100 um

Electrode arrays

Fine temporal resolution

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

Why two-photon microscopy • Deep penetration ~700 µm

• Low phototoxicity Hours of measurements

• Subcellular resolution ~400 nm

• Speed up to kHz

• Functional measurements

The two-photon effect One-photon absorption – Nonlinear effect – Small cross section – Square proportional with intensity Two-photon absorption

Mode-locked lasers Focusing

15

Pulsed laser increases 2p excitation Pulesd laser

kP0

CW laser

P0 1 𝑘

T

T 𝑃0 𝑇 = 𝑘𝑃0

Same average power:

1 𝑇 𝑘

Two-photon excitation:

CW

:

pulsed:

𝑇 = 10 𝑛𝑠

~ 𝑃0 2 𝑇 ~ (𝑘𝑃0 )2

1 𝑇 𝑘

+ 0 = 𝑘 𝑃0 2 𝑇

1 𝑇 = 100 𝑓𝑠 𝑘

→

𝑘 = 105

Focusing increases 2p excitation

distance

A ~ d2

Focal plane

Flux ~ d-2

2p Excitation ~ d-4

The two-photon effect One and two-photon excitation

The two-photon microscope Scanning mirrors

Mode-locked Ti:S laser

Infrared laser excitation MHz repetition rate

Detector

dichroic mirror

PMT

femtosecond impulse width

Visible fluorescent light Sample excitation localized to the focal point

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

One in vitro measurement 20 µm

0 -20

Vm (mV)

Rat hippocampal brain slice DIC image Patch-clamp Oregon Green BAPTA 1 Ca2+ concentration sensitive fluorescent dye Two-photon excitation measurements

40% dF/F

-40 -60

500 ms 21

-130

•

Scanline composed of any number of lines and curves

•

Arbitrary positions in the focal plane

•

1-30ms scan times, depending on the complexity of the scanline (≈ 3kHz bandwidth of the galvos)

500 ms

Line-scan

•

„Simultaneous” fluorescent measurement

spine1

•

Subsequent scanning over different curvatures

y(um)

-135

spine1

2 µm

-140 sp d sp

-145

sp d dendrite

5• mSynchronized digital outputs

1,0 0,8

(shutters, triggers, electric stimulation, drug application, etc...)

dF/F

0,6 0,4 0,2

0 dendrite

5

0,0 -0,2

10 x(um)

10 spine 1

0

500

1000

15 1500

time [ms]

2000

2500

20

Resolution of single synaptic inputs to a neuron No spine activated cell

Opt.

A

100 μm

One spine activated

More spine activated

-244

-246

-248

Action potential activated

y(um)

-250

-252

3 μm

-254

-256 0

0

Lorincz A, Rozsa B,-258 Katona G, Vizi ES, Tamas G (2007) Differential distribution of NCX1 contributes to spine-dendrite compartmentalization in CA1 pyramidal cells. Proc Natl Acad Sci U S A 104:1033-1038. A. Lorincz and B. Rozsa contributed equally to this work. -260 200

400

600

control

200

400

600

benzamil

ROIs are sparsely located

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

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!!!

There are many-many-many neurons to record in 3D

We need a special scanning solution: Acousto-optical scanning

Acousto-optical deflectors and lenses Acousto-optical deflection AcoustoOptic Medium

Diffracted light piezoelectric driver

RF sound enter the AO cell

Incident light

d· sin (αn) = 2n λ/2

Acousto-optic medium

Piezoelectric driver

Δf

Δ

Acousto-optical focusing

RF sound enters the AO cell

F

AcoustoOptic Medium

piezoelectric driver

 K f RF sound enter the AO cell

x Piezoelectric driver

3D AO microscope 2D-AO scanning and drift compensation

y

x

Beam expander

AO lens

AO lens

Tc3 Angular dispersion compensation

AO z-focusing q Beam stabilization

Tc4 PMT

m m

q

PMT

Dispersion compensation

in vivo Faraday isolator

Or in vitro

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

Custom developed electronics system

Digital IO module

Analog IO & PMT module

AO drive module (v5)

Motherboard

Driving functions and organization of the AO measurement Idő

AO cycle

X1 AOD frequency y1 AOD frequency x2 AOD frequency y2 AOD frequency Dead time Paramater upload

Focusing OK

I/O update to all AO cards

PMT data download Data omitted

Data stored

Driving functions

where

and

Control software

3D virtual reality Position sensors Shutter glasses

3D mouse „bird”

Virtual cursor

LCD monitor

b

Product developed…. Patents: WO2013102771, WO2013098568, WO2013098567, US2012044569, US2011279667, US2011279893, WO2010055362, US2011211254, EP2146234 Parameters • Large scanning volumes ( up to 1100 x 1100 x 3000 µm3 ) • Preserved resolution ( ~400 nm in the center ) • Max. scanning speed: ~ 53 kHz/point

Femto3D-AO Acousto-optic 3D-scanning two-photon microscope

Examples • 2000 regions (cells, dendrites ) @ 25 Hz – population imaging • 600 regions (cells, dendrites) @ 90 Hz • 5-10 regions (cells, dendrites) @ 6.6-3.3 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

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

Different scanning methods 1. Random access point scanning

1.

2. Multiple 3D trajectory scanning 3. Multiple 3D frame scanning 4. Multiple 3D folded frame scanning 5. Multiple 3D line scanning

2.

3. 5. 1 ROI

1s

4.

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

20

40

60 Cell #

80

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 #100

2000

6000 t[ms]

Random-access point scanning

Multiple 3D trajectory scanning 2 ms

30 µm

Point-by-point 2 ms

10 µm

Continuous trajectory scanning

4a

5a 7a 3a

2a 1a

measurement of dendritic spikes in PV neurons arrows indicate distance along dendrites

1a

2a

3a

4a

5a

7a

50 µm time, 200 ms

0.35 ΔF/F

6a

7b 6b 5b 4b 3b 2b 1b

SPW-EPSP

9b 8b

-0.1

SPW-EPSP = Sharp wave associated subthreshold EPSP Chiovini et. al (Neuron) 2014

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)

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

Multiple 3D frame scanning

Visual stimulus evoked network activity in vivo

Multiple 3D folded frame scanning

2

1

3

Raw data from a virtual 2D plane 20µm

Simultaneously measured dendritic regions - Data showed in dF/F

Multiple 3D folded frame scanning

1

4 5

3 6 2

7

13

8 11 12

9 10 50µm

Motion artefact correction Multiple 3D line scanning Heartbeat frequency movement

Responses from individual spines

1 ROI

time

1s

20 nm

2s 50% ΔF/F

2s

spine #2

spine #2

50% ΔF/F

spine #6 spine #9

spine #6

Szalay et l. (manusript in prep.)

spine #11

without motion correcting spine scans

spine #9 spine #11

with motion correction

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)

3x3D AO scanning methods New microscope able to measure multiple brain areas simultaneously Scan head #1 for imaging Scan head #2 for photo stimulation

2D-AO scanning and drift compensation

2D-AO scanning and drift compensation

y

x

Beam expander Beam expander

AO z-focusing

Tc3 Angular dispersion compensation

Beam stabilization

AO z-focusing q Beam stabilization

Tc4 Dispersion compensation

m

3D scan head #3

Detector PMT unit Ti:S laser Mai Tai DeepSee

AO lens AO lens

Faraday isolator

PMT

Input port #1

m Input port #2

Input port #3

Optical multiplexer Output port #1

Output port #2 & detectors

V1

LGN

Dispersion compensation

Output port #3 & detectors

Optical adapters

Laser amplifier

q

Faraday isolator

Laser amplifier Ti:S laser Mai Tai DeepSee

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

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!

Why neuroscience? „If brain transplantation were possible, that would be the only organ transplantation procedure where it is better to be a donor.”

Neuroscience... … 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?”

Thank You for your attention…

We look for new colleagues and students for both research and the company!

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

BME, Department of Atomic Physics Maák Pál

All employees of

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-604102-HBP, Magyary Zoltán (13-0314), TÁMOP-4.2.1. B-11/2/KMR-2011-0002, KTIA_NAP_12-2-2015-0006

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)

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

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