A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy

Kirkby, Paul A.; Nadella, K. M. Naga Srinivas; Silver, R. Angus

doi:10.1364/oe.18.013720

Cited by 114 publications

(123 citation statements)

References 28 publications

Supporting

Mentioning

117

Contrasting

Unclassified

Order By: Relevance

“…Számos technológia létezik már, amely képes gyors populációs és dendritikus aktivitás 3D-s rögzítésére [6,7,9,10,11,12,13,14,15,16,17,18], ám csak az akusztooptikai (AO) megoldásokon alapuló kétfoton-pásztázás volt képes a szükséges technikai paramétereket biztosítani, úgymint 20-nál több ROI pásztázása 30 μm-nél nagyobb z kiterjedésből, 100 Hz-nél nagyobb ismét-lési sebességgel [5,19,20,21]. A következőkben célunk az első olyan kétfoton-mikroszkóp megalkotása volt, amellyel lehetséges ROI-k véletlen elérésű pásztázása há-rom dimenzióban, leküzdve a korábbi megoldások korlátait.…”

Section: Akusztooptikai Pásztázásunclassified

Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintű idegsejtvizsgálatokhoz

Szalay¹,

Judák²,

Szadai³

et al. 2015

Orvosi Hetilap

View full text Add to dashboard Cite

Bevezetés: A kétfoton-mikroszkópia ideális eszköz annak tanulmányozására, hogy a különböző jeleket hogyan dolgozza fel az élő agyszövet. Kezdetben a pásztázó képalkotással teljes képeket készítettek, ami nem alkalmas gyors 3D-s jelek, mint például akciós potenciálok nyomon követésére. Célkitűzés: A szerzők célja különféle neuronalis aktivitás mintázatok mérése volt optikai úton. Módszer: A szerzők új lézerpásztázó módszereket dolgoztak ki és kifejlesztették az ezeket megvalósító mikroszkóphardvert. Eredmények: Többszörös vonal menti pásztázás lehetővé teszi, hogy a kísérletező több, számára érdekes mintarészletet mérjen, ennek eredményeképpen nemcsak a mintavételi sebesség nő, hanem a fl uoreszcens tranziensek jel-zaj viszonya is. Ezen elvet továbbvíve kifejlesztettek egy akusztooptikai eltérítő-kön alapuló 3D lézerpásztázó kétfoton-mikroszkópot, amely milliméteres mélységelérési tartománnyal és milliszekundum alatti időfelbontással rendelkezik. Használhatóságának bizonyítására visszaterjedő akciós potenciálok terjedését vizsgálták több száz mikrométer hosszú nyúlványokban, továbbá egérlátókéregben idegsejtek százaiban mérték szimultán a sejtek Ca 2+ -tranzienseit 3D véletlen címzésű üzemmódban, in vivo. Következtetések: A pásztázás leszűkí-tése az érdekes területekre nagy jel-zaj viszonyt és gyors ismétlési sebességet tesz lehetővé, miközben a kétfoton-mikroszkópok nagy behatolási mélysége is megtartott. Orv. Hetil., 2015, 156(52), 2120-2126. Kulcsszavak: mikroszkóp, háromdimenziós képalkotás, idegsejt-képalkotásFast three-dimensional two-photon scanning methods for studying neuronal physiology on cellular and network level Introduction: Two-photon microscopy is the ideal tool to study how signals are processed in the functional brain tissue. However, early raster scanning strategies were inadequate to record fast 3D events like action potentials. Aim: The aim of the authors was to record various neuronal activity patterns with high signal-to-noise ratio in an optical manner. Method: Authors developed new data acquisition methods and microscope hardware. Results: Multiple Line Scanning enables the experimenter to select multiple regions of interests, doing this not just increases repetition speed, but also the signal-to-noise ratio of the fl uorescence transients. On the same principle, an acousto-optical defl ector based 3D scanning microscope has been developed with a sub-millisecond temporal resolution and a millimeter z-scanning range. Its usability is demonstrated by obtaining 3D optical recordings of action potential backpropagation in several hundred micrometers long neuronal processes of single neurons and by 3D random-access

show abstract

Section: Akusztooptikai Pásztázásunclassified

Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintű idegsejtvizsgálatokhoz

Szalay¹,

Judák²,

Szadai³

et al. 2015

Orvosi Hetilap

View full text Add to dashboard Cite

show abstract

“…The drawback of AOD scanning, however, is that a certain amount of spatial and temporal dispersion is introduced by the AODs themselves, and while these effects can be compensated for, this adds to the complexity of system design (15,16). Kirkby et al have refined this approach considerably and have achieved a scan range of 137 μm in the z direction (17). Nonetheless, aberrations remain and we therefore suggest a useful compromise would be to combine these techniques in a hybrid arrangement that performs x-y positioning with AODs and z scanning using the ASU approach described in this paper.…”

Section: Discussionmentioning

confidence: 99%

Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates

Botcherby

Smith

Köhl

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

173

163

View full text Add to dashboard Cite

Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.fluorescence microscopy | multiphoton imaging | multiphoton microscopy | three-dimensional microscopy N eurons process information by integrating many thousands of synaptic inputs arriving at thin processes called dendrites and translating them into action potential output. In order to understand the neural code it is important to study both the activity in dendrites and the action potential outputs of large populations of neurons. However, direct electrical recordings from thin dendrites and simultaneous recordings from multiple identified neurons are difficult to achieve and often not feasible, especially in the in vivo setting. Instead, optical sectioning techniques using two-photon excitation of fluorescent indicators of neuronal activity have established themselves as the method of choice to monitor the activity in thin dendrites and large neuronal populations (1-6). Current technology, however, does not allow the spot of light to be scanned sufficiently fast in all three dimensions to fully address the questions of dendritic integration and neuronal population activity. While it is relatively straightforward to scan the focal spot in the x-y plane at kHz rates, scan rates along the z-axis are limited to approximately 20 Hz with conventional imaging approaches (7). This is due to the mechanical inertia of the objective lens and specimen during refocusing. Higher-speed refocusing was recently achieved using a carefully designed electrically tunable lens (8), but at the expense of the numerical aperture.Recently we overcame these fundamental problems and showed that neither speed nor numerical aperture needs to be compromised if we use a different microscope architecture (9). Using this method, the spot can now be scanned along the z axis at high speed while still maintaining diffraction-limited performance. In our design, scanning is carried out by moving a lightweight mirror instead of the objective and high-speed axial scanning is achieved without introducing significant spherical aberration. To demonstrate the practical feasibility of this principle, we have implemented our unique scan approach in a custombuilt two-photon microscope. With it, we monitored calcium transients both along the dendritic arbor of individual pyramidal neurons and in a population of bulk-loaded neurons from a large volum...

show abstract

“…However, complex mechanical devices are required for each scanning point, which made implementation difficult. Version #2 [35,37] and version #4 [36,[38][39][40] have also been described by other laboratories and have recently been used for functional measurements [35,36]. Versions #2-5 were analyzed by modeling and version #5 was found to be the best [33].…”

Section: New Concept For Angular Dispersion Compensationmentioning

confidence: 99%

Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo

Rózsa

Szalay

Katona

2016

Advanced Patch-Clamp Analysis for Neuroscientists

View full text Add to dashboard Cite

Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 Â 950 Â 3000 μm 3 ) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 Â 290 Â 200 μm 3 ) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSF x < 1.9 μm, PSF z < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca 2þ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca 2þ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest.

show abstract

A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy

Cited by 114 publications

References 28 publications

Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintű idegsejtvizsgálatokhoz

Háromdimenziós, gyors, kétfoton-pásztázó eljárások sejt- és hálózatszintű idegsejtvizsgálatokhoz

Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates

Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo

Contact Info

Product

Resources

About