Femto3D-AcoustoOptic of Two-Photon


Femto3D-AcoustoOptic microscope is the first 3D, real-time two-photon microscope on the market using electrically tunable lenses. With this microscope it is possible to focus the excitation point to any 3D location under the objective without mechanical restrictions reaching millimeter z-dimension scanning range and sub-millisecond temporal resolution. The microscope can provide about a million times faster scanning speed in accessing physiological data from extensive 3D structures as compared to other commercially available microscopes with mechanical z-focusing.




Femto3D-AcoustoOptic microscope is the first fast, 3D, two-photon microscope on the market. The microscope is capable of scanning neuronal, dendritic, and other neuropil activities by about one million faster speed as compared to previous realizations within a large (about cubic millimeter) scanning volume with preserved two-photon resolution. The microscope, using electrically tunable lenses, can focus the excitation point with up to 53 kHz speed to any 3D location under the objective without mechanical restrictions reaching sub-millisecond temporal resolution in a millimeter z-dimension scanning range.





   


Scanning modes of the microscope:

  • 3D random-access point scanning
  • 3D multiple trajectory scanning
  • 3D arbitrary frame scanning
  • All 2D scanning modes possible with galvanometer based scanners:
  • Frame scanning,
  • Folded/Multi-frame scanning
  • Line scanning,
  • Point scanning,
  • XYZ stack


With these features you can get

  • Ca2+ transient in 100-66 cells within 2 ms in the above written large volumes
  • Ca2+ transient in 200-132 cells within 4 ms in the above written large volumes
  • Ca2+ transient in 300-264 cells within 6 ms in the above written large volumes
  • You can get high resolution in the center ~200 μm x 200 μm x 200 μm volume therefore fine structures such as axon varicosities and dendritic spines can be resolved. Imaging long, tortuous dendritic segments (up to 250-300 μm) is possible.



With this microscope we are able to measure:

  • single-neuron imaging in vitro by obtaining 3D optical recordings of action potential backpropagation at sub-millisecond temporal resolution with random-access scanning
  • dendritic Ca2+ spike propagation in several-hundred-micrometer-long neuronal processes with the continuous 3D trajectory scanning mode
  • imaging neuronal populations in vivo by 3D random-access scanning of Ca2+ transients in several hundreds of neurons simultaneously in the mouse visual cortex at 80 Hz




Femto3D-AO is the only product capable of rapid, large-volume real time 3D scanning, even in in vivo conditions.

  • Random access scanning provides an increase of two to six orders of magnitude in the product of measurement speed and signal collection efficiency. No other currently available 3D scanning method, with high spatial resolution and deep penetration capability, can provide a similar increase
  • In a 290 μm × 290 μm × 200 μm core volume, the lateral and axial PSFs remained below 0.8 μm and 3 μm, respectively, and the device therefore could resolve dendritic spines or buttons.
  • Spatial resolution of the microscope: detection of single action potential–induced transients is possible in a near-cubic-millimeter scanning volume.
  • In 3D random-access scanning mode, 2–2,000 points can be scanned near-simultaneously, in one measurement cycle, with 20/N kHz to 50/N kHz, where N denotes the number of points.
  • Using the microscope it is possible to reach a temporal resolution of 54.7 μs in differentiating AP induced 3D Ca2+ responses, allowing the measurement of fast regenerative activity at the scale of small dendritic segments.
  • The position of the PSF can be finely adjusted to any spatial coordinates with 50-100 nm precision. Therefore, number of z-planes and coordinates are unlimited, which allows a very precise measurement control and precede targeting of the regions of interest to eliminate neuropil contamination.
  • Fully automated system, does not require engineering supervision. After installation the stability is monitored by a computer and it is automatically adjusted.


HOW IT WORKS


The system contains significant new design concepts:

  • it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range;
  • it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors;
  • it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures.

        




EXAMPLE OF MULTIPLE SITE DENDRITIC IMAGING



                                                                                                       

3D AO scanning (top, z stack): a 3D view of the dendritic arbor of a CA1 pyramidal cell imaged; spheres represent the measurement locations. Maximum intensity z-projection image of the same neuron; recorded dendrites are numbered. Schema of the apical trunk and the dendritic branches of the neuron is showing calcium transients recorded near-simultaneously in each of the dendrites, averaged from five traces. Repetition rate of the 3D point scanning was 80 Hz. Dendritic Ca2+ transients measured from the same dendritic point of the apical trunk (average of five traces) and corresponding somatic voltage traces (Vm).



EXAMPLE FOR 3D-AO TWO-PHOTON IN VIVO MEASUREMENTS 



Three dimensional random-access measurement of network activity in mouse V1. (a) In vivo experimental arrangement (sketch). Visual stimulation was induced by continuously moving bars (in 45-degree steps at eight directions of motion). (b) Maximal intensity side- and z-projection of the z-stack. 375 autodetected neuronal locations. (c) Spheres: from the same 375 neuronal location 3D Ca2+ responses was measured (visual stimulation: moving bar at +45°). Rows: single cells from a single 3D measurement. (d) Ca2+ transients examples from responding neurons in c. (e) Stimulation with 90° oriented stimulus  (at +135°) induced smaller responses from the same neurons with a different activation pattern.


MEASUREMENT OF PROPAGATING CA2+ WAVES

      
     

Left panel shows the 3D Ca2+ responses along a single dendrite during a sharpe-wave event. Note that the high scanning speed makes the propagation 

clearly distingusihable. The left panel shows the half maximum of the local transients versus distance from the soma. Propagation speed can be 

determined by linear fit.

 
See also:
B Chiovini, G F Turi, G Katona, A Kaszas, D Palfi, P Maak, G Szalay, M F Szabo, Z Szadai, Sz Kali and B Rozsa
Dendritic spikes induce ripples in parvalbumin interneurons during hippocampal sharp waves. 
Neuron (2014)



INTEGRATED EXTENSION OF THE MES SOFTWARE

The dedicated AO driving software module extends the functionalities of the MES software with the capabilities of the novel 3D AO hardware. 

  • All advantages of the MES software, integrated control of peripherals
  • Adds support to the AO measurement modes.
    •    High Speed Frame scanning
    •    3D rotated frame scanning
    •    Line scanning
    •    3D random access point scanning (3D RAMP)
    •    Z-stack acquisition
  • Multiple 3D line selection, according to background image stacks
  • Equal time or equal speed scanning of the 3D lines
  • Easy 3D editing and alignment of scan patterns
  • Flexible control of output channels, multiple scanning patterns (protocol architecture)
  • Integrated parallel data acquisition and analysis of electrical recordings
  • Real-time display of raw data
  • 3D spatial normalization and data projection
  • Control of laser intensity or PMT voltages according to depth
  • Depth change with objective movement or Z-scanner


All these functions integrated seamlessly into the MES software package, and are supplemented with the high number of analysis and accessory tools to achieve effective physiological measurements.







Contact us Address :Room A020, Floor 3, LianRi International Building, No.18 ,NanLangJiaYuan,ChaoYang District , Beijing City ,100022,China Telephone:010- 65129207 Fax :010- 65129207 Email :tim_liuyi@aliyun.com , tim@hktimwinter.com 京ICP备15043433