High-Speed, Random-Access Multiphoton Microscopy for Monitoring Synaptic and Neuronal Activity in 3D in Behaving Animals

  • Mate Marosi
  • Gergely Szalay
  • Gergely Katona
  • Balázs RózsaEmail author
Part of the Neuromethods book series (NM, volume 148)


Understanding neural computation requires methods for three-dimensional readout of neural activity simultaneously on somatic and dendritic scales. Random-access point scanning offers measurement speed increased by several orders of magnitude compared to classical raster scanning methods; however, it is highly sensitive to sample movements during behavior, when fluorescence information could be completely lost due to dislocation of the point of interest. Volume scanning, on the other hand, is insensitive to even relatively large movements, but the scanning speed can be as low as 0.1–1 Hz for full 3D volumes, which is insufficient for recording fast Ca2+ activity. The 3D Drift acousto-optical (AO) scanning method can extend each scanning point of random-access AO point scanning to small 3D line, surface, or volume elements for fast recording of complex neuronal activity in vitro and in vivo, allowing free selection of any portion of the volume measured that can help finding the optimal trade-off between point scanning and full-volume scans. The video-like information, of the volume or surface elements measured, allows offline motion correction even during behavior and brain movement.

In this chapter, fast 3D measurement of over 150 dendritic spines with 3D lines, over 100 somata with squares and cubes, or multiple spiny dendritic segments with surface and volume elements in awake and behaving animals will be demonstrated.


Two-photon microscopy Acousto-optical scanning Calcium imaging In vivo 3D functional imaging AO 3D Drift scanning 



The authors thank Linda Judák for the in vivo recordings and Alexa Bojdán for contributing to the figures.

Conflict of Interest

Dr. Gergely Katona and Dr. Balázs Rózsa are founders of Femtonics Ltd. Balázs Rózsa is a member of its scientific advisory board.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Mate Marosi
    • 1
  • Gergely Szalay
    • 1
  • Gergely Katona
    • 1
    • 2
  • Balázs Rózsa
    • 1
    • 2
    Email author
  1. 1.3D Functional Network and Dendritic Imaging, Institute of Experimental Medicine, Hungarian Academy of SciencesBudapestHungary
  2. 2.Two-Photon Measurement Technology Research Group, The Faculty of Information Technology, Pázmány Péter Catholic UniversityBudapestHungary

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