Holographic Two-Photon Microscopy for Simultaneous Multi-Plane Calcium Imaging

Yang W, Miller JE, Carrillo-Reid L, Pnevmatikakis E, Paninski L, Yuste R, Peterka DS. Simultaneous multi-plane imaging of neural circuits. Neuron 2016;89(2):269-284. doi: 10.1016/j.neuron.2015.12.012.

Background: Understanding brain function requires recording neuronal activity across multiple cortical layers simultaneously. Conventional two-photon microscopy relies on sequential scanning, limiting temporal resolution and volumetric coverage. To overcome these constraints, the authors developed a holographic two-photon imaging system enabling simultaneous multi-plane imaging for functional mapping of neuronal populations across cortical depths with single-cell precision.

Hypothesis: This study hypothesized that a holographically multiplexed two-photon microscope, combined with computational signal extraction, allows simultaneous high-speed calcium imaging across several cortical planes while maintaining accuracy and spatial resolution.

Methods: The authors modified a two-photon microscope by integrating a spatial light modulator (SLM) into the excitation path of a galvanometer-based scanner. Imaging was performed in awake mice expressing GCaMP6f in the visual cortex. The microscope was operated and data were acquired using ScanImage, which synchronized laser modulation and multi-plane scanning. Fluorescence signals were extracted using a constrained nonnegative matrix factorization (CNMF) algorithm to separate overlapping sources. The SLM enabled flexible focusing of multiple beamlets without moving the objective, permitting simultaneous imaging of laterally or axially displaced cortical regions.

Results: The system achieved dual- and triple-plane imaging of neuronal populations up to 500 µm deep. CNMF extraction improved signal-to-noise ratio by about 13% over traditional methods. Neurons imaged in different planes showed highly correlated dynamics and preserved orientation selectivity.

Conclusions: Combining holographic multiplexing with ScanImage-controlled two-photon microscopy enables fast, simultaneous multi-plane imaging with high fidelity, providing a versatile tool for studying three-dimensional neural circuit dynamics in vivo.