Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution.
We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.
Lillis
Kyle P
KP
Department of Biomedical Engineering, Boston University, Boston, MA 02215, United States.
Eng
Alfred
A
White
John A
JA
Mertz
Jerome
J
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology* / Q000502:physiology; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D009415:Nerve Net / Q000166:cytology* / Q000502:physiology; D009434:Neural Pathways / Q000166:cytology / Q000502:physiology; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D055095:Optics and Photonics / Q000295:instrumentation; D009924:Organ Culture Techniques; D017785:Photons; D051381:Rats; D020318:Rats, Long-Evans; D012984:Software; D013997:Time Factors
D005456:Fluorescent Dyes; D002118:Calcium