Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution

Abstract

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 100Hz, 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.

Somatic calcium level reports integrated spiking activity of cerebellar interneurons in vitro and in vivo

“…Scanning of large fields (600 ϫ 300 m) was used to identify structures and was performed at an effective pixel size of 0.5 m. For imaging individual MLIs or small groups of five to eight MLIs with a temporal resolution in the millisecond range we implemented the targeted path scanning strategy (TPS; Lillis et al 2008), in which the laser beam follows an optimized trajectory passing through the structures of interest. TPS protocols covered fields of ϳ200 ϫ 200 m with a total frame time of 10 -16 ms and a dwell time per MLI of 1-2 ms. To monitor single MLIs, smaller fields (10 -20 m wide) were scanned at an effective pixel size of 0.25 m and dwell times of 30 -60 ms.…”

Neuronal network analyses: premises, promises and uncertainties

“…These limits are being addressed (e.g. Lillis et al 2008), but the dream of using voltage sensitive dyes to monitor activity in large numbers of network neurons simultaneously at single cell resolution (that 'the photon will replace the electron for probing neuronal function'; Scanziani & Hausser 2009) still seems far away.…”

Method to quantify accuracy of position feedback signals of a three-dimensional two-photon laser-scanning microscope

“…Therefore, an accurate monitoring of the absolute position of the focal volume over time is the main requirement accomplishing a reliable reconstruction of the spatial information of targeted cells. So far, internal feedback signals of the microscope's positioning devices were routinely used to achieve this goal [2,4,10,17]. Using fluorescent beads embedded in agarose, the feasibility of reconstructing morphological information was shown [2].…”