Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy

There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio.

[1]  C. Stosiek,et al.  In vivo two-photon calcium imaging of neuronal networks , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Squier,et al.  Simultaneous imaging of multiple focal planes using a two-photon scanning microscope. , 2007, Optics letters.

[3]  Hyungsik Lim,et al.  Shaping field for 3D laser scanning microscopy. , 2015, Optics letters.

[4]  S. Shoham,et al.  Remotely scanned multiphoton temporal focusing by axial grism scanning. , 2012, Optics letters.

[5]  Philipp J. Keller,et al.  Whole-brain functional imaging at cellular resolution using light-sheet microscopy , 2013, Nature Methods.

[6]  Benjamin F. Grewe,et al.  Two-Photon Imaging of Neuronal Network Dynamics in Neocortex , 2014 .

[7]  Rainer W Friedrich,et al.  High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device , 2012, Nature Protocols.

[8]  O. Paulsen,et al.  Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates , 2012, Proceedings of the National Academy of Sciences.

[9]  Keith J. Kelleher,et al.  Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity , 2008, Nature Neuroscience.

[10]  Karl Deisseroth,et al.  Neuronal filtering of multiplexed odour representations , 2011, Nature.

[11]  Detlev Schild,et al.  Activity correlation imaging: visualizing function and structure of neuronal populations. , 2009, Biophysical journal.

[12]  Karel Svoboda,et al.  ScanImage: Flexible software for operating laser scanning microscopes , 2003, Biomedical engineering online.

[13]  Balázs Rózsa,et al.  Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes , 2012, Nature Methods.

[14]  Jerome Mertz,et al.  Axial range of conjugate adaptive optics in two-photon microscopy. , 2015, Optics express.

[15]  T. Wilson,et al.  An optical technique for remote focusing in microscopy , 2008 .

[16]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[17]  F. Helmchen,et al.  Imaging cellular network dynamics in three dimensions using fast 3D laser scanning , 2007, Nature Methods.

[18]  E. Stelzer.  The Intermediate Optical System of Laser-Scanning Confocal Microscopes , 2006 .

[19]  Rainer W Friedrich,et al.  Processing of Odor Mixtures in the Zebrafish Olfactory Bulb , 2004, The Journal of Neuroscience.

[20]  Jerome Mertz,et al.  Two-photon microscopy in brain tissue: parameters influencing the imaging depth , 2001, Journal of Neuroscience Methods.

[21]  Charles P. Lin,et al.  Continuous volumetric imaging via an optical phase-locked ultrasound lens , 2015, Nature Methods.

[22]  Joseph P. Zinter,et al.  Maximizing fluorescence collection efficiency in multiphoton microscopy , 2011, Optics express.

[23]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[24]  Sreekanth H. Chalasani,et al.  Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators , 2009, Nature Methods.

[25]  K. Svoboda,et al.  Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience , 2006, Neuron.

[26]  D. Raible,et al.  A targeted gene expression system using the tryptophan repressor in zebrafish shows no silencing in subsequent generations , 2014, Development.

[27]  Benjamin F. Grewe,et al.  Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens , 2011, Biomedical optics express.

[28]  E. Boyden,et al.  Simultaneous whole-animal 3D-imaging of neuronal activity using light-field microscopy , 2014, Nature Methods.

[29]  Rainer W Friedrich,et al.  Neuronal computations in the olfactory system of zebrafish. , 2013, Annual review of neuroscience.

[30]  Tony Wilson,et al.  Aberration-free optical refocusing in high numerical aperture microscopy. , 2007, Optics letters.

[31]  Warren R. Zipfel,et al.  Comparison of objective lenses for multiphoton microscopy in turbid samples. , 2015, Biomedical optics express.

[32]  Jeffrey N. Stirman,et al.  Wide field-of-view, twin-region two-photon imaging across extended cortical networks , 2014 .

[33]  Benjamin F. Grewe,et al.  High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision , 2010, Nature Methods.

[34]  Stefan R. Pulver,et al.  Ultra-sensitive fluorescent proteins for imaging neuronal activity , 2013, Nature.