Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates.

Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 02/2012; 109(8):2919-24. DOI: 10.1073/pnas.1111662109
Source: PubMed

ABSTRACT Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues.
    Scientific Reports 12/2014; 4(7253). DOI:10.1038/srep07253 · 5.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement. However, for many commercial objective lenses, it can be difficult to accurately locate the position of the back focal plane. This paper investigates the impact of this limitation on the fidelity of three-dimensional data sets of living cardiac tissue, specifically the introduction of distortions. These distortions limit the accuracy of sarcomere measurements taken directly from raw volumetric data. The origin of the distortion is first identified through simulation of a remote focussing microscope. Using a novel three-dimensional calibration specimen it was then possible to quantify experimentally the size of the distortion as a function of objective misalignment. Finally, by first approximating and then compensating the distortion in imaging data from whole heart rodent studies, the variance of sarcomere length (SL) measurements was reduced by almost 50%.
    Frontiers in Physiology 09/2014; 5(384). DOI:10.3389/fphys.2014.00384
  • [Show abstract] [Hide abstract]
    ABSTRACT: Light-sheet fluorescence microscopy (LSFM) affords highly parallelized 3D imaging with optical sectioning capability and minimal light exposure. However, using Gaussian beams for light-sheet generation results in a trade-off between beam waist thickness and the area over which the beam can approximate a light-sheet. Here, we present a novel form of LSFM that uses incoherent extended focusing to produce divergence free light-sheets with near diffraction-limited resolution and uniform intensity distribution along the propagation direction. We demonstrate the imaging performance of the new technique by volumetric imaging of beads, collagen fibers, and melanoma cancer cells with sub-cellular resolution.
    Optics Express 10/2014; 22(21). DOI:10.1364/OE.22.026141 · 3.53 Impact Factor

Full-text (2 Sources)

Available from
May 30, 2014

Michael M Kohl