Microprisms for In Vivo Multilayer Cortical Imaging

Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA.
Journal of Neurophysiology (Impact Factor: 2.89). 06/2009; 102(2):1310-4. DOI: 10.1152/jn.91208.2008
Source: PubMed


Cortical slices allow for simultaneous imaging of multiple cortical layers. However, slices lack native physiological inputs and outputs. Although in vivo, two-photon imaging preserves the native context, it is typically limited to a depth of <500 microm. In addition, simultaneous imaging of multiple cortical layers is difficult due to the stratified organization of the cortex. We demonstrate the use of 1-mm microprisms for in vivo, two-photon neocortical imaging. These prisms enable simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. Images were collected from the mouse motor and somatosensory cortex and show a nearly 900-microm-wide field of view. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution is sufficient to resolve dendritic spines on layer V neurons. Images collected using the microprism are comparable to images collected from a traditional slice preparation. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. H&E staining shows the surrounding tissue remains in its native, stratified organization. Estimation of neuronal damage using propidium iodide and a fluorescent Nissl stain reveals cell damage is limited to <100 microm from the tissue-glass interface. Microprisms are a straightforward tool offering numerous advantages for into neocortical tissue.

Download full-text


Available from: Michael J Levene
  • Source
    • "Another challenge to using NW electrodes in vivo is recording beyond the layer of dead cells and protective glia that surround implanted electrodes. Studies have reported the thickness of the dead cell layers to be approximately 40 microns (Chia and Levene, 2009). Therefore, to access healthy cells, the NWs electrodes must penetrate through this dead layer. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Brain-machine interfaces (BMIs) that can precisely monitor and control neural activity will likely require new hardware with improved resolution and specificity. New nanofabricated electrodes with feature sizes and densities comparable to neural circuits may lead to such improvements. In this perspective, we review the recent development of vertical nanowire (NW) electrodes that could provide highly parallel single-cell recording and stimulation for future BMIs. We compare the advantages of these devices and discuss some of the technical challenges that must be overcome for this technology to become a platform for next-generation closed-loop BMIs.
    Full-text · Article · Mar 2013 · Frontiers in Neural Circuits
  • Source
    • "They suffer from strong spherical and chromatic aberrations [9–12]. When combined with a refractive glass lens, GRIN lenses can form objectives with limited monochromatic aberration correction and increased NA [13] while maintaining outer diameters as small as or smaller than a biopsy needle [14–16]. However, without axial chromatic aberration correction in the imaging system, dyes with different emission spectra cannot be imaged without one or more appearing blurred. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Currently, researchers and clinicians lack achromatized endomicroscope objectives that are as narrow as biopsy needles. We present a proof-of-concept prototype that validates the optical design of an NA0.4 objective. The objective, built with plastic lenses, has a 0.9 mm clear aperture and is achromatized from 452 nm to 623 nm. The objective's measured Strehl ratio is 0.74 ± 0.05 across a 250 μm FOV. We perform optical sectioning via structured illumination through the objective while capturing fluorescence images of breast carcinoma cells stained with proflavine and cresyl violet. This technology has the potential to improve optical biopsies and provide the next step forward in cancer diagnostics.
    Full-text · Article · Feb 2013 · Biomedical Optics Express
  • Source
    • "Another application may be the understanding of the relationships between neuronal activation and local cerebral vascular dynamics via the astrocytic network [44]. Transfected calcium indicators [45], transgenic mice with fluorescent neurons [46] and additional iv injections of FITC-dextran and sulforhodamine for the staining of respectively the cerebral vasculature and astrocytes might be used to avoid intracerebral injections. The iv method leads to a more exhaustive labeling of astrocytes in comparison to transgenic animals. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.
    Full-text · Article · Apr 2012 · PLoS ONE
Show more