Abstract
Dendritic signals play an essential role in processing visual information in the retina. To study them in neurites too small for electrical recording, we developed an instrument that combines a multi-photon (MP) microscope with a through-the-objective high-resolution visual stimulator. An upright microscope was designed that uses the objective lens for both MP imaging and delivery of visual stimuli to functionally intact retinal explants or eyecup preparations. The stimulator consists of a miniature liquid-crystal-on-silicon display coupled into the optical path of an infrared-excitation laser-scanning microscope. A pair of custom-made dichroic filters allows light from the excitation laser and three spectral bands ('colors') from the stimulator to reach the retina, leaving two intermediate bands for fluorescence imaging. Special optics allow displacement of the stimulator focus relative to the imaging focus. Spatially resolved changes in calcium-indicator fluorescence in response to visual stimuli were recorded in dendrites of different types of mammalian retinal neurons.
🔬 Techniques
💻 Software
✨ Fluorophores
🧪 Sample Preparation
🏭 Microscope Brands
🧪 Reagent Suppliers
📷 Detectors
🔎 Objectives
🎨 Filters
💻 Software Details
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📊 Figures
Fig. 1
Retinal cross-section and representative fluorescence images of two cell types
a Drawing of a retinal cross-section illustrating layering ( NFL nerve fiber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nu...
Fig. 2
Schematic overview of the Eyecup scope
Drawing illustrating the different sections of the Eyecup scope; indicated are the optical paths for laser excitation light (entering from the top right ; the source, a mode-locked Ti/Sapphire laser, ...
Fig. 3
Technical drawings of the Eyecup scope
a CAD drawing illustrating the mechanical arrangement of the Eyecup scope, including 2P excitation/scanning arm, visual stimulator arm and detector head (compare Fig. 2 ; for abbreviations, see Fig. 2...
Fig. 4
Technical drawings of alternative mirror arrangement
a CAD drawing showing the alternative mirror arrangement (between tube lens and objective) used in a modified version of the Eyecup scope (see u201cMechanical designu201d) as well as in the in vivo sc...
Fig. 5
Visual through-the-objective (TTO) stimulator and stimulation light path
a Spectrum illustrating arrangement of fluorescent filters and dichroic mirror (DM Obj ) bands. Approximate photoreceptor absorption peaks in nm from [ 20 , 21 ]. b Optical path of the stimulator ligh...
Fig. 6
Images of the retina
a A living rabbit retina whole-mount viewed with the objective-side CCD camera using through-the-condenser IR illumination. Tissue surface ( 1 ), with a patch electrode over a hole in the inner limiti...
Fig. 7
Laser-evoked responses in the absence of fluorescent dye
a Schematic cross-section of the retina illustrating scan size and focal planes (both for 2P imaging and light stimulation) relative to the recorded ganglion cell. b Spiking responses of two unidentif...
Fig. 8
Laser-evoked responses with fluorescence dye in tissue/recorded cell
a Voltage response of a direction-selective ON/OFF ganglion cell, which was recorded and filled with OGB-1 via a patch-clamp electrode. The cell spikes periodically to its own somatic fluorescence whe...
Fig. 9
Recording light stimulus-evoked activity
a Simultaneously recorded dendritic Ca 2+ ( red trace , from time-lapsed image scan with 64u00d78 pixels/frame and 2 ms/line) and somatic voltage ( black trace ) in an ON DS ganglion cell ( a 1 ). Res...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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