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T. Euler, P.B. Detwiler, D.J. Margolis, S.E. Hausselt, W. Denk; Eyecup Scope – Optophysiological Recordings of Light–Stimulus Evoked Fluorescence Signals in the Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5394.
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© ARVO (1962-2015); The Authors (2016-present)
Dendritic signals play an essential role in processing visual information in the retina. To study signals in neurites too small for electrical recording we developed an instrument that combines a two–photon microscope with a through–the–objective (TTO) visual stimulator.
An upright microscope was designed that uses an objective lens (water immersion, 20x, approx. 1 mm field of view) for both two–photon micro–fluorimetry and delivery of visual stimuli to functionally intact flat–mounted retinal explants or eyecup preparations. The TTO stimulator consists of a miniature liquid–crystal–on–silicon (LCoS) display coupled into the optical path of a 930 nm laser scanning microscope. A pair of custom–made dichroic filters allows light from the excitation laser as well as three bands ('colors') from the stimulator to reach the retina, leaving two intermediate bands for fluorescence imaging. The laser–scanner offset is used to image different regions of a selected neuron while the XY–position of the objective lens remains fixed to prevent lateral shifts in the TTO stimulus pattern. Focus–compensation optics are used to keep the stimulus focused on the photoreceptors while imaging dendrites at different focal planes.
Spatially–resolved visually–evoked calcium signals were recorded in different types of mammalian retinal neurons after filling them with fluorescent calcium indicator. The stimulus resolution at the level of the photoreceptors reached approx. 2 µm/pixel. Intensity range and contrast, which depended on the LCoS type and illumination LED(s), were 10 to 40 kilophotons·s–1·µm–2 (at 578 nm) and 72%, respectively. We tested stimulus refresh rates up to 80 Hz, however, rates up to 300 Hz (monochromatic) are possible.
The 'eyecup scope' combines a fast and high–resolution visual stimulator and a two–photon laser scanning fluorescence microscope to study dendritic signals in retinal neurons.
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