High-speed line-scan confocal imaging of stimulus-evoked intrinsic optical signals in the retina

Abstract

A rapid line-scan confocal imager was developed for functional imaging of the retina. In this imager, an acousto-optic deflector was employed to produce mechanical vibration- and inertia-free light scanning, and a high-speed (68,000 Hz) linear CCD camera was used to achieve subcellular and submillisecond spatiotemporal resolution imaging. Two imaging modalities, i.e., frame-by-frame and line-by-line recording, were validated for the reflected light detection of intrinsic optical signals (IOSs) in visible light stimulus activated frog retinas. Experimental results indicated that fast IOSs were tightly correlated with retinal stimuli and could track visible light flicker stimulus frequency up to at least 2 Hz.

Fig. 1

(a) Schematic diagram of line-scan confocal system. CO: collimator; CL: cylindrical lens BS: beam splitter; AOD: acousto-optic deflector; DM: dichroic mirror; Lx: spherical lenses. Focal length of the CL is 50 mm. Focal length of the lenses L1–L5 are 80mm, 60mm, 120mm, 60mm, and 100mm, respectively. (b) Confocal image showing clear cellular structure of individual photoreceptors. In the x direction (i.e., parallel to the focused scanning line), theoretical resolution of the imager is ~ 1.9 µm (0.61λ/NA). In the y direction (i.e., perpendicular to the focused scanning line), theoretical resolution of the imager is ~ 1.3 µm (0.4λ/NA). (c) Axial resolution of line-scan confocal imaging system was tested to be ~ 12 µm.

Fig. 2

Frame-by-frame imaging of IOSs. (a) Raw image of photoreceptors. (b) Pre-stimulus IOS image. (c) Post-stimulus IOSs image. The raw confocal images were acquired at the speed of 100 frames/s, with NIR imaging light focused at photoreceptor layer. Both pre-stimulus and post-stimulus IOSs images were an average over 250 ms. (d) Tracings 1–6 revealed the IOSs variation property of local areas pointed by arrowheads 1–6 in (c). Tracing 7 represents integral IOSs by averaging all the pixels of each IOS image. Vertical line indicates the onset of the stimulus. (e) The percentage statistics of activated retinal areas with positive (> 5% ΔI/I) and negative (< −5% ΔI/I) IOSs. The threshold (5%ΔI/I) was used to reduce the effect of background noise on the statistics. Trace 1 (positive IOSs) and 2 (negative IOSs) are statistic results of experiment trial with stimulus delivered. Trace 3 (positive IOSs) and 4 (negative IOSs) are statistic results of control trial without stimulus.

Fig. 3

Line-by-line imaging of IOSs. (a) Line-by-line raw image; (b) Line-by-line IOS image; (c) Line-by-line DIOS image. (d) Representative localized IOSs (arrows 1–4). S: visible light stimulus. (e) The statistics of activated retinal area ratios with positive (black) and negative (grey) IOSs. Inset panel shows enlarged picture of early optical response marked by the gray bar. Rapid IOSs occurred within 5ms after stimulus onset (inset panel). (f) Representative localized DIOSs (arrows 1–4).