KEYWORDS: Information operations, Retina, In vivo imaging, Confocal microscopy, Near infrared, Image resolution, Line scan image sensors, Intrinsic optical signal, Imaging systems, Eye
Intrinsic optical signal imaging (IOS) promises a noninvasive method for high resolution examination of retinal function.
Using freshly isolated animal retinas, we have conducted a series of experiments to test fast IOSs which have time
courses comparable to electrophysiological kinetics. In this article, we demonstrate the feasibility of in vivo imaging of
fast IOSs correlated with retinal activation in anesthetized frog (Rana Pipiens). A rapid (68,000 lines/s) line-scan
confocal ophthalmoscope was constructed to achieve high-speed (200 frames/s) near infared (NIR) recording of fast
IOSs. By rejecting out-of-focus background light, the line-scan confocal imager provided enough resolution to
differentiate individual photoreceptors in vivo. With visible light stimulation, NIR confocal images disclosed transient
IOSs with time courses comparable to retinal ERG kinetics. High-resolution IOS images revealed both positive
(increasing) and negative (decreasing) light responses, with sub-cellular complexity, in the activated retina.
KEYWORDS: Information operations, Retina, Neurons, Visible radiation, Near infrared, Retinal scanning, Imaging systems, Visualization, Signal processing, Intrinsic optical signal
Simultaneous imaging of different retinal neurons functioning together can benefit better understanding of visual signal
processing mechanisms. In this paper we demonstrate that intrinsic optical signal (IOS) imaging allows simultaneous
monitoring of stimulus-evoked responses propagating from the photoreceptors to inner retinal neurons. High resolution
imaging revealed robust IOSs at the photoreceptor, inner plexiform and ganglion cell layers. While IOSs of the
photoreceptor layer were mainly confined to the area directly stimulated by the visible light; IOSs of inner retinal layers
spread from the stimulus site into relatively large areas.
High resolution monitoring of stimulus-evoked retinal neural activities is important for understanding retinal neural
mechanisms, and for diagnosis of retinal disease and evaluation of treatment. Fast intrinsic optical signals (IOSs), which
have the time courses comparable to retinal electrophysiological responses, hold the promise for high resolution imaging
of retinal neural activities. However, application of fast IOS imaging has been hindered by contamination of slow, high
magnitude, optical responses associated with transient hemodynamic and metabolic changes. We recently demonstrated
the feasibility of separating fast IOSs from slow optical responses by combined dynamic differential imaging and high
frequency flicker stimulation.
A rapid line-scan confocal imager was developed for reflected light measurement of fast IOSs correlated with retinal
activation. This functional imager provides two, i.e., frame-by-frame and line-by-line, imaging modalities. While
frame-by-frame imaging allows dynamic visualization of IOSs over a two-dimensional retinal area at a frame speed
> 100 Hz; line-by-line recording can provide ultrafast (> 10 KHz) monitoring of a fixed line area of the retina. A
series of experiments was conducted to characterize reflected IOSs in frog retinas, and simultaneous
electrophysiological responses were measured. Our experiments indicated that reflected IOSs were tightly correlated
with retinal stimuli. Because of effective rejection of out-of-focus background light, rapid confocal imaging
typically disclosed fast IOSs with magnitude peak > 30% ▵I/I, where ▵I was dynamic optical change and I was
background light intensity. We anticipate that further development of the IOS imaging technology will pave the way
toward noninvasive, high resolution evaluation of retinal function.
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