Transient intrinsic optical responses associated with neural activation offer an attractive strategy for dynamic imaging of neural activity, and may provide a noninvasive methodology for imaging of retinal function. Here we demonstrate the feasibility of near infrared imaging of fast intrinsic optical changes in isolated frog retina activated by visible light. Using a photodiode detector in a transmitted light geometry, we routinely measured dynamic transmitted optical responses in single passes, at the level of one part in 10(4) of background light. Rapid CCD image sequences acquired with transmitted light (bright field) illumination disclosed larger fractional responses and showed evidence of multiple response components with both negative- and positive-going signals with different timecourses. Dark field imaging further enhanced the contrast and sensitivity of optical measures of neural activation. High-resolution imaging disclosed optical responses in single pixels often exceeding 5%, of background light, allowing dynamic imaging at the resolution of single cells, in single passes. Fast optical signals are closely related to identified response components of the electroretinogram. Optical responses showed complex but consistent spatial organization from frame to frame. Our experimental results and theoretical analysis suggest that the optical responses may result from dynamic volume changes corresponding to ion and water flow across the cell membrane, directly associated with the electrophysiological response.
"In oxygenated Ringer’s solution, freshly isolated retinas are viable and excitable, at least for a few hours. We have recently using freshly isolated retinas, including both sliced  and flat-mounted [32–39] retinas, to investigate stimulus-evoked retinal neural activities. In this study, the same retinal preparation, i.e., isolated but living retinas, to characterize cellular sources of retinal autofluorescence. "
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.
"A number of proposed sources of this functional signal, including photoreceptors (Kahlert et al., 1990), membrane depolarization (Stepnoski et al., 1991), and altered metabolism have been attributed to the changes in the local optical properties of the retinal tissue that are detectable by measurement of light-scattering signals. In an effort to isolate the source of this signal, several ex vivo studies of retinal samples (Yao and George, 2006) have measured stimulusinduced intrinsic NIR signals that have been recorded from retinal layers, including the inner retina and the region of the optic disc (Ts'o et al., 2009). "
[Show abstract][Hide abstract] ABSTRACT: Independent component analysis (ICA) is a statistical technique that estimates a set of sources mixed by an unknown mixing matrix using only a set of observations. For this purpose, the only assumption is that the sources are statistically independent. In many applications, some information about the nature of the unknown signals is available. In this paper we show a method for incorporating prior information about the mixing matrix to increase the levels of detection of responses to visual stimuli. Experimentally, our method matches the performance of known ICA algorithms for high SNR and can greatly improve the performance for low levels of SNR or low levels of signal-to-background ratio (SBR). For the problem of signal extraction, we have achieved detection for signals as small as 0.01% (-40 dB SBR) in hybrid live/synthetic data simulations. In experiments using a functional imager of the retina, measured changes in reflectance in response to visual stimulus are in the order of 0.1-1% of the total pixel intensity value, which makes the functional signal difficult to detect by standard methods. The results of the analysis show that using ICA-P signal levels of 0.1% can be detected. The approach also generalizes the standard Infomax algorithm which can be thought of as a special case of ICA-P when the confidence parameter or a tolerance value is zero. For in vivo animal experiments, we show that signal detection agreement over a range of confidence values parameters can be used to establish reflectance changes in response to the visual stimulus.
Medical image analysis 02/2011; 15(1):35-44. DOI:10.1016/j.media.2010.06.009 · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Optical methods offer a number of advantages for the study of neural systems. Optical techniques are relatively noninvasive,
and offer wide field of view, in addition to high resolution in time and in space. Improvements in optical sensor technologies
and imaging techniques continually enhance imaging performance, and extend resolution into three dimensions. Digital signal
processing strategies allow increasingly subtle signals to be extracted and visualized. Imaging methods allow large populations
of cells to be examined simultaneously, while resolving individual cells. Differential absorption or fluorescence emission
by endogenous biochemicals or exogenous reporters allows characterization of specific aspects of the chemical and physical
environment of cells, and produces signals that are highly correlated with neural activation. Fast intrinsic optical signals,
which appear to be tightly coupled to the biophysical processes of neural activation, hold great promise for dynamic imaging
of function in large populations of neurons. Coupled with multi-channel electrophysiological and computational modeling techniques,
optical imaging enables powerful new understanding of the function of the brain.
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