[Study on rat intestinal absorption characteristics of total flavonoids from Coreopsis tinctoria].

The rat everted intestinal sac model was adopted to investigate the absorption of total flavonoids from Coreopsis tinctoria in different intestinal segments. Cyaniding-3-O-ß-D-glucoside, chlorogenic acid, flavanomarein, quercetagetin-7-O-ß-D-glucoside, iso-okanin, marein and 3,5-dicaffeoylquinic acid which as the major chemical components of total flavonoids from C. tinctoria were selec-ted as the study objects to evaluate the absorption characteristics of each component in different intestinal segments. The results showed that the absorption of seven components of total flavonoids at different intestinal segments was in consistent with zero order absorption rate. The K_a of chlorogenic acid, flavanomarein, quercetagetin-7-O-ß-D-glucoside, isookanin and 3,5-dicaffeoylquinic acid increased with increasing of concentration of total flavonoids(P<0.05), indicating that the intestinal absorption of these five components was passive transport. The K_a of cyaniding-3-O-ß-D-glucoside and marein showed a weak concentration dependence, suggesting that the absorption of them may be an positive and passive co-existing mode. The result of absorption in different intestinal segments showed that cyaniding-3-O-ß-D-glucoside, chlorogenic acid, flavanomarein, quercetagetin-7-O-ß-D-glucoside, marein and 3,5-dicaffeoylquinic acid were mainly absorbed in ileum, while isookanin was mainly absorbed in jejunum. The total flavonoids of C. tinctoria are selectively absorbed in intestinal tract, the rat everted intestinal sac model can be used to evaluate the multi-component intestinal absorption characteristics of total flavonoids from C. tinctoria.

Comparative intrinsic optical signal imaging of wild-type and mutant mouse retinas.

Functional measurement is important for retinal study and disease diagnosis. Transient intrinsic optical signal (IOS) response, tightly correlated with functional stimulation, has been previously detected in normal retinas. In this paper, comparative IOS imaging of wild-type (WT) and rod-degenerated mutant mouse retinas is reported. Both 2-month and 1-year-old mice were measured. In 2-month-old mutant mice, time course and peak value of the stimulus-evoked IOS were significantly delayed (relative to stimulus onset) and reduced, respectively, compared to age matched WT mice. In 1-year-old mutant mice, stimulus-evoked IOS was totally absent. However, enhanced spontaneous IOS responses, which might reflect inner neural remodeling in diseased retina, were observed in both 2-month and 1-year-old mutant retinas. Our experiments demonstrate the potential of using IOS imaging for noninvasive and high resolution identification of disease-associated retinal dysfunctions. Moreover, high spatiotemporal resolution IOS imaging may also lead to advanced understanding of disease-associated neural remodeling in the retina.

Intrinsic optical signal imaging of glucose-stimulated insulin secreting ß-cells.

Simultaneous monitoring of many functioning ß-cells is essential for understanding ß-cell dysfunction as an early event in the progression to diabetes. Intrinsic optical signal (IOS) imaging has been shown to allow high resolution detection of stimulus-evoked physiological responses in the retina and other neural tissues. In this paper, we demonstrate the feasibility of using IOS imaging for functional examination of insulin secreting INS-1 cells, a popular model for investigating diabetes associated ß-cell dysfunction. Our experiments indicate that IOS imaging permits simultaneous monitoring of glucose-stimulated physiological responses in multiple cells with high spatial (sub-cellular) and temporal (sub-second) resolution. Rapid IOS image sequences revealed transient optical responses that had time courses tightly correlated with the glucose stimulation.

Circular polarization intrinsic optical signal recording of stimulus-evoked neural activity.

Linear polarization intrinsic optical signal (LP-IOS) measurement can provide sensitive detection of neural activities in stimulus-activated neural tissues. However, the LP-IOS magnitude and signal-to-noise ratio (SNR) are highly correlated with the nerve orientation relative to the polarization plane of the incident light. Because of the complexity of orientation dependency, LP-IOS optimization and outcome interpretation are time consuming and complicated. In this study, we demonstrate the feasibility of circular polarization intrinsic optical signal (CP-IOS) measurement. Our theoretical modeling and experimental investigation indicate that CP-IOS magnitude and SNR are independent from the nerve orientation. Therefore, CP-IOS promises a practical method for polarization IOS imaging of complex neural systems.

In vivo confocal imaging of fast intrinsic optical signals correlated with frog retinal activation.

Using freshly isolated animal retinas, we have conducted a series of experiments to test fast intrinsic optical signals (IOSs) that have time courses comparable to electrophysiological kinetics. In this Letter, we demonstrate the feasibility of in vivo imaging of fast IOSs in intact frogs. A rapid line-scan confocal ophthalmoscope was constructed to achieve high-speed IOS recording. By rejecting out-of-focus background light, the line-scan confocal imager provided the resolution to differentiate individual photoreceptors in vivo. Rapid confocal imaging disclosed robust IOSs with time courses comparable to retinal electroretinogram kinetics. High-resolution IOS images revealed both positive (increasing) and negative (decreasing) light responses, with subcellular complexity.

High spatiotemporal resolution imaging of fast intrinsic optical signals activated by retinal flicker stimulation.

High resolution monitoring of stimulus-evoked retinal neural activities is important for understanding retinal neural mechanisms, and can be a powerful tool for retinal disease diagnosis and treatment outcome evaluation. Fast intrinsic optical signals (IOSs), which have the time courses comparable to that of electrophysiological activities in the retina, hold the promise for high resolution imaging of retinal neural activities. However, application of fast IOS imaging has been hindered by the contamination of slow, high magnitude optical responses associated with transient hemodynamic and metabolic changes. In this paper we demonstrate the feasibility of separating fast retinal IOSs from slow optical responses by combining flicker stimulation and dynamic (temporal) differential image processing. A near infrared flood-illumination microscope equipped with a high-speed (1000 Hz) digital camera was used to conduct concurrent optical imaging and ERG measurement of isolated frog retinas. High spatiotemporal resolution imaging revealed that fast IOSs could follow flicker frequency up to at least 6 Hz. Comparable time courses of fast IOSs and ERG kinetics provide evidence that fast IOSs are originated from stimulus activated retinal neurons.

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

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.

Microlens array recording of localized retinal responses.

We designed a rapid functional imager for the parallel recording of localized intrinsic optical signals (IOSs). This imager used a microlens array (MLA)-based illuminator to deliver visible stimulus light and near-infrared (NIR) recording light simultaneously. The parfocal configuration of the stimulus and recording light illumination enabled confocal recording of the stimulus-evoked IOSs. Because the MLA stimulation/recording spots were widely separated on the retina, and only the photoreceptors within the MLA stimulation/recording spots were stimulated, the potential IOS cross talk effect among neighboring retinal areas was minimized. Our experiments revealed robust IOS activities tightly correlated with localized retinal responses.

Parallel optical monitoring of visual signal propagation from the photoreceptors to the inner retina layers.

Understanding of visual signal processing can benefit from simultaneous measurement of different types of retinal neurons working together. In this Letter, we demonstrate that intrinsic optical signal (IOS) imaging of frog retina slices allows simultaneous observation of stimulus-evoked responses propagating from the photoreceptors to the inner neurons. High-resolution imaging revealed robust IOSs at the photoreceptor, the inner plexiform, and the ganglion cell layers. While IOSs of the photoreceptor layer were mainly confined to the area directly stimulated by the visible light, IOSs of the inner retinal layers spread from the stimulus site into relatively large areas with a characteristic near-to-far time course.

Optical dissection of stimulus-evoked retinal activation.

Better understanding of stimulus-evoked intrinsic optical signals (IOSs) in the retina promises new methodology for study and diagnosis of retinal function. Using a flood-illumination near infrared (NIR) light microscope equipped with high-speed CCD (80 Hz) and CMOS (1000 Hz) cameras, we validated depth-resolved enface imaging of fast IOSs in isolated retina of leopard frog. Both positive (increasing) and negative (decreasing) IOSs were observed at the photoreceptor and inner layers of the retina. The distribution of IOSs with opposite polarities showed a center-surround pattern. At the photoreceptor layer, negative IOSs dominated the center area illuminated by the stimulus light spot, while positive signals dominated the surrounding area. In contrast, at inner retinal layers, positive IOSs dominated the center area covered by the stimulus light spot, and negative IOSs were mainly observed in the surrounding area. Fast CMOS imaging disclosed rapid IOSs within 5 ms after the stimulus onset, and both ON and OFF optical responses were observed associated with a step light stimulus.

Near-infrared imaging of fast intrinsic optical responses in visible light-activated amphibian retina.

High performance functional imaging is needed for dynamic measurements of neural processing in retina. Emerging techniques for visual prosthesis also require advanced methodology for reliable validation of electromagnetic stimulation of the retina. Imaging of fast intrinsic optical responses associated with neural activation promises a variety of technical advantages over traditional single and multichannel electrophysiological techniques for these purposes, but the application of fast optical signals for neural imaging has been limited by low signal-to-noise ratio and high background light intensity. However, by using an optimized near-infrared probe light and improved optical system, we improve the optical signals substantially, allowing single pass measurements with approximately micron resolution. We image fast intrinsic optical responses with different optical modalities, i.e., bright field, dark field, and cross-polarization, from isolated retina activated by visible light stimulation. At single cell resolution, bright-field imaging discloses the maxima of optical responses approximately 5% dI/I, where dI is the dynamic optical change and I is the baseline light intensity. Dark-field imaging techniques further enhance the sensitivity of optical measurements, and show the maxima of optical responses exceeding 10% dI/I. Cross-polarized imaging provides optical sensitivity similar to dark-field imaging, but different patterns of neural activation are observed.

Super-resolution scanning laser microscopy through virtually structured detection.

High resolution microscopy is essential for advanced study of biological structures and accurate diagnosis of medical diseases. The spatial resolution of conventional microscopes is light diffraction limited. Structured illumination has been extensively explored to break the diffraction limit in wide field light microscopy. However, deployable application of the structured illumination in scanning laser microscopy is challenging due to the complexity of the illumination system and possible phase errors in sequential illumination patterns required for super-resolution reconstruction. We report here a super-resolution scanning laser imaging system which employs virtually structured detection (VSD) to break the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost and phase-artifact free strategy to achieve super-resolution in scanning laser microscopy.

In vivo optical coherence tomography of light-driven melanosome translocation in retinal pigment epithelium.

Optical coherence tomography (OCT) may revolutionize fundamental investigation and clinical management of age-related macular degeneration and other eye diseases. However, quantitative OCT interpretation is hampered due to uncertain sub-cellular correlates of reflectivity in the retinal pigment epithelium (RPE) and photoreceptor. The purpose of this study was twofold: 1) to test OCT correlates in the RPE, and 2) to demonstrate the feasibility of longitudinal OCT monitoring of sub-cellular RPE dynamics. A high resolution OCT was constructed to achieve dynamic imaging of frog eyes, in which light-driven translocation of RPE melanosomes occurred within the RPE cell body and apical processes. Comparative histological examination of dark- and light-adapted eyes indicated that the RPE melanin granule, i.e., melanosome, was a primary OCT correlate. In vivo OCT imaging of RPE melanosomes opens the opportunity for quantitative assessment of RPE abnormalities associated with disease, and enables longitudinal investigation of RPE kinetics correlated with visual function.

Functional imaging of retinal photoreceptors and inner neurons using stimulus-evoked intrinsic optical signals.

Retinal development is a dynamic process both anatomically and functionally. High-resolution imaging and dynamic monitoring of photoreceptors and inner neurons can provide important information regarding the structure and function of the developing retina. In this chapter, we describe intrinsic optical signal (IOS) imaging as a high spatiotemporal resolution method for functional study of living retinal tissues. IOS imaging is based on near infrared (NIR) light detection of stimulus-evoked transient change of inherent optical characteristics of the cells. With no requirement for exogenous biomarkers, IOS imaging is totally noninvasive for functional mapping of stimulus-evoked spatiotemporal dynamics of the photoreceptors and inner retinal neurons.

In vivo confocal intrinsic optical signal identification of localized retinal dysfunction.

PURPOSE: The purposes of this study were to investigate the physiological mechanism of stimulus-evoked fast intrinsic optical signals (IOSs) recorded in dynamic confocal imaging of the retina, and to demonstrate the feasibility of in vivo confocal IOS mapping of localized retinal dysfunctions. METHODS: A rapid line-scan confocal ophthalmoscope was constructed to achieve in vivo confocal IOS imaging of frog (Rana pipiens) retinas at cellular resolution. In order to investigate the physiological mechanism of confocal IOS, comparative IOS and electroretinography (ERG) measurements were made using normal frog eyes activated by variable-intensity stimuli. A dynamic spatiotemporal filtering algorithm was developed to reject the contamination of hemodynamic changes on fast IOS recording. Laser-injured frog eyes were employed to test the potential of confocal IOS mapping of localized retinal dysfunctions. RESULTS: Comparative IOS and ERG experiments revealed a close correlation between the confocal IOS and retinal ERG, particularly the ERG a-wave, which has been widely used to evaluate photoreceptor function. IOS imaging of laser-injured frog eyes indicated that the confocal IOS could unambiguously detect localized (30 µm) functional lesions in the retina before a morphological abnormality is detectable. CONCLUSIONS: The confocal IOS predominantly results from retinal photoreceptors, and can be used to map localized photoreceptor lesion in laser-injured frog eyes. We anticipate that confocal IOS imaging can provide applications in early detection of age-related macular degeneration, retinitis pigmentosa, and other retinal diseases that can cause pathological changes in the photoreceptors.

Functional imaging of glucose-evoked rat islet activities using transient intrinsic optical signals.

We demonstrate intrinsic optical signal (IOS) imaging of intact rat islet, which consists of many endocrine cells working together. A near-infrared digital microscope was employed for optical monitoring of islet activities evoked by glucose stimulation. Dynamic NIR images revealed transient IOS responses in the islet activated by low-dose (2.75mM) and high-dose (5.5mM) glucose stimuli. Comparative experiments and quantitative analysis indicated that both glucose metabolism and calcium/insulin dynamics might contribute to the observed IOS responses. Further investigation of the IOS imaging technology may provide a high resolution method for ex vivo functional examination of the islet, which is important for advanced study of diabetes associated islet dysfunctions and for improved quality control of donor islets for transplantation.

Dynamic intrinsic optical signal monitoring of electrically stimulated inner retinal neural response.

Dynamic monitoring of stimulus-evoked inner neural response is important for functional validation of stimulation protocols of retinal prosthetic devices. In this paper, we demonstrate label-free intrinsic optical signal (IOS) imaging of electrically stimulated inner neural response in freshly isolated mouse retinas. While single-pulse stimulation evoked rapid IOS within 20 ms, pulse-train stimulation indicated that the fast IOS response can follow frequency stimulation up to at least 8 Hz. Fast IOS imaging promises a noninvasive method for high resolution examination of electrically evoked retinal response, without artifact contamination of electrical stimulus.

Investigation of the hyper-reflective inner/outer segment band in optical coherence tomography of living frog retina.

This study is to test anatomic correlates, including connecting cilium (CC) and inner segment (IS) ellipsoid, to the hyper-reflective band visualized by optical coherence tomography (OCT) and commonly attributed to the photoreceptor inner/outer segment (IS/OS) junction. A line-scan OCT (LS-OCT) was constructed to achieve sub-cellular resolution (lateral: ≈ 2 µm; axial: ≈ 4 µm) of excised living frog retinas. An electro-optic phase modulator was employed for rapid and vibration-free phase modulation. Comparison of normalized distance measurements between LS-OCT images and histological images revealed that the dominant source of the signal reported as the IS/OS OCT band actually originates from the IS.

Two-photon excited autofluorescence imaging of freshly isolated frog retinas.

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.

Intrinsic optical signal imaging of retinal activation.

Fast intrinsic optical signals (IOSs) correlated with stimulus-activated retinal responses are reviewed. Fast IOSs have a time course comparable to the stimulus-evoked electrophysiological kinetics of the retina, and thus promise a new methodology for high-resolution evaluation of the physiological health of the retina. However, practical application of fast IOSs for retinal study and diagnosis is challenging because of their low sensitivity and limited specificity. Using isolated amphibian retinas, a series of experiments to optimize and characterize fast IOSs has been conducted. Fast, high-resolution nearinfrared light imaging disclosed both positive (increasing) and negative (decreasing) optical responses in adjacent retinal areas, which satisfied spatial resolution essential to the differentiation of IOSs from opposite polarities. At the subcellular (approximately microm) level, fast IOSs often exceeded 5% DeltaI/I, where I is the dynamic optical change, and I is the background light intensity. Experiments with isolated frog retinas suggest that negative IOSs stem primarily from the photoreceptor layer, while positive IOSs come from inner retinal layers.