Ingo Schiessl

Neurovascular coupling investigated with two-dimensional optical imaging spectroscopy in rat whisker barrel cortex

Optical imaging slit spectroscopy is a powerful method for estimating quantitative changes in cerebral haemodynamics, such as deoxyhaemoglobin, oxyhaemoglobin and blood volume (Hbr, HbO2 and Hbt, respectively). Its disadvantage is that there is a large loss of spatial data as one image dimension is used to encode spectral wavelength information. Single wavelength optical imaging, on the other hand, produces high‐resolution spatiotemporal maps of brain activity, but yields only indirect measures of Hbr, HbO2 and Hbt. In this study we perform two‐dimensional optical imaging spectroscopy (2D‐OIS) in rat barrel cortex during contralateral whisker stimulation to obtain two‐dimensional maps over time of Hbr, HbO2 and Hbt. The 2D‐OIS was performed by illuminating the cortex with four wavelengths of light (575, 559, 495 and 587 nm), which were presented sequentially at a high frame rate (32 Hz). The contralateral whisker pad was stimulated using two different durations: 1 and 16 s (5 Hz, 1.2 mA). Control experiments used a hypercapnic (5% CO2) challenge to manipulate baseline blood flow and volume in the absence of corresponding neural activation. The 2D‐OIS method allowed separation of artery, vein and parenchyma regions. The magnitude of the haemodynamic response elicited varied considerably between different vascular compartments; the largest responses in Hbt were in the arteries and the smallest in the veins. Phase lags in the HbO2 response between arteries and veins suggest that a process of upstream signalling maybe responsible for dilating the arteries. There was also a consistent increase in Hbr from arterial regions after whisker stimulation.

Blind signal separation from optical imaging recordings with extended spatial decorrelation

Optical imaging is the video recording of two-dimensional patterns of changes in light reflectance from cortical tissue evoked by stimulation. We derived a method, extended spatial decorrelation (ESD), that uses second-order statistics in space for separating the intrinsic signals into the stimulus related components and the nonspecific variations. The performance of ESD on model data is compared to independent component analysis algorithms using statistics of fourth and higher order. Robustness against sensor noise is scored. When applied to optical images, ESD separates the stimulus specific signal well from biological noise and artifacts.

Four projection streams from primate V1 to the cytochrome oxidase stripes of V2

In the primate visual system, areas V1 and V2 distribute information they receive from the retina to all higher cortical areas, sorting this information into dorsal and ventral streams. Therefore, knowledge of the organization of projections between V1 and V2 is crucial to understand how the cortex processes visual information. In primates, parallel output pathways from V1 project to distinct V2 stripes. The traditional tripartite division of V1-to-V2 projections was recently replaced by a bipartite scheme, in which thin stripes receive V1 inputs from blob columns, and thick and pale stripes receive common input from interblob columns. Here, we demonstrate that thick and pale stripes, instead, receive spatially segregated V1 inputs and that the interblob is partitioned into two compartments: the middle of the interblob projecting to pale stripes and the blob/interblob border region projecting to thick stripes. Double-labeling experiments further demonstrate that V1 cells project to either thick or pale stripes, but rarely to both. We also find laminar specialization of V1 outputs, with layer 4B contributing projections mainly to thick stripes, and no projections to one set of pale stripes. These laminar differences suggest different contribution of magno, parvo, and konio inputs to each V1 output pathway. These results provide a new foundation for parallel processing models of the visual system by demonstrating four V1-to-V2 pathways: blob columns-to-thin stripes, blob/interblob border columns-to-thick stripes, interblob columns-to-palelateral stripes, layer 2/3–4A interblobs-to-palemedial stripes.

Spatial and Spectral Imaging of Retinal Laser Photocoagulation Burns

PURPOSE To correlate in vivo spatial and spectral morphologic changes of short- to long-pulse 532 nm Nd:YAG retinal laser lesions using Fourier-domain optical coherence tomography (FD OCT), autofluorescence (AF), fluorescein angiography (FA), and multispectral imaging. METHODS Ten eyes with treatment-naive preproliferative or proliferative diabetic retinopathy were studied. A titration grid of laser burns at 20, 100, and 200 milliseconds was applied to the nasal retina and laser fluence titrated to produce four grades of laser lesion visibility: subvisible (SV), barely visible (BV, light-gray), threshold (TH, gray-white), and suprathreshold (ST, white). The AF, FA, FD-OCT, and multispectral imaging were performed 1 week before laser, and 1 hour, 4 weeks, and 3 and 6 months post-laser. Multispectral imaging measured relative tissue oxygen concentration. RESULTS Laser burn visibility and lesion size increased in a linear relationship according to fixed fluence levels. At fixed pulse durations, there was a semilogarithmic increase in lesion size over 6 months. At 20 milliseconds, all grades of laser lesion were reduced significantly in size after 6 months: SV, 51%; BV, 54%; TH, 49%; and ST, 50% (P < 0.001), with retinal pigment epithelial proliferation and photoreceptor infilling. At 20 milliseconds, there was healing of photoreceptor inner segment/outer segment junction layers compared with 100- and 200-millisecond lesions. Significant increases in mean tissue oxygenation (range, four to six units) within the laser titration area and in oxygen concentration across the laser lesions (P < 0.01) were detected at 6 months. CONCLUSIONS For patients undergoing therapeutic laser, there may be improved tissue oxygenation, higher predictability of burn morphology, and more spatial localization of healing responses of burns at 20 milliseconds compared with longer pulse durations over time.

Orientation selectivity in the common marmoset (Callithrix jacchus): the periodicity of orientation columns in V1 and V2.

Orientation selectivity is a ubiquitous property of the primary visual cortex of mammals. Within the primate, orientation selectivity is arranged into vertical columns that are organized into a regular patchy pattern. Previous studies, in old world primates, have noted an anisotropy in this arrangement that appears to be due to the presence of ocular dominance columns within the same tissue. In addition, orientation selective responses appear to be arranged into bands of activity within the adjoining extrastriate region V2. Little is known about the precise arrangement of orientation columns within V2. In this study, we examined the layout of orientation columns within both V1 and V2 of a new world primate, the common marmoset, using optical imaging. New world primates have the advantage that, unlike the macaque, V2 exists on the cortical surface, a requirement for this form of optical mapping. We found the arrangement of orientation columns to be isotropic within marmoset V1 with an average repeat distance of around 575 mum, smaller than the repeat distance previously reported for the macaque. We found no evidence of ocular dominance within the animals tested supporting the claim that ocular dominance columns when present distort the mapping of orientation in V1. In V2 we found that orientation columns were larger and as in other primates were represented in discrete bands throughout V2. Orientation columns were spaced on average around 1 mm apart. This suggests that, at least in the marmoset, the visual system maps orientation at a different scale within V1 and V2.

High-resolution hyperspectral imaging of the retina with a modified fundus camera

PURPOSE to examine the practical feasibility of developing a hyperspectral camera from a Zeiss fundus camera and to illustrate its use in imaging diabetic retinopathy and glaucoma patients. METHODS the original light source of the camera was replaced with an external lamp filtered by a fast tunable liquid-crystal filter. The filtered light was then brought into the camera through an optical fiber. The original film camera was replaced by a digital camera. Images were obtained in normals and patients (primary open angle glaucoma, diabetic retinopathy) recruited at the Manchester Royal Eye Hospital. RESULTS a series of eight images were captured across 495- to 720-nm wavelengths, and recording time was less than 1.6s. The light level at the cornea was below the ANSI limits, and patients judged the measurement to be very comfortable. Images were of high quality and were used to generate a pixel-to-pixel oxygenation map of the optic nerve head. Frame alignment is necessary for frame-to-frame comparison but can be achieved through simple methods. CONCLUSIONS we have developed a hyperspectral camera with high spatial and spectral resolution across the whole visible spectrum that can be adapted from a standard fundus camera. The hyperspectral technique allows wavelength-specific visualization of retinal lesions that may be subvisible using a white light source camera. This hyperspectral technique may facilitate localization of retinal and disc pathology and consequently facilitate the diagnosis and management of retinal disease.

Optical imaging of the retinotopic organization of V1 in the common marmoset

We examined the retinotopic mapping of the visual world in the primary visual cortex of the marmoset monkey using differential optical imaging. Two sets of complementary stripe-like locations were visually stimulated in turn. Their difference depicts the cortical representations of continuous bands of visual space. By rotating the sets of stripe-like locations it is possible to map different spatial axes. Analogous to the macaque we found that the V1/V2 border represented the vertical meridian, while horizontal, 45-, and 135-degree angled stripes of space were also represented in a continuous manner. We developed a new automatic method of calculating local measures of cortical magnification from our optical retinotopic maps. Using this method we found no evidence of any local anisotropies in cortical representation. Overall our results indicate that space is mapped isotropically in the primary visual cortex of the common marmoset.

Delayed reperfusion deficits after experimental stroke account for increased pathophysiology.

Cerebral blood flow and oxygenation in the first few hours after reperfusion following ischemic stroke are critical for therapeutic interventions but are not well understood. We investigate changes in oxyhemoglobin (HbO2) concentration in the cortex during and after ischemic stroke, using multispectral optical imaging in anesthetized mice, a remote filament to induce either 30 minute middle cerebral artery occlusion (MCAo), sham surgery or anesthesia alone. Immunohistochemistry establishes cortical injury and correlates the severity of damage with the change of oxygen perfusion. All groups were imaged for 6 hours after MCAo or sham surgery. Oxygenation maps were calculated using a pathlength scaling algorithm. The MCAo group shows a significant drop in HbO2 during occlusion and an initial increase after reperfusion. Over the subsequent 6 hours HbO2 concentrations decline to levels below those observed during stroke. Platelets, activated microglia, interleukin-1α, evidence of BBB breakdown and neuronal stress increase within the stroked hemisphere and correlate with the severity of the delayed reperfusion deficit but not with the ΔHbO2 during stroke. Despite initial restoration of HbO2 after 30 min MCAo there is a delayed compromise that coincides with inflammation and could be a target for improved stroke outcome after thrombolysis.

Relationships between visual field sensitivity and spectral absorption properties of the neuroretinal rim in glaucoma by multispectral imaging.

PURPOSE To investigate the relationship between neuroretinal rim (NRR) differential light absorption (DLA, a measure of spectral absorption properties) and visual field (VF) sensitivity in primary open-angle glaucoma (POAG). METHODS Patients diagnosed with (n = 22) or suspected of having (n = 7) POAG were imaged with a multispectral system incorporating a modified digital fundus camera, 250-W tungsten-halogen lamp, and fast-tuneable liquid crystal filter. Five images were captured sequentially within 1.0 second at wavelengths selected according to absorption properties of hemoglobin (range, 570-610 nm), and a Beer-Lambert law model was used to produce DLA maps of residual NRR from the images. Patients also underwent VF testing. Differences in NRR DLA in vertically opposing 180° and 45° sectors either side of the horizontal midline were compared with corresponding differences in VF sensitivity on both decibel and linear scales by Spearmans rank correlation. RESULTS The decibel VF sensitivity scale showed significant relationships between superior-inferior NRR DLA difference and sensitivity differences between corresponding VF areas in 180° NRR sectors (Spearman ρ = 0.68; P < 0.0001), superior-/inferior-temporal 45° NRR sectors (ρ = 0.57; P < 0.002), and superior-/inferior-nasal 45° NRR sectors (ρ = 0.59; P < 0.001). Using the linear VF sensitivity scale significant relationships were found for 180° NRR sectors (ρ = 0.62; P < 0.0002) and superior-inferior-nasal 45° NRR sectors (ρ = 0.53; P < 0.002). No significant difference was found between correlations using the linear or decibel VF sensitivity scales. CONCLUSIONS Residual NRR DLA is related to VF sensitivity in POAG. Multispectral imaging may provide clinically important information for the assessment and management of POAG.

Orientation-Cue Invariant Population Responses to Contrast-Modulated and Phase-Reversed Contour Stimuli in Macaque V1 and V2

Visual scenes can be readily decomposed into a variety of oriented components, the processing of which is vital for object segregation and recognition. In primate V1 and V2, most neurons have small spatio-temporal receptive fields responding selectively to oriented luminance contours (first order), while only a subgroup of neurons signal non-luminance defined contours (second order). So how is the orientation of second-order contours represented at the population level in macaque V1 and V2? Here we compared the population responses in macaque V1 and V2 to two types of second-order contour stimuli generated either by modulation of contrast or phase reversal with those to first-order contour stimuli. Using intrinsic signal optical imaging, we found that the orientation of second-order contour stimuli was represented invariantly in the orientation columns of both macaque V1 and V2. A physiologically constrained spatio-temporal energy model of V1 and V2 neuronal populations could reproduce all the recorded population responses. These findings suggest that, at the population level, the primate early visual system processes the orientation of second-order contours initially through a linear spatio-temporal filter mechanism. Our results of population responses to different second-order contour stimuli support the idea that the orientation maps in primate V1 and V2 can be described as a spatial-temporal energy map.