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Brain activity has been investigated by several methods with different principles, notably …

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- Optical and electrical recording of neural activity evoked by graded contrast visual stimulus

A. Measuring principle

Near infrared spectroscopy and electroencephalography have a complementary role in extracting spatial and temporal information on direct (electrical) or indirect (metabolic) neural activity. Near infrared spectroscopy, which measures the light absorption spectra of hemoglobin, directly measures changes in blood volume and blood oxygenation supporting neural activity [4]. Electroencephalographic technique is sensitive to variations in electrical potential in brain tissue caused by neuron depolarization and repolarization [2]. These methods reflect different aspects of the underlying physiological mechanisms, which include both vascular metabolic and electrophysiological processes mutually interacting in the visual cortex during visual stimulation.

B. Near infrared spectroscopy

Near Infrared Spectroscopy (NIRS) is a well-known optical technique first proposed by Franz Jöbsis in 1977 for in-vivo investigation of tissue oxygenation [6]. NIRS is based on the transparency of tissue to light in the spectral range between 700 and 1000 nm combined with the chromophore content of tissues: mainly water, deoxyhemoglobin and oxyhemoglobin.

It has been well documented that NIRS is a useful technique for investigating cerebral hemodynamic changes in humans [4]. However, due to the relatively high scattering of the skull and white matter coefficients, it is difficult for NIR photons to penetrate the head for a depth greater than few centimetres. For this reason, NIRS is essentially limited to assessing cortical function [7,8].

The hemodynamic changes measured with NIRS are often called slow signals because they occur within seconds after brain activity begins. It has also known that NIRS has the capability to non-invasively measure neural activity [9-11]. Compared with the slow hemodynamic response, the neural signal (named "fast signal") occurs within milliseconds after stimulation. Researchers suggest that this signal originates from action potentials and the consequent swelling of the neural cells or from a brief period of anaerobic metabolism that somehow alters tissue transparency [12].

Theory describing the migration of NIR photons through tissue is widely discussed in the literature [13-16]. The most accurate model is based on the photon diffusion equation that can be solved assuming that photons in tissue perform a random walk dominated by the high value of the scattering coefficient. This assumption is reasonable in near-infrared spectral region since the transport scattering coefficient μ's(λ) is about 50 times larger than the absorption coefficient μa(λ) in human tissues.

In our previous paper [17], we proposed to combine this model with the knowledge of water concentration in human tissue. This approach allows the absolute variations of μa(λ) and μ's(λ) as well as changes in oxygenated and deoxygenated hemoglobin concentration (Δ[HbO2] and Δ[HHb], respectively) to be measured according to the following system of equations:


where λ1, λ2 and λ3 represent the wavelengths of the photons used to explore the tissue, whereas αHbO2 and αHHb are the extinction coefficients of oxyhemoglobin and deoxyhemoglobin, respectively. The solution of this system is achieved by minimizing the least square errors.

C. Electroencephalography

ElectroEncephaloGraphy (EEG) is a common technique for recording the scalp surface electrical signal originating from electrical activity of the underlying brain structures. It includes complex information coming from all cerebral areas, and the signal amplitude, due to various somato-sensorial stimulation, is maximal for electrodes located around the {associated/underlying} brain area. Essentially, the occipital area produces a response, as an EEG variation, for visual stimulation. Consequently, an international standardization for electrode placement has been introduced with the 10–20 System: the electrode positions O1 and O2 correspond to the left and right occipital hemisphere, respectively. A total visual field stimulation induces a similar response in both locations, which can be used to study visual functioning.

However, when attempting to extract the visual response signal from the EEG, the very low signal-to-noise ratio imposes the need to acquire several EEG recordings to obtain reliable data and to process the pooled signals by means of averaging techniques to detect and evaluate specific evoked signals. Therefore, to improve the signal-to-noise ratio, the brain response must not be corrupted by other synchronous neural activities during boxcar averaging. When the sensory system involved in stimulation is the visual system, the response obtained from EEG through averaging is defined Visual Evoked Potential (VEP). The averaging algorithm implies the need for repetitive stimulation; depending on the stimulation intensity, normally 40 to 100 stimulations are needed to recover the VEP. The stimulation intensity is normally denoted according to some parameter of the stimulus applied; in our case we chose contrast, defined as Math, where L1 and L2 are the luminance values of adjacent areas of the stimulus. The VEP is interpreted as a function of time, VEP(t); its RMS value is defined as:


where VEPk (k = 1,..., N) are the discrete values of the VEP after A/D conversion. VEPrms is the parameter strictly related to variation of the stimulus contrast, as we will see.

D. System description

1. Block diagram and basic functions

The block diagram of the developed measuring setup is shown in Figure 1. It consists of four main blocks: (a) optical unit, (b) electrical unit, (c) visual stimulation and electro-optical recording interface, and (d) control unit.

2. Optical unit

In our previous paper [17], to overcome coupling between the scattering and absorption coefficients, we described a NIR-CWS instrument that allows quantitative assessments exploiting precise absorption measurements close to 975 nm, the absorption peak of water. Moreover, this system exploits an original detection scheme based on a time variant filter that approaches optimum shaping (in terms of signal to noise ratio) and has good rejection of photodetector offset, ambient light, and signal fluctuations due to probe movements. In this study we use the commercial release of our system produced by a spin-off company of our University: NIROX srl.

To monitor two types of blood chromophores, we employed three laser diodes emitting different wavelengths in the near-infrared spectral region. The emitted radiation from each laser diode source is collimated into a single fiber. The back-diffused light is collected by a 3 mm diameter liquid optical guide (VIS-NIR 77634, Oriel Instruments, Germany).

The receiver unit was designed to maximize the signal-to-noise ratio (SNR), reject continuous and alternate ambient light and reduce the effects of artefacts induced by optical head movement. It included a band-pass optical filter (650–1000 nm), high-sensitivity large area avalanche photodiode module (C5460-01, Hamamatsu, Japan), shaping network described in our previous paper [18], A/D converter (27.7 Hz) and USB interface to the PC.

3. Electrical unit

The EEG signals were acquired using a cap with Ag/AgCl electrodes, amplified through a custom analog conditioning system (0.1–100 Hz bandpass filter at 12 dB/octave, gain of 50.000) and digitized at 250 Hz per channel, through an A/D NI card (AT-MIO16E) plugged into the PC. The software, based on the abWindows/CVI NI platform, enables control and display of the acquired EEG and processed VEP signals in real time.

4. Electro-optical recording interface and visual stimulation

Great care was devoted to develop a suitable and compliant optical head. The design guidelines were focused on obtaining optimal fiber-to-skin coupling to avoid excessive pressure and motion artefacts. Moreover, since the optimal measuring position and fiber placement are not known a priori, we designed a variable fiber harness that allows different source-detector configurations and precise placement on the brain region of interest. The developed optical head consists of two aluminum elements: a Hollow Cube (HC) fixed on a polystyrene helmet and a mobile Fiber Holder (FH). The HC offers a matrix of 8 × 8 different horizontal and vertical locations. This geometry allows 16 different vertical sites and 16 horizontal sites to be measured over the occipital area, with a distance between two consecutive locations of 0.6 cm. This hollow cube was designed to orient the fiber harness and thus fibers orthogonal to the skin surface. The FH element can be positioned and fixed to the HC element using a pair of screws.

The FH can hold up to four illumination multimode fibers and a collection liquid guide, as illustrated in Figure 2. Its surface is black to prevent unwanted back reflected light. To prevent discharge of the fibers from the optical head once in contact with the tissue, we inserted an o-ring for each fiber between the FH and a fixing Aluminum Plate (AP in Figure 2).

As shown in Figure 3, a modified EEG-cap was designed with a hole in the right side to house the CW-NIRS optical head over the occipital area.

Stimuli are generated through a VSG2/2 (CRS ltd) video card driven by custom software enabling control of luminance, contrast, spatial frequency and reversal frequency.

The stimulus selected for the experiment is a windmill pattern, presenting decreasing spatial frequency content from the center to periphery, to match retinal functional organization. This pattern is projected on a large screen (1152 × 864 mm) by an LCD projector at an angle of 30 degrees to the subject's eyes. Optical stimulation consists of the reversal, at constant overall luminance, of a black and white (b/w) checkered pattern at a rate of 8 Hz.

5. Control unit and signal processing

The supervisor software provides timing signals for the start and duration of the stimulation and rest periods and controls the signal acquisition of both the CW-NIRS and EEG systems. The system is composed of two modules. One module is dedicated to visual stimulation management; through a series of panels it enables the user to drive a Cambridge Instruments VSG/2 card installed on a PC to display the spatial frequency alternating at 8 Hz with appropriate intensity and contrast on a monitor. The second module runs CW-NIRS and EEG acquisitions and performs digital signal filtering.

E. Experimental protocol

Nine subjects ranging in age from 20 to 57 years were examined. Each volunteer gave informed consent to participate in our study and was adequately informed on the protocol details.

All measurements were performed while subjects were lying in a quiet room with dimmed light; after visual adaptation, the experimental procedure was started as soon as a stable baseline for both oxyhemoglobin and deoxyhemoglobin concentration was reached.

Subjects were given different visual patterns. Because of the symmetry of the pattern stimulus in the visual field, similar VEP responses are obtained in the right and left brain hemisphere among healthy subjects [19]. Thus the EEG-cap was positioned under the polystyrene helmet and the occipital EEG channel was placed in the standard O1 location, whereas the CW-NIRS optical head was positioned over the standard O2 location. This recording layout is shown in Figure 3, considering that the calcarine sulcus varies widely in relation to cranial landmarks [20]. Therefore, the optical probe was placed vertically over the right occipital region at the level of the calcarine sulcus. The emitting fiber was positioned 1 cm to the right of the midline to avoid the sagittal sinus, and 3.5 cm from the collection liquid guide. Exploiting the different locations available in the HC fixed in the helmet, we determined that this was the best geometry to record signals associated with visual cortex activity.

All measurements were performed while subjects were lying in front of the screen. During the resting periods, a grey screen with the same total luminance of the stimulation pattern and with a central fixation cross was displayed. The subjects were asked to focus on the center of the monitor throughout the experiment.

The experimental protocol consisted in four measurement epochs. Each epoch included a twofold presentation: a 60 second "rest" period without stimulation, followed by the inverting windmill pattern, lasting 60 seconds at a frequency of 8 Hz. Figure 4 illustrates the four measurement epochs and the corresponding visual stimulation.

The relationship between stimulus contrast and VEP amplitude is well known [21]. Our aim was to verify if a similar relationship holds between hemodynamic response and contrast parameter; so we presented each subject three stimulus-contrast levels. We assumed the maximum contrast achievable from our LCD projector to be 100%; the contrast value could be varied at 1% and 10% by the commands of the VSG/2 graphic card software.

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