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Exp Neurobiol 2013; 22(3): 158-166
Published online September 30, 2013
© The Korean Society for Brain and Neural Sciences
Shin Ae Kim1 and Sang Beom Jun2,3*
1School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA30332, United States, Departments of 2Electronics Engineering, 3Brain and Cognitive Sciences, Ewha Womans University, Seoul 120-750, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-3277-3892, FAX: 82-2-3277-3494
The optical neural recording techniques are promising tools in recent years. Compared to the traditional electrophysiological recording, the optical means offer several advantages including no inclusion of electrical noise, simultaneous imaging of a large number of neurons, or selective recording from genetically-targeted neurons. Overall the optical neural recording technique comprises the intrinsic and the extrinsic optical recordings. The methods for intrinsic neural recording employ the change of optical properties in brains such as blood flow/oxygenation, cellular volume change, or refractive index change without addition of external indicators. Those properties can be detected using various optical techniques including laser Doppler flowmetry (LDF), near-infrared (NIR) spectrometer, functional optical coherence tomography (fOCT), and surface plasmon resonance (SPR). The extrinsic monitoring techniques use fluorescence signals reflecting neuronal activity via chemical or genetic modification of the neuronal cells. Two most popular activity-dependent fluorescent probes, calcium indicators and voltage-sensitive fluorescent proteins will be examined in this review. The principles, the instrumentations and
Keywords: optical neural recording, intrinsic optical recording, extrinsic optical recording, calcium indicators, voltage-sensitive, fluorescent protein (VSFP)
Mammalian brains process complex information including sensations, movements and complicated cognitive functions using sequences of action potentials in a large population of neurons. To process multiple signals simultaneously, brains take advantage of neural networks consisting of more than ten billion neurons connecting to one another. The individual neurons are known to have synaptic connections to hundreds or thousands of other neurons . These intricate connections make it difficult to study functions of individual neurons or connections. Furthermore, some of the functional regions are located in the deep brain regions such as thalamus, hypothalamus, hippocampus and so on [2-4].
Various technologies have been introduced for neural recording from the mammalian brain
Fiber-optic technique allows minimally invasive imaging from the cortex to the deep-brain. The fibers typically include a transparent core surrounded by a transparent cladding material with a lower index of refraction and the difference of the index induces the total internal reflection. The total internal reflection causes the fibers to act as a light-guide to transmit light between the two separate ends of the fiber. Use of the fibers for
Here, we briefly review the representative fiber optic techniques for
Laser doppler flowmetry (LDF) is a method measuring cerebral blood flow. Cerebral blood flow correlates well with brain activity enabling for functional neuroimaging target. When low-power laser light (780 nm, ~1.6 mW) is emitted on the brain, the light is backscattered in dynamic red blood cells as well as in static tissues [27-29]. The light in the measuring volume of tissue is a mixture of unshifted and Doppler-shifted signal from static tissue and dynamic blood cells, respectively. The fraction of the shifted signal corresponds to the moving blood cells in the measuring region. Therefore, the magnitude and frequency distribution of the backscattered light relates to the number and velocity of moving blood cells within the measuring volume of tissue.
The first fiber-optic LDF system was presented in 1977 by Holloway and Watkins , and various improved versions have been successfully applied to brain imaging in various species such as rodent [27, 31], pig , and primate [33, 34]. In measurements of the brain function, the LDF takes advantage of the real-time continuous imaging [27, 35]. The LDF is also non-invasive technique, not disturbing the nature physiological state of microcirculation because the LDF does not require a tight contact with target tissue. Moreover, by implementing LDF with optic fibers, it is available to access the brain without fine alignment control for same light-pathway between two measuring points.
Near-infrared (NIR) spectroscopy is performed for the non-invasive brain research because the brain is relatively transparent in the NIR range between 700~1,000 nm [19, 36-38]. The transparency comes from relatively small absorption of both water and hemoglobin within this wavelength region, so called 'optical window'. The increase in the cerebral blood flow exceeding the increase in oxygen consumption leads to an increase of intravascular hemoglobin oxygenation during brain activity. The NIR light has a characteristic absorption in both oxygenated hemoglobin and deoxygenated hemoglobin [19, 36-39]. Therefore based on the NIR absorption measurements, concentration changes of oxygenated hemoglobin and deoxygenated hemoglobin can be measured during functional brain activation.
The concept that brain activity is related to intrinsic microvascular changes was asserted more than a century ago by Roy and Sherrington , and their assertion was demonstrated through modern imaging techniques [1, 19, 26, 36-39, 41, 42]. Jöbsis firstly demonstrated the possibility of NIR spectroscopy to detect changes of cortical oxygenation during hyperventilation on an adult brain . Even though the lack of spatial resolution and attenuated NIR signal by the layering from the cortical surface remain major problems to be overcome [38, 43], the NIR images can be obtained in undemanding set-ups without invasive surgery and applicable to bedside use for human brain research. In addition, the fiber-optic NIR spectroscopy systems are commercially available. Further detail of recent NIR spectroscopy in terms of features, strengths, advantages, and limitations are reviewed in Quaresima et al. .
Optical coherence tomography (OCT) is an interferometric technique that measures scattering light from the tissue with NIR. Although standard OCT measures architectural tissue morphology, functional optical coherence tomography (fOCT) detects optical changes in cells and tissues during physiological events related to neuronal activation. The fOCT provides two-dimensional, depth-integrated activation maps of brain activity by imaging the scattering changes during the neuronal activation [25, 44, 45]. The scattering changes contain two types of characterized signals: (1) a fast signal (less than a millisecond) from the refractive index changes at neuronal membranes by ionic composition changes; (2) a slow signal (few seconds) from the geometry changes of neuronal compartments or organelles by osmotic volume changes [25, 36]. Both types of signals are reported with fOCT during neuronal activation in a brain.
fOCT can be used in noninvasive imaging with high spatiotemporal resolution. Because fOCT implements NIR as its light source, large penetration depth of NIR allows noninvasive imaging of neural activity in the brain. In addition, fOCT has better spatial resolution for detecting scattered signals than NIR spectroscopy with high sensitivity to small optical changes . Combination with other technologies such as polarization sensitive OCT (PS-OCT) and multi-functional OCT (MF-OCT) has been providing more various information on tissue properties than standard OCT imaging alone.
A surface plasmon resonance (SPR) sensor has been also demonstrated as a recording tool of neural activity-dependent signals through the experimental approaches [18, 24, 47] as well as numerical approaches [48, 49]. SPR sensors have a high sensitivity about the refractive index change on a thin metal layer because they employ surface electromagnetic evanescent waves at the metaldielectric interface. The SPR signal results from the integrated refractive index changes within a very small measurement volume and the refractive index can be altered by a change in the ionic composition and/or fractional volume change between different organelles. Although the SPR mechanism underlying these fast intrinsic optical responses is not clearly understood yet, it is proven that the optical SPR signals were originated from neural activities evoked by stimulation with simultaneous electrical recording and pharmacological analysis [18, 24].
SPR technique was also demonstrated for
Activity-related intrinsic changes associated with neural activities or metabolisms have been used for cortical functional mapping since the 1980s. Aside from previous four imaging techniques various IOS imaging techniques have been developed (e.g., functional photoacoustic microscopy, NIR spectrometry, diffuse optical tomography, etc.) and the popularity of IOS imaging is increased together with technical advancement.
IOS offers distinct advantages over extrinsic signal imaging. By not requiring any chemicals or virus insertion for labeling, IOS imaging is ideally suited to study chronic preparations and clinical applications. While high-level long-term expression of fluorescent probes can lead to photo-toxicity , IOS address the problems by measuring the natural physiology of the brain activities. Moreover, IOS imaging techniques do not require the removal of chemicals or micro-beads after imaging from the brain, and they are thereby clinically applicable . Therefore, IOS imaging for intraoperative mapping in humans provides an unparalleled opportunity to examine the basic physiology and organization of the functioning human brain. The opportunity exists to study questions that are impossible in other species, such as language organization and higher cognitive functions. However, integrated signal over the measuring volume is a limit of IOS. Because the reflected signals are averaged over the volume, IOS does not allow specific target cell detection without precise manipulation. Also, since IOS is low in signal to noise ratio, higher sensitive systems are required for better interpretation of the brain activity.
In the nervous system, the intracellular calcium concentration plays an important role in a variety of means. In presynaptic terminals, exocytosis of neurotransmitter is triggered by calcium influx. In postsynaptic regions in dendritic spines, the calcium transient is involved in synaptic plasticity. More importantly, calcium signaling in nucleus is known to regulate the gene transcriptions . At the resting state, the intracellular calcium concentration is maintained less than 100 nM in most neurons while the concentration rises around 10 to 100 times higher during the generation of action potentials . Therefore, the recording of somatic calcium signal is commonly used to monitor the action potentials of the neurons.
In order to monitor the neural activity-dependent calcium signal, there have been a number of attempts to develop appropriate optical probes expressing the intracellular calcium signals as fluorescence changes. The first calcium indicator was employing calcium-binding photoproteins such as aequorin . Later, more sensitive fluorescent calcium indicators were developed using the binding reaction between a fluorescent chromophore and calcium-selective chelators like EGTA or BAPTA . Based on this mechanism, to date, several calcium indicators with different excitation spectra and different affinities to calcium have been introduced including quin-2, fura-2, fluo-3, fluo-4, Oregon Green Bapta and so on [58-61]. Since these dyes are easy to use and have large signal to nose ratio, these dyes are now widely used in neurophysiological studies.
In order to load the calcium indicators into the intracellular domain, in early days, the dyes were delivered inside the individual neurons using a sharpened microelectrode, glass patch micropipette or electroporation. These techniques are still widely used for basic studies of calcium signaling in neurons. To load the dyes into a population of neurons simultaneously, the most popular method is the use of membrane-permeable acetoxymethyl (AM) ester forms. The AM calcium dye molecules are trapped inside the cells after the hydrophobic eater residues are removed during the entry into the cytosol. As the application for
The true breakthrough of the use of calcium indicator as an optical probe for neural activity was the introduction of genetically encoded calcium indicators (GECIs). GECIs enable the measurement of the cytosolic calcium concentration from the genetically identified neurons. Among several different methods to express GECIs, the most popular one is currently the use of viral transduction using stereotaxic injection into the targeted brain areas. Among various viral vectors, lenti-, adeno- and adeno-associated viral vectors are commonly used to deliver the GECI constructs into the neurons of interest [62, 63]. More importantly, the specific expression is achieved via the use of the specific promoters or the transgenic animals with the Cre recombinase driver . In addition, the production of transgenic mice with GECI expression has been also attempted recently because it would facilitate the experiment procedure tremendously . Even though there have been a few successes, the generation of GECI transgenic animal is not always possible for the specific types of neurons.
Voltage-sensitive probes are the fluorescence dyes which change their intensity in response to the exerted voltage changes . Since lots of physiological processes including action potentials are accompanied with changes in the membrane potential, the fluorescence measurement can be used as an optical indicator if the dye molecules are properly anchored at the membrane. In principle, the voltage-sensitive fluorescent protein (VSFP) has a paired structure of hydrocarbon chains as the anchors and a hydrophilic group for alignment of the chromophore. It is believed that the chromophore shows an electrochromic mechanism during change of the electric field. Compared to calcium indicators, voltage-sensitive fluorescence proteins were first introduced more lately and highlighted due to the fast response time and the linear measurement of membrane potential.
Recently, the successful genetically encoded voltage-sensitive probes were reported based on the insertion of VSFP into voltage-gated potassium or sodium channels . Even though those probes were able to express fluorescence change proportional to the membrane potential, the efficiency of targeting specific membrane was not stable and the signal to noise ratio was not high enough to be detected
The instrumentation for the extrinsic fluorescence detection requires the light-sensing device and the appropriate light source for the excitation. Unlike
Other than the methods mentioned above, various optical techniques can be applied for