This PDF file contains the editorial “Neurophotonics Pioneers and News” for NPh Vol. 2 Issue 02

This PDF file contains the editorial “Neurophotonics 2014 List of Reviewers” for NPh Vol. 2 Issue 02

Noninvasive imaging of brain function has become an indispensable pillar of neuroscience and clinically motivated research on brain disease. Despite the undisputed strength ofMRI-basedtechniques,lessdemandingandbroadlyaffordable methods may be necessary to complement and expand the scope of neuroimaging from a bulky scanner to the bedside or into ecologically meaningful environments. Nearinfrared spectroscopy has clearly developed into one of the most exciting options increasingly used by cognitive and developmental researchers, neonatologists, anesthesiologists, neurologists, and psychiatrists. In concert with electrophysiological and other vascular-based imaging techniques, NIRS may well serve to link basic research to clinical challenges

Functional near-infrared spectroscopy (fNIRS) is an optical imaging method that is used to noninvasively measure cerebral hemoglobin concentration changes induced by brain activation. Using structural guidance in fNIRS research enhances interpretation of results and facilitates making comparisons between studies. AtlasViewer is an open-source software package we have developed that incorporates multiple spatial registration tools to enable structural guidance in the interpretation of fNIRS studies. We introduce the reader to the layout of the AtlasViewer graphical user interface, the folder structure, and user files required in the creation of fNIRS probes containing sources and detectors registered to desired locations on the head, evaluating probe fabrication error and intersubject probe placement variability, and different procedures for estimating measurement sensitivity to different brain regions as well as image reconstruction performance. Further, we detail how AtlasViewer provides a generic head atlas for guiding interpretation of fNIRS results, but also permits users to provide subject-specific head anatomies to interpret their results. We anticipate that AtlasViewer will be a valuable tool in improving the anatomical interpretation of fNIRS studies.

This PDF file contains the editorial “Pioneers in Neurophotonics: Special Section Honoring Professor Lawrence B. Cohen” for NPh Vol. 2 Issue 02

This PDF file contains the editorial “Personal recollections: regarding the pioneer days of optical recording of membrane potential using voltage-sensitive dyes” for NPh Vol. 2 Issue 02

This PDF file contains the editorial “Larry Cohen, indomitable pioneer” for NPh Vol. 2 Issue 02

This PDF file contains the editorial “Larry Cohen—50 ways to DYE your science” for NPh Vol. 2 Issue 02

Dynamic calcium and voltage imaging is a major tool in modern cellular neuroscience. Since the beginning of their use over 40 years ago, there have been major improvements in indicators, microscopes, imaging systems, and computers. While cutting edge research has trended toward the use of genetically encoded calcium or voltage indicators, two-photon microscopes, and in vivo preparations, it is worth noting that some questions still may be best approached using more classical methodologies and preparations. In this review, we highlight a few examples in neurons where the combination of charge-coupled device (CCD) imaging and classical organic indicators has revealed information that has so far been more informative than results using the more modern systems. These experiments take advantage of the high frame rates, sensitivity, and spatial integration of the best CCD cameras. These cameras can respond to the faster kinetics of organic voltage and calcium indicators, which closely reflect the fast dynamics of the underlying cellular events.

In neocortical pyramidal neurons, action potentials (APs) propagate from the axon into the dendritic tree to influence distal synapses. Traditionally, AP backpropagation was studied in the thick apical trunk. Here, we used the principles of optical imaging developed by Cohen to investigate AP invasion into thin dendritic branches (basal, oblique, and tuft) of prefrontal cortical L5 pyramidal neurons. Multisite optical recordings from neighboring dendrites revealed a clear dichotomy between two seemingly equal dendritic branches belonging to the same cell (“sister branches”). We documented the variable efficacy of AP invasion in basal and oblique branches by revealing their AP voltage waveforms. Using fast multisite calcium imaging, we found that trains of APs are filtered differently between two apical tuft branches. Although one dendritic branch passes all spikes in an AP train, another branch belonging to the same neuron, same cortical layer, and same path distance from the cell body, experiences only one spike. Our data indicate that the vast differences in dendritic voltage and calcium transients, detected in dendrites of pyramidal neurons, arise from a nonuniform distribution of A-type K+ conductance, an aggregate number of branch points in the path of the AP propagation and minute differences in dendritic diameter.

Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity. By shaping light onto neighboring axons and dendrites, we observed that dendritic back-propagating action potentials were broader and slowly rising compared with axonal action potentials, differences not measured in the same structures illuminated with a large “pseudowidefield” (pWF) spot of the same excitation density. Shaped illumination trials showed reduced baseline fluorescence, higher baseline noise, and fractional fluorescence transient amplitudes two times greater than trials acquired with pWF illumination of the same regions.

The invention of membrane voltage protein indicators widens the reach of optical voltage imaging in cell physiology, most notably neurophysiology, by enabling membrane voltage recordings from genetically defined cell types in chronic and life-long preparations. While the last years have seen a dramatic improvement in the technical performance of these indicators, concomitant innovations in optogenetics, optical axon tracing, and high-speed digital microscopy are beginning to fulfill the age-old vision of an all-optical analysis of neuronal circuits, reaching beyond the limits of traditional electrode-based recordings. We will present our personal account of the development of protein voltage indicators from the pioneering days to the present state, including their applications in neurophysiology that has inspired our own work for more than a decade.

Investigating the developmental organization of the embryonic nervous system is one of the major challenges in the field of neuroscience. Despite their significance, functional studies on the vertebrate embryonic central nervous system (CNS) have been hampered by the technical limitations associated with conventional electrophysiological methods. The advent of optical techniques using voltage-sensitive dyes, which were developed by Dr. Cohen and his colleagues, has enabled electrical activity in living cells to be monitored noninvasively and also facilitated the simultaneous recording of neural responses from multiple regions. Using optical recording techniques, it is now possible to follow the functional organization of the embryonic CNS and image the spatiotemporal dynamics involved in the formation of this neural network. We herein briefly reviewed optical studies on the embryonic CNS with a special emphasis on methodological considerations and the study of neuronal circuit formation, which demonstrates the utility of fast voltage-sensitive dye imaging as a powerful tool for elucidating the functional organization of the embryonic CNS.

The combination of fluorescence measurements of membrane potential and intracellular Ca²⁺ concentration allows correlating the electrical and calcium activity of a cell with spatial precision. The technical advances allowing this type of measurement were achieved only recently and represent an important step in the progress of the voltage imaging approach pioneered over 40 years ago by Lawrence B. Cohen. Here, we show how this approach can be used to investigate the function of Ca²⁺ channels using the foreseen possibility to extract Ca²⁺ currents from imaging experiments. The kinetics of the Ca²⁺ current, mediated by voltage-gated Ca²⁺ channels, can be accurately derived from the Ca²⁺ fluorescence measurement using Ca²⁺ indicators with K_D>10  μM that equilibrate in <1  ms. In this respect, the imaging apparatus dedicated to this application is described in detail. Next, we illustrate the mathematical procedure to extract the current from the Ca²⁺ fluorescence change, including a method to calibrate the signal to charge flux density. Finally, we show an example of simultaneous membrane potential and Ca²⁺ optical measurement associated with an action potential at a CA1 hippocampal pyramidal neuron from a mouse brain slice. The advantages and limitations of this approach are discussed.

We explore the feasibility of obtaining a spatially resolved picture of Ca²⁺ inward currents (I_Ca ) in multicellular cardiac tissue by differentiating optically recorded Ca²⁺ transients that accompany propagating action potentials. Patterned growth strands of neonatal rat ventricular cardiomyocytes were stained with the Ca²⁺ indicators Fluo-4 or Fluo-4FF. Preparations were stimulated at 1 Hz, and Ca²⁺ transients were recorded with high spatiotemporal resolution (50  μm, 2 kHz analog bandwidth) with a photodiode array. Signals were differentiated after appropriate digital filtering. Differentiation of Ca²⁺ transients resulted in optically recorded calcium currents (ORCCs) that carried the temporal and pharmacological signatures of L-type Ca²⁺ inward currents: the time to peak amounted to ∼2.1  ms (Fluo-4FF) and ∼2.4  ms (Fluo-4), full-width at half-maximum was ∼8  ms, and ORCCs were completely suppressed by 50  μmol/LCdCl₂. Also, and as reported before from patch-clamp studies, caffeine reversibly depressed the amplitude of ORCCs. The results demonstrate that the differentiation of Ca²⁺ transients can be used to obtain a spatially resolved picture of the initial phase of I_Ca in cardiac tissue and to assess relative changes of activation/fast inactivation of I_Ca following pharmacological interventions.

Several genetically encoded fluorescent sensors of voltage were created by systematically truncating the length of the linker sequence between the voltage-sensing domain and the position of the fluorescent protein, Super Ecliptic A227D. In addition to varying the length, the amino acid composition at the fusion site for the fluorescent protein was modified. Both linker length and amino acid composition affected the size and voltage sensitivity of the optical signal. The truncation mutants revealed a potential structural periodicity with a maximum signal three amino acids from the voltage-sensing domain and another maximum 11 amino acids from the voltage-sensing domain. These results confirm that the linker length and composition can fine tune the size and voltage range of the sensor. The potential periodicity suggests that the orientation of the fluorescent protein could be important for improving the signal size implicating dimerization of the fluorescent protein.

We describe an experimental approach that uses light to both control and detect neuronal activity in mouse barrel cortex slices: blue light patterned by a digital micromirror array system allowed us to photostimulate specific layers and columns, while a red-shifted voltage-sensitive dye was used to map out large-scale circuit activity. We demonstrate that such all-optical mapping can interrogate various circuits in somatosensory cortex by sequentially activating different layers and columns. Further, mapping in slices from whisker-deprived mice demonstrated that chronic sensory deprivation did not significantly alter feedforward inhibition driven by layer 5 pyramidal neurons. Further development of voltage-sensitive optical probes should allow this all-optical mapping approach to become an important and high-throughput tool for mapping circuit interactions in the brain.

Imaging calcium transients associated with neuronal activity has yielded important insights into neural physiology. Genetically encoded calcium indicators (GECIs) offer conspicuous potential advantages for this purpose, including exquisite targeting. While the catalogue of available GECIs is steadily growing, many newly developed sensors that appear promising in vitro or in model cells appear to be less useful when expressed in mammalian neurons. We have, therefore, evaluated the performance of GECIs from two of the most promising families of sensors, G-CaMPs [ Nat. Biotechnol. 19(2), 137–141 (2001)] and GECOs [ Science 333(6051), 1888–1891 (2011)], for monitoring action potentials in rat brain. Specifically, we used two-photon excitation fluorescence microscopy to compare calcium transients detected by G-CaMP3; GCaMP6f; G-CaMP7; Green-GECO1.0, 1.1 and 1.2; Blue-GECO; Red-GECO; Rex-GECO0.9; Rex-GECO1; Carmine-GECO; Orange-GECO; and Yellow-GECO1s. After optimizing excitation wavelengths, we monitored fluorescence signals associated with increasing numbers of action potentials evoked by current injection in CA1 pyramidal neurons in rat organotypic hippocampal slices. Some GECIs, particularly Green-GECO1.2, GCaMP6f, and G-CaMP7, were able to detect single action potentials with high reliability. By virtue of greatest sensitivity and fast kinetics, G-CaMP7 may be the best currently available GECI for monitoring calcium transients in mammalian neurons.

Brain–computer interfaces (BCIs) allow individuals to use only cognitive activities to interact with their environment. The widespread use of BCIs is limited, due in part to their lack of user-friendliness. The main goal of this work was to develop a more user-centered BCI and determine if: (1) individuals can acquire control of an online near-infrared spectroscopy BCI via usability and performance-informed selection of mental tasks without compromising classification accuracy and (2) the combination of usability and performance-informed selection of mental tasks yields subjective ease-of-use ratings that exceed those attainable with prescribed mental tasks. Twenty able-bodied participants were recruited. Half of the participants served as a control group, using the state-of-the-art prescribed mental strategies. The other half of the participants comprised the study group, choosing their own personalized mental strategies out of eleven possible tasks. It was concluded that users were, in fact, able to acquire control of the more user-centered BCI without a significant change in accuracy compared to the prescribed task BCI. Furthermore, the personalized BCI yielded higher subjective ease-of-use ratings than the prescribed BCI. Average online accuracies of 77±12.9% and 73±12.9% were achieved by the personalized and prescribed mental task groups, respectively.

Infrared neural stimulation (INS) is a method to depolarize neurons with infrared light. While consensus exists that heating of the target structure is essential, subsequent steps that result in the generation of an action potential are controversially discussed in the literature. The question of whether cochlear INS is an acoustic event has not been clarified. Results have been published that could be explained solely by an acoustic event. However, data exist that do not support an acoustical stimulus as the dominant factor in cochlear INS. We review the different findings that have been suggested for the mechanism of INS. Furthermore, we present the data that clarify the role of an acoustical event in cochlear INS. Masking experiments have been performed in hearing, hearing impaired, and severely hearing impaired animals. In normal hearing animals, the laser response could be masked by the acoustic stimulus. Once thresholds to acoustic stimuli were elevated, the ability to acoustically mask the INS response gradually disappeared. Thresholds for acoustic stimuli were significantly elevated in animals with compromised cochlear function, while the thresholds for optical stimulation remained largely unchanged. The results suggest that the direct interaction between the radiation and the target structure dominates cochlear INS.

Noninvasive localization of an epileptogenic zone is a fundamental step for presurgical evaluation of epileptic patients. Here, we applied long-term simultaneous functional near-infrared spectroscopy (fNIRS)/electroencephalogram (EEG) monitoring for focus diagnosis in patients with mesial temporal lobe epilepsy (MTLE). Six MTLE patients underwent long-term (8–16 h per day for 4 days) fNIRS/EEG monitoring for the occurrence of spontaneous seizures. Four spontaneous seizures were successfully recorded out of the six patients. To determine oxy-Hb amplitude, the period-average values of oxy-Hb across 20 s from the EEG- or clinically defined epileptic onset were calculated for both hemispheres from the simultaneously recorded fNIRS data. The average oxy-Hb values for the temporal lobe at the earlier EEG- or clinically defined epileptic onsets were greater for the epileptic side than for the contralateral side after EEG activity suppression, spike train, and clinical seizure in all four cases. The true laterality was determined based on the relief of seizures by selective amygdalo-hippocampectomy. Thus, oxy-Hb amplitude could be a reliable measure for determining the epileptic focus side. Long-term simultaneous fNIRS/EEG measurement serves as an effective tool for recording spontaneous seizures. Cerebral hemodynamic measurement by fNIRS would serve as a valuable supplementary noninvasive measurement method for presurgical evaluation of MTLE.

Near-infrared spectroscopy (NIRS) is a noninvasive neuroimaging technique used to assess cerebral hemodynamics. Its portability, ease of use, and relatively low operational cost lend itself well to the long-term monitoring of hemodynamic changes, such as those in epilepsy, where events are unpredictable. Long-term monitoring is associated with challenges including alterations in behaviors and motion that can result in artifacts. Five patients with epilepsy were assessed for interictal hemodynamic changes and alterations in behavior or motion. Based on this work, visual inspection was used to identify NIRS artifacts during a period of interest, specifically prior to seizures, in four patients. A motion artifact reduction algorithm (MARA, also known as the spline interpolation method) was tested on these data. Alterations in the NIRS measurements often occurred simultaneously with changes in motion and behavior. Occasionally, sharp shift artifacts were observed in the data. When artifacts appeared as sustained baseline shifts in the data, MARA reduced the standard deviation of the data and the appearance improved. We discussed motion and artifacts as challenges associated with long-term monitoring of cerebral hemodynamics in patients with epilepsy and our group’s approach to circumvent these challenges and improve the quality of the data collected.

In recent years, it has been demonstrated that using functional near-infrared spectroscopy (fNIRS) channels with short separations to explicitly sample extra-cerebral tissues can provide a significant improvement in the accuracy and reliability of fNIRS measurements. The aim of these short-separation channels is to measure the same superficial hemodynamics observed by standard fNIRS channels while also being insensitive to the brain. We use Monte Carlo simulations of photon transport in anatomically informed multilayer models to determine the optimum source–detector distance for short-separation channels in adult and newborn populations. We present a look-up plot that provides (for an acceptable value of short-separation channel brain sensitivity relative to standard channel brain sensitivity) the optimum short-separation distance. Though values vary across the scalp, when the acceptable ratio of the short-separation channel brain sensitivity to standard channel brain sensitivity is set at 5%, the optimum short-separation distance is 8.4 mm in the typical adult and 2.15 mm in the term-age infant.

Changes in blood perfusion in highly interconnected pial arterioles provide important insights about the vascular response to ischemia within brain. The functional role of arteriolo-arteriolar anastomosis (AAA) in regulating blood perfusion through penetrating arterioles is yet to be discovered. We apply a label-free optical microangiography (OMAG) technique to evaluate the changes in vessel lumen diameter and red blood cell velocity among a large number of pial and penetrating arterioles within AAA abundant region overlaying the penumbra in the parietal cortex after a middle cerebral artery occlusion (MCAO). In comparison with two-photon microscopy, the OMAG technique makes it possible to image a large number of vessels in a short period of time without administering exogenous contrast agents during a time-constrained MCAO experiment. We compare vasodynamics in penetrating arterioles at various locations. The results show that the MCA connected penetrating arterioles close to a strong AAA dilate, while those belonging to a region away from AAAs constrict in various degrees. These results suggest AAAs play a major role in supporting the active dilation of the penetrating arterioles, thus compensating a significant amount of blood to the ischemic region, whereas the poor blood perfusion occurs at the regions away from AAA connections, leading to ischemia.