Temporal mirror-symmetry in functional signals recorded from rat barrel cortex with optical coherence tomography.

Functional optical coherence tomography (fOCT) detects activity-dependent light scattering changes in micro-structures of neural tissue, drawing attention as in vivo volumetric functional imaging technique at a sub-columnar level. There are 2 plausible origins for the light scattering changes: (i) hemodynamic responses such as changes in blood volume and in density of blood cells and (ii) reorientation of dipoles in cellular membrane. However, it has not been clarified which is the major contributor to fOCT signals. Furthermore, previous studies showed both increase and decrease of reflectivity as fOCT signals, making interpretation more difficult. We proposed combination of fOCT with Fourier imaging and adaptive statistics to the rat barrel cortex. Active voxels revealed barrels elongating throughout layers with mini-columns in superficial layers consistent with physiological studies, suggesting that active voxels revealed by fOCT reflect spatial patterns of activated neurons. These voxels included voxels with negative changes in reflectivity and those with positive changes in reflectivity. However, they were temporally mirror-symmetric, suggesting that they share common sources. It is hard to explain that hemodynamic responses elicit positive signals in some voxels and negative signals in the other. On the other hand, considering membrane dipoles, polarities of OCT signals can be positive and negative depending on orientations of scattering particles relative to the incident light. Therefore, the present study suggests that fOCT signals are induced by the reorientation of membrane dipoles.

3D topology of orientation columns in visual cortex revealed by functional optical coherence tomography.

Orientation tuning is a canonical neuronal response property of six-layer visual cortex that is encoded in pinwheel structures with center orientation singularities. Optical imaging of intrinsic signals enables us to map these surface two-dimensional (2D) structures, whereas lack of appropriate techniques has not allowed us to visualize depth structures of orientation coding. In the present study, we performed functional optical coherence tomography (fOCT), a technique capable of acquiring a 3D map of the intrinsic signals, to study the topology of orientation coding inside the cat visual cortex. With this technique, for the first time, we visualized columnar assemblies in orientation coding that had been predicted from electrophysiological recordings. In addition, we found that the columnar structures were largely distorted around pinwheel centers: center singularities were not rigid straight lines running perpendicularly to the cortical surface but formed twisted string-like structures inside the cortex that turned and extended horizontally through the cortex. Looping singularities were observed with their respective termini accessing the same cortical surface via clockwise and counterclockwise orientation pinwheels. These results suggest that a 3D topology of orientation coding cannot be fully anticipated from 2D surface measurements. Moreover, the findings demonstrate the utility of fOCT as an in vivo mesoscale imaging method for mapping functional response properties of cortex in the depth axis. NEW & NOTEWORTHY We used functional optical coherence tomography (fOCT) to visualize three-dimensional structure of the orientation columns with millimeter range and micrometer spatial resolution. We validated vertically elongated columnar structure in iso-orientation domains. The columnar structure was distorted around pinwheel centers. An orientation singularity formed a string with tortuous trajectories inside the cortex and connected clockwise and counterclockwise pinwheel centers in the surface orientation map. The results were confirmed by comparisons with conventional optical imaging and electrophysiological recordings.

Highly sensitive optical interferometric technique reveals stress-dependent instantaneous nanometric growth fluctuations of Chinese chive leaf under heavy metal stress.

Plant growth apart from being a complex and highly dynamic is dependent on its immediate environment. Leaf expansion measurements using Statistical Interferometry Technique, a sensitive interferometric technique at nanometric accuracy and at sub-second levels revealed the presence of characteristic nanometric intrinsic fluctuations [Plant Biotechnology 31, 195 (2014)]. In this paper, we demonstrate that the nanometric intrinsic fluctuations are sensitive enough that they change under exposure of heavy metals, essential micronutrient zinc and non-essential element cadmium, at relatively low concentrations in the leaves of Chinese chive (Allium tuberosum). The nanometric intrinsic fluctuations of leaves were observed for 4h under three cadmium concentrations or two zinc concentrations. Results showed significant reduction of nanometric intrinsic fluctuations for all cadmium concentrations, and in contrast significant increase of nanometric intrinsic fluctuations for all zinc concentrations. There was significant reduction of nanometric intrinsic fluctuations for cadmium exposure of concentrations of 0.001mM for even an hour, and significant increment of nanometric intrinsic fluctuations under 0.75mM zinc from 1hr exposure. For comparison, antioxidative enzymes and metal uptake were also measured under 4hr exposure of cadmium or zinc. However, no significant changes could be seen in antioxidative enzymes within 4h under the smaller concentration of 0.001mM cadmium as seen for nanometric intrinsic fluctuations. The results imply that nanometric intrinsic fluctuations can be not only used as a measure for heavy metal stress but also it can be more sensitive to detect the toxic as well as positive effects of smaller amounts of heavy metal on plants at an early stage.

The synergy of microplastics with the heavy metal zinc has resulted in reducing the toxic effects of zinc on lentil (Lens culinaris) seed germination and seedling growth.

There is growing recognition of the impact of the rising presence of microplastics (MPs) on terrestrial plant growth and, in general, the terrestrial ecosystem. Simultaneously, there is growing heavy metal accumulation in agricultural lands at an astonishing rate owing to the overwhelming use of chemical fertilizers, herbicides, and weedicides. Thus, there is a need to investigate the synergetic effect of MPs along with heavy metals on the inducing combined toxicity. This study investigates effects at smaller exposure periods of a few hours using a novel optical imaging technique, Biospeckle Coherence Tomography. Biospeckle Optical Coherence Tomography (bOCT) is a novel optical imaging technique that we successfully demonstrated earlier in visualizing the internal activity of plants. Previous studies of authors using the bOCT technique have demonstrated its potential in the independent application of polyethylene microplastic (PEMPs) as well as zinc within 6 h after their treatments. The strong inhibitory effect of 100 mg L-1, Zn, and PEMPs alone on the germination of Lens culinaris could be visualized with bOCT. The current study demonstrated that against expectation, combined effects of Zn toxicity were reduced when combined with MPs. This is suggested due to the significant reduction of Zn uptake by the seedlings through the interaction of Zn and MPs in an aqueous solution. Mass-spectrometry results also indicate a reduced intake of Zn. Our findings suggest that PEMPs could be able to reduce the over-availability of Zn, thus mitigating the Zn toxicity on lentils.

Effects of microplastics on lentil (Lens culinaris) seed germination and seedling growth.

Widespread use of plastics and mishandling has resulted in severe environmental issues affecting seed germination and seedling growth. This study investigates the effect of polyethylene microplastics (740-4990 nm PEMPs) on lentil (Lens culinaris) seed germination and seedling growth using Biospeckle Optical Coherence Tomography (bOCT), a technique that we successfully demonstrated earlier in visualizing the internal activity of plants. Lentil seeds were exposed to PEMPs bioassay for seven days with 10, 50, and 100 mg L-1 concentrations. The average speckle contrast was calculated after 0 h, 6 h, 12 h, and 24 h of exposure, and statistically significant differences were observed just after 6 h of exposure under all the treatments. However, with conventional parameters, germination viability, germination rate, root and shoot lengths, fresh and dry seedling weights, and antioxidative enzymes, no significant effect was observed until 2 d of exposure. The results revealed that the presence of PEMPs significantly reduced the internal activity at the initial stages that could be visualized only by the use of bOCT, which has never been observed till now. Our results demonstrated for the first time the effect that microplastics indeed could hinder the internal activity during germination of the seeds, possibly resulting from the physical blockage of pores leading to stunted growth at later stages.

A simple adaptive difference algorithm with CO2 measurements for evaluating plant growth under environmental fluctuations.

OBJECTIVE: The aim of this study is to demonstrate an adaptive method that is robust toward environmental fluctuations and provides a real-time measure of plant growth by measuring CO2 consumption. To verify the validity of the proposed method, the relation between the plant growth and variation in light conditions with a closed experimental system was investigated. RESULTS: The proposed method was used to measure the photosynthetic rate induced by photosynthetic photon flux density (PPFD) and to evaluate plant growth under continuous and pulsed light in arugula plants. The PPFD-dependent change in photosynthetic rate was measured. And in the condition range of 200-10,000 µs pulse period and 50% duty ratio of pulsed light, there was no change in the growth rate of plants assuming the same PPFD as continuous light. These experiments showed the validity of the adaptive method in removing environmental fluctuations without precise control of temperature and humidity.

Demonstration of laser biospeckle method for speedy in vivo evaluation of plant-sound interactions with arugula.

In recent years, it is becoming clearer that plant growth and its yield are affected by sound with certain sounds, such as seedling of corn directing itself toward the sound source and its ability to distinguish stuttering of larvae from other sounds. However, methods investigating the effects of sound on plants either take a long time or are destructive. Here, we propose using laser biospeckle, a non-destructive and non-contact technique, to investigate the activities of an arugula plant for sounds of different frequencies, namely, 0 Hz or control, 100 Hz, 1 kHz, 10 kHz, including rock and classical music. Laser biospeckles are generated when scattered light from biological tissues interfere, and the intensities of such speckles change in time, and these changes reflect changes in the scattering structures within the biological tissue. A leaf was illuminated by light from a laser light of wavelength 635 nm, and the biospeckles were recorded as a movie by a CMOS camera for 20 sec at 15 frames per second (fps). The temporal correlation between the frames was characterized by a parameter called biospeckle activity (BA)under the exposure to different sound stimuli of classical and rock music and single-frequency sound stimuli for 1min. There was a clear difference in BA between the control and other frequencies with BA for 100 Hz being closer to control, while at higher frequencies, BA was much lower, indicating a dependence of the activity on the frequency. As BA is related to changes from both the surface as well as from the internal structures of the leaf, LSM (laser scanning microscope) observations conducted to confirm the change in the internal structure revealed more than 5% transient change in stomatal size following exposure to one minute to high frequency sound of 10kHz that reverted within ten minutes. Our results demonstrate the potential of laser biospeckle to speedily monitor in vivo response of plants to sound stimuli and thus could be a possible screening tool for selecting appropriate frequency sounds to enhance or delay the activity of plants. (337 words).

Biospeckle optical coherence tomography in speedy visualizing effects of foliar application of plant growth hormone to Chinese chives leaves.

OBJECTIVE: The aim of this study is to demonstrate the potential of applying the contrast of the speckles obtained as noise in optical coherence tomography (OCT) images to monitor short term activity changes during foliar application of phytohormones to a plant leaf. Plant growth hormone, gibberellic acid (GA3) was sprayed onto the leaf of Chinese chives and after 60 min, OCT images (1 frame: 512 × 2048 pixels) were recorded at ten frames per second for a few tens of seconds. RESULTS: Contrast across the temporal axis was calculated for each pixel of the structural images and biospeckle OCT contrast images were obtained under the conditions of before and after application of GA3 for different concentrations 0, 40, and 100 µM. Application of 40 µM GA3 failed to show any differences in the OCT structural images. However, bOCT contrast image was clearly different. Changes were found to be statistically significant. Although the mechanism for the contrast difference is not clear, it can be said there is a large change across the temporal scale with the application of GA3. Demonstration of OCT utilizing the speckle contrast is believed to have the potential as a promising tool in plant physiology.

Using the light scattering component of optical intrinsic signals to visualize in vivo functional structures of neural tissues.

Visualization of changes in reflected light from in vivo brain tissues reveals spatial patterns of neural activity. An important factor which influences the degree of light reflected includes the change in light scattering elicited by neural activation. Microstructures of neural tissues generally cause light scattering, and neural activities are associated with some changes in the microstructures. Here, we show that the optical properties unique to light scattering enable us to visualize spatial patterns of retinal activity non-invasively (FRG: functional retinography), and resolve functional structures in depth (fOCT: functional optical coherence tomography).

Functional optical coherence tomography of rat olfactory bulb with periodic odor stimulation.

In rodent olfactory bulb (OB), optical intrinsic signal imaging (OISI) is commonly used to investigate functional maps to odorant stimulations. However, in such studies, the spatial resolution in depth direction (z-axis) is lost because of the integration of light from different depths. To solve this problem, we propose functional optical coherence tomography (fOCT) with periodic stimulation and continuous recording. In fOCT experiments of in vivo rat OB, propionic acid and m-cresol were used as odor stimulus presentations. Such a periodic stimulation enabled us to detect the specific odor-responses from highly scattering brain tissue. Swept source OCT operating at a wavelength of 1334 nm and a frequency of 20 kHz, was employed with theoretical depth and lateral resolutions of 6.7 µm and 15.4 µm, respectively. We succeeded in visualizing 2D cross sectional fOCT map across the neural layer structure of OCT in vivo. The detected fOCT signals corresponded to a few glomeruli of the medial and lateral parts of dorsal OB. We also obtained 3D fOCT maps, which upon integration across z-axis agreed well with OISI results. We expect such an approach to open a window for investigating and possibly addressing toward inter/intra-layer connections at high resolutions in the future.

An optical interferometric technique for assessing ozone induced damage and recovery under cumulative exposures for a Japanese rice cultivar.

Exposure to ozone (O3) causes reduction both in the growth and yield of rice (Oriza sativa L.). Commonly used Chlorophyll fluorescent measurements are not sensitive enough for short term exposure of O3 aiming an immediate assessments. Such a conventional method typically needs exposure over a few days to detect the influence. As an alternative method, we proposed a novel non-invasive, robust, real-time, optical Statistical Interferometric Technique (SIT) to measure growth at an accuracy of 0.1 nm with a commonly consumed Japanese rice cultivar, Koshihikari. In the present study, we have conducted a repetitive O3 exposure experiment for three days under three different concentrations of 0 nl l(-1) (control), 120 nl l(-1), and 240 nl l(-1), to investigate the damage and recovery strengths. As a measure to assess the effect and recovery from three consecutive day exposures of O3, we measured the elongation rate (nm mm(-1) sec(-1)) every 5.5 sec for 7 hours, and it revealed nanometric elongation rate fluctuations or Nanometric Intrinsic Fluctuations (NIF). Comparing the standard deviation (SD) of normalized nanometric intrinsic fluctuations (NNIF), which was normalized by that before the exposure, we found that drastic reductions under both 120 nl l(-1) and 240 nl l(-1) O3 concentrations. Reduction percentages were large under high O3 concentration of 240 nl l(-1) indicating the possibility of irreversible effect. However exposure to 120 nl l(-1) of O3 showed recovery on the 2(nd) and 3(rd) days. While SIT did reveal immediate effect based on an observation for a few hours, the visible foliar effect could be observed only after a week. Hence, the technique could provide a way for fast assessment of effect and recovery due to cumulative exposure of O3 and hence the tolerance as well as the vitality of plant.

In vivo layer visualization of rat olfactory bulb by a swept source optical coherence tomography and its confirmation through electrocoagulation and anatomy.

Here, we report in vivo 3-D visualization of the layered organization of a rat olfactory bulb (OB) by a swept source optical coherence tomography (SS-OCT). The SS-OCT operates at a wavelength of 1334 nm with respective theoretical depth and lateral resolutions of 6.7 µm and 15.4 µm in air and hence it is possible to get a 3D structural map of OB in vivo at the micron level resolution with millimeter-scale imaging depth. Up until now, with methods such as MRI, confocal microscopy, OB depth structure in vivo had not been clearly visualized as these do not satisfy the criterion of simultaneously providing micron-scale spatial resolution and imaging up to a few millimeter in depth. In order to confirm the OB's layered organization revealed by SS-OCT, we introduced the technique of electrocoagulation to make landmarks across the layered structure. To our knowledge this is such a first study that combines electrocoagulation and OCT in vivo of rat OB. Our results confirmed the layered organization of OB, and moreover the layers were clearly identified by electrocoagulation landmarks both in the OCT structural and anatomical slice images. We expect such a combined study is beneficial for both OCT and neuroscience fields.

Swept source optical coherence tomography as a tool for real time visualization and localization of electrodes used in electrophysiological studies of brain in vivo.

In studies of in vivo extracellular recording, we usually penetrate electrodes almost blindly into the neural tissue, in order to detect the neural activity from an expected target location at a certain depth. After the recording, it is necessary for us to determine the position of the electrodes precisely. Generally, to identify the position of the electrode, one method is to examine the postmortem tissue sample at micron resolution. The other method is using MRI and it does not have enough resolution to resolve the neural structures. To solve such problems, we propose swept source optical coherence tomography (SS-OCT) as a tool to visualize the cross-sectional image of the neural target structure along with the penetrating electrode. We focused on a rodent olfactory bulb (OB) as the target. We succeeded in imaging both the OB layer structure and the penetrating electrode, simultaneously. The method has the advantage of detecting the electrode shape and the position in real time, in vivo. These results indicate the possibility of using SS-OCT as a powerful tool for guiding the electrode into the target tissue precisely in real time and localizing the electrode tip during electrophysiological recordings.

Functional optical coherence tomography reveals localized layer-specific activations in cat primary visual cortex in vivo.

Surface neural activity has been widely visualized using optical intrinsic signal imaging (OISI) from various cortical sensory areas. OISI of the cortical surface with a CCD camera gives integrated information across a depth of a few hundred micrometers. We visualize depth-resolved activation patterns of cat primary visual cortex by functional optical coherence tomography (fOCT). A comparison of the depth-integrated results of fOCT maps with the optical intrinsic signal profiles shows fairly good agreement. Our results reveal layer-specific activation patterns and indicate that the activation was not homogeneous.

Localization of activity-dependent changes in blood volume to submillimeter-scale functional domains in cat visual cortex.

We have examined whether blood volume changes induced by neural activation are controlled precisely enough for us to visualize the submillimeter-scale functional structure in anesthetized and awake cat visual cortex. To activate the submillimeter-scale functional structures such as iso-orientation domains in the cortex, visual stimuli (gratings) were presented to the cats. Two methods were used to examine the spatial precision of blood volume changes including changes in total hemoglobin content and changes in plasma volume: (i) intrinsic signal imaging at the wavelength of hemoglobin's isosbestic point (569 nm) and (ii) imaging of absorption changes of an intravenously injected dye. Both measurements showed that the visual stimuli elicited stimulus-nonspecific and stimulus-specific blood volume changes in the cortex. The former was not spatially localized, while the latter was confined to iso-orientation domains. From the measurement of spatial separation of the iso-orientation domains, we estimated the spatial resolution of stimulus-specific blood volume changes to be as high as 0.6 mm. The changes in stimulus-nonspecific and -specific blood volume were not linearly correlated. These results suggest the existence of fine blood volume control mechanisms in the capillary bed in addition to global control mechanisms in arteries.