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.

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).

Expansion of the dynamic range of statistical interferometry and its application to extremely short- to long-term plant growth monitoring.

In this study, we propose a method to expand the dynamic range of expansion or strain measurement using statistical interferometry. Statistical interferometry is a very accurate interferometric technique that is applicable to practical rough surface objects [Opt. Lett. 16, 883 (1991); J. Opt. Soc. Am. A 18, 1267 (2001)]. It is based on the statistical stability of a fully developed speckle field and was successfully applied to measure the growth of plants in our previous study [Environ. Exp. Bot. 64, 314 (2008); J. For. Res. 12, 393 (2007)]. However, the measurable range of the expansion of the object was restricted to less than one wavelength of the light used. Improvement of the dynamic range is confirmed experimentally in this work by introducing a large expansion up to 300 µm while keeping the precision of measurement high. Next, the improved system is applied to monitor plant growth from the subnanometric scale to several hundreds of micrometers under some environmental conditions. These features of the method make it especially worthwhile in botanical and agricultural studies.

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.

Nanometer accuracy statistical interferometric technique in monitoring the short-term effects of exogenous plant hormones, auxin and gibberellic acid, on rice plants.

Statistical interferometric technique (SIT) is a highly sensitive, high speed non-contact, and non-destructive optical technique developed by our group capable of measuring instantaeoues sub-nanometer displacements. SIT applied to plant leaf elongation revealed nanometric intrinsic fluctuaitons (NIF) that are robust and sensitive to variations in the environment making NIF as a measure of healthiness of the plants. In this study, exogenous plant hormones, auxin (2,4-dichlorophenoxyacetic acid-2,4-D), and gibberellic acid (GA3), along with an auxin transport inhibitor 2,3,5-triiodobenzoic acid-TIBA, that affect plant growth were used to investigate their effects on NIF. Rice (Oriza sativa) seedlings were used, and their roots were exposed to 1, 2, and 4 µM 2,4-D, and the auxin transport inhibitor, TIBA, of 10, and 20 µM for 22 h and GA3 solution of different concentrations of 10, 40, and 100 µM for 5 h. Results showed significant increment in NIF for 1 µM and reduction for 4 µM 2,4-D while applicaiton of both 10, and 20 µM TIBA led to reduction in NIF. On the other hand, significant increment in NIF for 40 µM, and a significant reduction at a higher concentration of 100 µM for 5 hours of GA3 were also observed in comparison to those of control. Our results indicate that NIF as revealed by SIT could show both the positive and negative effects depending on the concentration of exogenous hormones, and transport inhibitors. Results suggest that SIT could be a valuable tool being sensitive enough to speedily assess the effects of plant growth hormones.

Fractal based complexity analysis of wheat root system under different heavy metals.

In this study, fractal geometry was applied to characterize the complexity of the root system morphology of wheat plants under the exposure of heavy metals, namely cadmium (Cd), copper (Cu) and zinc (Zn). We proposed a measure called, relative complexity index (RCI), a ratio based on fractal dimension (FD) before and after exposure to heavy metals. FDs were calculated by box-counting method with digitized and skeletonized images of roots of wheat plants cultivated in hydroculture system. RCI, and relative weight were mesuared under different concentrations of Cd (0.001, 0.01 and 0.05 mM), Cu (0.016, 0.4 and 1.2 mM) and Zn (0.3 and 0.75 mM). Results showed significant reduction of RCI for Cd stress with 0.01 and 0.05, all Cu concentrations and promotion at all zinc concentrations. In comparison, no statistically significant changes were found in conventional relative weight measurement at low concentrations of Cu, Cd and Zn. RCI were more sensitive and were reliable in reflecting the influence of heavy metals than the conventional measure. These results imply that RCI can be an effective measure of the negative and positive effects of heavy metals on the development of complexity of root system under heavy metal exposures.

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.

Dynamic electronic speckle pattern interferometry (DESPI) phase analyses with temporal Hilbert transform.

In this study, we propose the Hilbert transform (HT) method for phase analysis of a Dynamic ESPI signal. The data processing is performed in the temporal domain, using the temporal history of the interference signal at every single pixel. The final results give a temporal development of the two-dimensional deformation field. To reduce the influence of the fluctuations of bias intensity on the calculated phase, it was removed prior to performing the HT. This method was demonstrated for defects distinction and the determination of the sign change in the deformation field in two different experiments. The range of measurement lies between submicrons and tens of microns and the spatial resolution is better when compared to the fringe analysis method and the spatial carrier method.

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.

Nanometer-scale elongation rate fluctuations in the Myriophyllum aquaticum (Parrot feather) stem were altered by radio-frequency electromagnetic radiation.

The emission of radio-frequency electromagnetic radiation (EMR) by various wireless communication base stations has increased in recent years. While there is wide concern about the effects of EMR on humans and animals, the influence of EMR on plants is not well understood. In this study, we investigated the effect of EMR on the growth dynamics of Myriophyllum aquaticum (Parrot feather) by measuring the nanometric elongation rate fluctuation (NERF) using a statistical interferometry technique. Plants were exposed to 2 GHz EMR at a maximum of 1.42 Wm(-2) for 1 h. After continuous exposure to EMR, M. aquaticum plants exhibited a statistically significant 51 ± 16% reduction in NERF standard deviation. Temperature observations revealed that EMR exposure did not cause dielectric heating of the plants. Therefore, the reduced NERF was due to a non-thermal effect caused by EMR exposure. The alteration in NERF continued for at least 2.5 h after EMR exposure and no significant recovery was found in post-EMR NERF during the experimental period.

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.

Use of dynamic electronic speckle pattern interferometry with the Hilbert transform method to investigate thermal expansion of a joint material.

A dynamic electronic speckle pattern interferometry method is applied to investigate thermal expansion of a joint material (ceramic-stainless steel) as a practical industrial object. The speckle interference signal is considered in the temporal domain and the phase is analyzed by the Hilbert transform method. Errors caused by the bias and modulation variations over the phase values are first examined by numerical simulation. Two experiments are performed with in-plane and out-of-plane sensitive systems to study the 3D deformation field thoroughly. The deformation field showed clearly the difference between the thermal expansions of the stainless steel and ceramic. It was also revealed that the boundary of materials and its vicinity suffer very large thermal strain due to the significantly large difference in the linear coefficient of thermal expansions.