Roberto A. Braga

Dynamic laser speckle and applications

Preface The Author Contributors List The Speckle Phenomenon N. G. Gaggioli and J. A. Pomarico Dynamic Speckle Origin and Features M. Trivi Speckle and Dynamic Speckle phenomena Theoretical and Numerical Models M. F. Ruiz Gale, E. N. Hogert, N. G. Gaggioli, H. J. Rabal, Dr., and A. T. da Costa Junior Methods of Dynamic Speckle Analysis-Statistical Analysis R. Arizaga, Lic. Activity Images Generalized Differences, Fujiis, Lasca, and Related Methods H. J. Rabal, Ph.D. Frequency Analysis L. I. Passoni, G. H. Sendra, and C. M. Arizmendi Granular Computing in THSP Fuzzy Granular Analysis A. L. Dai Pra and L. I. Passoni Applications in Biological Samples R. A Braga Jr, Ph.D., G. F Rabelo, Ph.D., J. B. Barreto Fo.,Ph.D., F. M Borem, Ph.D., J. Pereira, Ph.D., M. Muramatsu, Ph.D.,and I. M. Dal Fabbro, Ph.D. Applications in Nonbiological Materials H. Jorge Rabal and R. A. Braga Jr. Didactic Dynamic Speckle Software M. Marcon and R.A. Braga Jr. Perspectives H. Jorge Rabal, Ph.D., and R. A. Braga Jr., Ph.D.

Live biospeckle laser imaging of root tissues

Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.

Continuous, high-resolution biospeckle imaging reveals a discrete zone of activity at the root apex that responds to contact with obstacles.

BACKGROUND AND AIMS Shining a laser onto biological material produces light speckles termed biospeckles. Patterns of biospeckle activity reflect changes in cell biochemistry, developmental processes and responses to the environment. The aim of this work was to develop methods to investigate the biospeckle activity in roots and to characterize the distribution of its intensity and response to thigmostimuli. METHODS Biospeckle activity in roots of Zea mays, and also Jatropha curcas and Citrus limonia, was imaged live and in situ using a portable laser and a digital microscope with a spatial resolution of 10 μm per pixel and the ability to capture images every 0.080 s. A procedure incorporating a Fujii algorithm, image restoration using median and Gaussian filters, image segmentation using maximum-entropy threshold methods and the extraction of features using a tracing algorithm followed by spline fitting were developed to obtain quantitative information from images of biospeckle activity. A wavelet transform algorithm was used for spectral decomposition of biospeckle activity and generalized additive models were used to attribute statistical significance to changes in patterns of biospeckle activity. KEY RESULTS The intensity of biospeckle activity was greatest close to the root apex. Higher frequencies (3-6 Hz) contributed most to the total intensity of biospeckle activity. When a root encountered an obstacle, the intensity of biospeckle activity decreased abruptly throughout the root system. The response became attenuated with repeated thigmostimuli. CONCLUSIONS The data suggest that at least one component of root biospeckle activity resulted from a biological process, which is located in the zone of cell division and responds to thigmostimuli. However, neither individual cell division events nor root elongation is likely to be responsible for the patterns of biospeckle activity.

Estimation of total leaf area in perennial plants using image analysis

One feature of most horticultural crop plants that is biologically relevant to their yield and productivity is total leaf area. However, direct methods of estimation of the leaf area cause damage to the plants, whereas indirect methods such as based on light measurement, demand accuracy in the setup of the measurement procedure, which is specific to each crop. Coffee is one of the most important perennial plants related to worldwide trade, and this demands some ability to estimate the productivity of the crop, as well as all the perennial plants involved in production of agricultural products. This study aims to build a model based on indirect measures to estimate the leaf area in coffee plants using image analysis. Two models were evaluated, one based on the height and width of the canopies, and other based on the area of the digital image of a tree. The results of the models have been compared with the real area of the leaves using the destructive approach with measurement of area of all the leaves using a digital scanner. Comparisons between the models and the real values indicated values of adjusted R2 of about 0.82 with a model using the height and the width values, and about 0.91 in the second model which used the area projection. The robustness of the model using the height and the width values were tested using data presented in the literature to other cultivars and achieved R2 = 0.54 with an outlier point and 0.91 without it.

Monitoring of the action of drugs in melanoma cells by dynamic laser speckle.

Abstract. This work presents the development of a protocol based on the dynamic laser speckle designed to monitor the reaction of cancer cells of line MEL-RC08 to the application of the drug Colcemid in two different concentrations: 0.2 and 0.4  μg/mL. The protocol was designed using the forward scattering approach with an He-Ne laser of 632.8 nm illuminating the samples, a control, and two variations of Colcemid, being monitored along 8 h. The data were analyzed numerically in the time and in the frequency domain, and the results presented the ability of the technique to monitor the action of the drug, particularly Colcemid (0.4  μg/mL).

Accuracy in dynamic laser speckle: optimum size of speckles for temporal and frequency analyses

Abstract. The dynamic laser-speckle phenomenon has been used as a potential tool to monitor the activity of many biological and nonbiological samples; however, a key tailoring of the experimental configuration must be taken into account to avoid wrong measurements, since the general rules addressed to speckle as information cannot be directly adopted in dynamic laser-speckle monitoring. The speckle/pixel size ratio is provided by the f-number and by the magnification of the macro lens, and attention is mainly directed toward adjusting a speckle/pixel ratio higher than 1. However, a speckle/pixel ratio much higher than one does not mean an optimum adjustment. This work tested different apertures with fixed magnification yielding to monitor a drying paint process. The outcomes were evaluated in the time and frequency domains. The highest speckle/pixel size ratio was not the best to monitor the process using the dynamic laser speckle under frequency analysis. Tailoring of the devices must take into account the optimum speckle/pixel size ratio, which could vary depending on the application, and the known Nyquist theorem cannot be considered as a sufficient condition since the setup of the optical camera with its macro and iris must also be adjusted in accordance with the frequency response.

Dynamic laser speckle analyzed considering inhomogeneities in the biological sample

Abstract. Dynamic laser speckle phenomenon allows a contactless and nondestructive way to monitor biological changes that are quantified by second-order statistics applied in the images in time using a secondary matrix known as time history of the speckle pattern (THSP). To avoid being time consuming, the traditional way to build the THSP restricts the data to a line or column. Our hypothesis is that the spatial restriction of the information could compromise the results, particularly when undesirable and unexpected optical inhomogeneities occur, such as in cell culture media. It tested a spatial random approach to collect the points to form a THSP. Cells in a culture medium and in drying paint, representing homogeneous samples in different levels, were tested, and a comparison with the traditional method was carried out. An alternative random selection based on a Gaussian distribution around a desired position was also presented. The results showed that the traditional protocol presented higher variation than the outcomes using the random method. The higher the inhomogeneity of the activity map, the higher the efficiency of the proposed method using random points. The Gaussian distribution proved to be useful when there was a well-defined area to monitor.

Leaf area estimation of medium size plants using optical metrology

The total leaf area (TLA) estimation is an important feature of the crops and their assessment a challenge, since the direct methods of obtaining it are destructive and time consuming. Non-destructive methods have been explored to obtain the TLA by indirect approaches, in turn creating other features, as the leaf area index. The development of non-destructive technology to access the TLA of a plant has been the subject of much research, and the optical metrology is an promising approach. In this work, some indirect methods associated with optical approaches were evaluated as an alternative to obtain the TLA of the coffee plant. Commercial equipment were used, such as a camera with a fish eye lens and lux meters, associated to the sizes of the canopies were tested and compared to another non-destructive method and with methods proposed in the literature. The association between production and the TLA estimated was also evaluated. The results showed that the commercial equipment, generally used in forestry, was not the best approach in coffee plants, and that the methods related to the size and lux values of the plants were the best alternatives to estimate the TLA of the coffee plant.

Alternative protocols on dynamic speckle laser analysis

Biospeckle or dynamic laser speckle is a phenomenon developed when a dynamic process occurs in a material under laser illumination. This phenomenon contains considerable information related to both biological and non-biological activity of the material under study. As a non-invasive, non-destructive and low cost technique, biospeckle laser (BSL) has been an outstanding tool for monitoring biological properties. Thus, its application in optical instrumentation has grown over the years, especially in the areas of biology, medicine and agriculture. High sensitivity of biospeckle laser (BSL) technique and variability of biological material combined with the large number of variables involved in speckle pattern formation have brought great challenges to the search for safer, more robust analysis techniques. Therefore, this study aimed to develop and refine methodologies for the BSL analysis and tested alternative protocols for the prime analysis. Particularly, we present a protocol to obtain requirements before the main analysis, in an attempt to eliminate image quality based on subjectivism or research experience. This protocol was based on the creation of requirements to achieve the best speckle patterns such as evaluating the saturation, the homogeneity and the contrast of the grains. The alternative protocol offered a testing approach before the main experiment to increase the certainty to get image and data in an accepted quality, avoiding the loose of time and samples in analysis of a row of a questionable data. The tests were performed in well-known BSL data and presented as a feasible step before the main experiments.

A Portable Dynamic Laser Speckle System for Sensing Long-Term Changes Caused by Treatments in Painting Conservation

Dynamic laser speckle (DLS) is used as a reliable sensor of activity for all types of materials. Traditional applications are based on high-rate captures (usually greater than 10 frames-per-second, fps). Even for drying processes in conservation treatments, where there is a high level of activity in the first moments after the application and slower activity after some minutes or hours, the process is based on the acquisition of images at a time rate that is the same in moments of high and low activity. In this work, we present an alternative approach to track the drying process of protective layers and other painting conservation processes that take a long time to reduce their levels of activity. We illuminate, using three different wavelength lasers, a temporary protector (cyclododecane) and a varnish, and monitor them using a low fps rate during long-term drying. The results are compared to the traditional method. This work also presents a monitoring method that uses portable equipment. The results present the feasibility of using the portable device and show the improved sensitivity of the dynamic laser speckle when sensing the long-term process for drying cyclododecane and varnish in conservation.