Emery C. Lins

Detection of citrus canker in citrus plants using laser induced fluorescence spectroscopy.

Citrus canker is a serious disease caused by Xanthomonas citri subsp. citri bacteria, which infects citrus plants (Citrus spp.) leading to a large economic loss in citrus production worldwide. In Brazil citrus canker control is done by an official eradication campaign, therefore early detection of such disease is important to prevent greater economic losses. However, detection is difficult and so far it has been done by visual inspection of each tree. Suspicious leaves from citrus plants in the field are sent to the laboratory to confirm the infection by laboratory analysis, which is a time consuming. Our goal was to develop a new optical technique to detect and diagnose citrus canker in citrus plants with a portable field spectrometer unit. In this paper, we review two experiments on laser induced fluorescence spectroscopy (LIF) applied to detect citrus canker. We also present new data to show that the length of time a leaf has been detached is an important variable in our studies. Our results show that LIF has the potential to be applied to citrus plants.

Increased viability of odontoblast-like cells subjected to low-level laser irradiation

Studies have shown that the increase of cell metabolism depends on the low level laser therapy (LLLT) parameters used to irradiate the cells. However, the optimal laser dose to up-regulate pulp cell activity remains unknown. Consequently, the aim of this study was to evaluate the metabolic response of odontoblast-like cells (MDPC-23) exposed to different LLLT doses. Cells at 20000 cells/cm2 were seeded in 24-well plates using plain culture medium (DMEM) and were incubated in a humidified incubator with 5% CO2 at 37°C. After 24 h, the culture medium was replaced by fresh DMEM supplemented with 5% (stress by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to different laser doses from a near infrared diode laser prototype designed to provide a uniform irradiation of the wells. The experimental groups were: G1: 1.5 J/cm2 + 5% FBS; G2: 1.5 J/cm2 + 10% FBS; G3: 5 J/cm2 + 5% FBS; G4: 5 J/cm2 + 10% FBS; G5: 19 J/cm2 + 5% FBS; G6: 19 J/cm2 + 10% FBS. LLLT was performed in 3 consecutive irradiation cycles with a 24-hour interval. Non-irradiated cells cultured in DMEM supplemented with either 5 or 10% FBS served as control groups. The analysis of the metabolic response was performed by the MTT assay 3 h after the last irradiation. G1 presented an increase in SDH enzyme activity and differed significantly (Mann-Whitney test, p < 0.05) from the other groups. Analysis by scanning electron microscopy showed normal cell morphology in all groups. Under the tested conditions, LLLT stimulated the metabolic activity of MDPC-23 cultured in DMEM supplemented with 5% FBS and exposed to a laser dose of 1.5 J/cm2. These findings are relevant for further studies on the action of near infrared lasers on cells with odontoblast phenotype.

A novel 785-nm laser diode-based system for standardization of cell culture irradiation.

OBJECTIVE The purpose of this study was to develop a novel device that concatenates alignment of infrared lasers and parallel procedure of irradiation. The purpose of this is to seek standardization of in vitro cell irradiation, which allows analysis and credible comparisons between outcomes of different experiments. BACKGROUND DATA Experimental data obtained from infrared laser therapies have been strongly dependent upon the irradiation setup. Although further optical alignment is difficult to achieve, in contact irradiation it usually occurs. Moreover, these methods eventually use laser in a serial procedure, extending the time to irradiate experimental samples. METHODS A LASERTable (LT) device was designed to provide similar infrared laser irradiation in 12 wells of a 24 well test plate. It irradiated each well by expanding the laser beam until it covers the well bottom, as occurs with unexpanded irradiation. To evaluate the effectiveness of this device, the spatial distribution of radiation was measured, and the heating of plain culture medium was monitored during the LT operation. The irradiation of LT (up to 25 J/cm(2) - 20 mW/cm(2); 1.250 sec) was assessed on odontoblast-like cells adhered to the bottom of wells containing 1 mL of plain culture medium. Cell morphology and metabolism were also evaluated. RESULTS Irradiation with LT presented a Gaussian-like profile when the culture medium was not heated >1°C. It was also observed that the LT made it 10 times faster to perform the experiment than did serial laser irradiation. In addition, the data of this study revealed that the odontoblast-like cells exposed to low-level laser therapy (LLLT) using the LT presented higher metabolism and normal morphology. CONCLUSIONS The experimental LASERTable assessed in this study provided parameters for standardization of infrared cell irradiation, minimizing the time spent to irradiate all samples. Therefore, this device is a helpful tool that can be effectively used to evaluate experimental LLLT protocols.

Thermography Applied During Exercises With or Without Infrared Light-Emitting Diode Irradiation: Individual and Comparative Analysis

OBJECTIVE The aim of our study was to evaluate the cutaneous temperature during an exercise on a treadmill with or without infrared light-emitting diode (LED) irradiation in postmenopausal women. BACKGROUND DATA Thermography is an imaging technique in which radiation emitted by a body in the middle and far infrared spectrum is detected and associated with the temperature of the bodys surface. MATERIALS AND METHODS Eighteen postmenopausal women were randomly divided into two groups: (1) the LED group, which performed the exercises on a treadmill associated with phototherapy (n=9) and; (2) the exercise group, which performed the exercises on a treadmill without additional phototherapy (n=9). The irradiation parameters for each womens thigh were: array of 2000 infrared LEDs (850 nm) with an area of 1,110 cm(2), 100 mW, 39 mW/cm(2), and 108 J/cm(2) for 45 min. The submaximal constant-speed exercise on the treadmill at intensities between 85% and 90% maximal heart rate (HRmax) with or without phototherapy were performed during 45 min, to perform the thermographic analysis. Thermography images were captured before the exercise (t=0), after 10, 35, and 45 min of exercising (t=10, t=35, and t=45) and at 5 min post-exercising (t=50). RESULTS The LED group showed an increased cutaneous thigh temperature during the exercise (from 33.5±0.8°C to 34.6±0.9°C, p=0.03), whereas the exercise group showed a reduced cutaneous temperature (from 33.5±0.6 to 32.7±0.7°C, p=0.02). The difference between the groups was significant (p<0.05) at t=35, t=45, and t=50. CONCLUSIONS These data indicate an improved microcirculation, and can explain one possible mechanism of action of phototherapy associated with physical exercises.

Optical fiber laser induced fluorescence spectroscopy as a citrus canker diagnostic

Citrus canker is a serious disease caused by Xanthomonas citri subsp. citri bacteria, which infects citrus plants (Citrus spp.) leading to large economic losses in citrus production worldwide. In this work, laser induced fluorescence spectroscopy (LIF) was investigated as a diagnostic technique for citrus canker disease in citrus trees at an orchard using a portable optical fiber based spectrometer. For comparison we have applied LIF to leaves contaminated with citrus canker, citrus scab, citrus variegates chlorosis, and Huanglongbing (HLB, Greening). In order to reduce the noise in the data, we collected spectra from ten leaves with visual symptoms of diseases and from five healthy leaves per plant. This procedure is carried out in order to minimize the environmental effect on the spectrum (water and nutrient supply) of each plant. Our results show that this method presents a high sensitivity (approximately 90%), however it does present a low specificity (approximately 70%) for citrus canker diagnostic. We believe that such poor performance is due to the fact that the optical fiber collects light from only a small part of the leaf. Such results may be improved using the fluorescence imaging technique on the whole leaf.

Assembly, Calibration and Application of a Hyperspectral Image System for Biomedical Imaging

The use of light-mater interaction for the detection of chemical-physical processes is not a new concept in biology, though it is not fully developed for tissue diagnosis. It is already known that reflectance as well as fluorescence images can provide important information on biochemical composition and metabolic processes in biological samples. In spite of this potential, the experimental available image systems usually are complex making clinical implementation difficult. In this study, we describe the construction and characterization of an experimental device to produce hyperspectral images between 400 and 1.000 nm. The system is composed by a spectrograph, a set of lenses for image formation and a CCD camera for acquisition. We describe in details also the calibration procedure to determine system parameters as image width, field of vision, spectral and space resolution. The illumination system uses high power light emitting diodes, either at white light or centered at 470 and 405 nm. We demonstrate that our system is able to obtain reflectance as well as fluorescence images of biological samples. As example of application it was used in Dentistry. Data from spectral images of violetinduced autofluorescence of bovine tooth were evaluated in order to detect demineralization process. Our results demonstrate that this system is operating as projected and it has enough sensitivity to detect changes in low-intensity optical signals as fluorescence.

Study of Kinetics and Distribution of Photogem ® in Animal Liver Through Fluorescence Imaging

In this work the pharmacokinetics and spatial distribution of Photogem in rat liver have been evaluated using in vivo fluorescence imaging. The imaging system is basically composed by a violet-blue light source, a CCD color camera connected to a computer, and filters. During the total investigation time (90 minutes), RGB color images were acquisitioned at 15 min interval. For image analysis a Matlab routine was set to separate the RGB component and to plot the mean intensity of a selected region of interest. The results have shown that the major liver autofluorescence signal (G component) remained constant. The Photogem fluorescence detected at liver surface (R component) presents a peak around 30 min. The spatial distribution of Photogem shows heterogeneity and is still under analysis and discussion.

Organic dye penetration quantification into a dental composite resin cured by LED system using fluorescence spectroscopy

A major characteristic of LEDs systems is the lower heat emission related with the kind of light generation and spectral emission band. Material temperature during photoactivation can promote different photocuring performance. Organic dye penetration could be a trace to identify the efficacy of photocured composite resin. A new method using fluorescent spectroscopy through digital image evaluation was developed in this study. In order to understand if there is a real influence of material temperature during the photoactivation procedure of a dental restorative material, a hybrid composite resin (Z250, 3M-Espe, USA) and 3 light sources, halogen lamp (510 mW/cm2) and two LED systems 470±10nm (345 and 1000 mW/cm2) under different temperatures and intensities were used. One thousand and five hundred samples under different associations between light sources and temperatures (0, 25, 50, 75 and 100oC were tested and immediately kept in 6G rodamin dye solution. Dye penetration was evaluated through fluorescent spectroscopy recorded by digital image data. Pixels in gray scale showed the percentage penetration of organic dye into the composite resin mass. Time and temperature were statistically significant (p<0.05) through the ANOVA statistical test. The lowest penetration value was with 60 seconds and 25oC. Time and temperature are important factors to promote a homogeneous structure polymerized composite resin more than the light source type, halogen or LEDs system.

Fluorescence Spectroscopy for Detection of Citrus Canker in Orange Plantation

In this work, we investigate the use of induced fluorescence for the detection of Citrus Canker in orange plantations in Brazil. The spectrum allows us to detect the disease in a very early stage.

Observation of Climacteric-Like Behavior of Citrus Leaves Using Fluorescence Spectroscopy

Observation of climacteric-like behavior in citrus leaves depends on the detection of ethylene. However, such detection requires a gas chromatographer and complex sample preparation procedures. In this work, fluorescence spectroscopy was investigated as a diagnostic technique for climacteric-like behavior in citrus leaves. Our results indicate that the chlorophyll fluorescence presents a time evolution consistent with the ethylene evolution. Therefore, fluorescence spectroscopy may be used to observe the climacteric-like behavior in citrus leaves.