Optical improvement of the dynamic laser speckle for seed analysis using portable digital cameraABSTRACT
The biological activity assessment using the dynamic laser speckle (DLS) technique is recognized as a feasible methodology to analyze seeds vigor. Nevertheless, the technique presents the accessibility as a restriction to final users, and the large size of the equipment is a key limitation to allow the portability of the DLS. This work aimed to propose a reduced and portable configuration of the image capture system adopted in the DLS technique for seed analysis applications. A compacted digital camera with an embedded magnification, known by mini microscope, was tested associated with adapted diaphragm as an alternative to the traditional CCD camera, macro zoom and integrated diaphragm. The tests of the proposed adapted mini microscope, in comparison to the traditional configuration, were carried out in the monitoring of drying a paint and in the separation of high-vigor from early-aged seeds. Diaphragms with three different apertures (3, 4 and 5 mm) were evaluated. The results using the 3 mm diameter aperture presented the best performance, like in the case of the traditional CCD camera set. It was possible to follow the drying of a paint activity compared to its loss of weight and to distinguish statistically high-vigor from early-aged seeds at a significance level of 5%. The reliable results of the proposed configuration offered a compact and portable version for using the DLS in seed analysis laboratories.
Index terms:
Analysis; seeds; germination
RESUMO
A avaliação da atividade biológica em sementes utilizando a técnica do speckle laser dinâmico (DLS) é reconhecida como uma metodologia viável para a análise do vigor de sementes. Todavia, a técnica apresenta uma restrição de acesso para usuários finais, e a dimensão dos equipamentos é uma limitação chave para permitir a portabilidade do DLS. Este trabalho objetivou propor uma configuração portátil e compacta do sistema de captura de imagens, adotado pela técnica do DLS aplicada à análise de sementes. Uma compacta câmera digital com magnificação integrada, conhecida como mini microscópio, foi testada com um diafragma adaptado como alternativa ao conjunto CCD, macro zoom e diafragma embutido. Os testes do mini microscópio adaptado, comparados à configuração tradicional, foram conduzidos no monitoramento de secagem de pintura e na separação de sementes de alto vigor e envelhecidas artificialmente. Diafragmas com três diferentes aberturas (de 3, 4 e 5 mm) foram testados. Os resultados usando a abertura de 3 mm apresentaram a melhor performance como no caso do sistema tradicional do conjunto de câmera CCD. Assim, foi possível seguir a atividade da secagem de pintura comparada a sua perda de peso, bem como, a distinção estatística de sementes de alto vigor de sementes envelhecidas artificialmente a um nível de significância de 5%. Os resultados confiáveis da configuração proposta oferecem uma versão compacta e portátil para uso do DLS para uso em laboratório de análise de sementes.
Termos para indexação:
Análises; sementes; germinação
Introduction
Dynamic laser speckle (DLS) is a valuable noninvasive optical technique that has been applied in various fields, including medicine (Konovalov et al., 2023; Rodriguez-Loya, Lerma, & Gardea-Torresdey, 2023; Sdobnov et al., 2024) and agriculture (González-Peña et al., 2014; O’Callaghan et al., 2019; Zdunek et al., 2014; Zilpelwar et al., 2022). This method relies on the temporal variation in the speckle pattern produced when a laser beam interacts with a sample. Therefore, the dynamic speckle pattern can be used to extract information from the activity linked to the motion of dispersers of light within a biological sample (González-Peña et al., 2016; Ramírez-Miquet et al., 2017; Chen et al., 2023; Lu et al., 2023); thus, this technique is referred to as the biospeckle laser (BSL) method. The level of the “boiling effect” presented by the speckle pattern over time can be linked to the biological activity related to many phenomena inside the sample.
The traditional setup used to apply the BSL is commonly restricted to optical laboratories (Vieria & Braga, 2020) and in a non-portable way, with the image being acquired by a CCD camera associated with macro zoom lenses that usually come with a diaphragm integrated (between the CCD sensor and the macro); the CCD camera with a macro zoom lens compromises the portability of the system since both present a large volume of motion of the equipment close to the applications. The size reduction of the equipment (BSL setup) is a challenge, and it can be performed in a reliable way using solid-state lasers instead of He-Ne devices (González-Peña, Braga, & Pujaico-Rivera, 2018).
In addition, size reduction must be performed for the optical devices responsible for image acquisition. One alternative to the traditional CCD, with macro zoom lenses and a diaphragm, is the CMOS camera built in a digital mini microscope with an integrated macro zoom lens. However, the issue is the absence of a diaphragm, while the traditional macro zoom lens usually involves the diaphragm being integrated and placed between the camera and the macro-camera, yielding good results with the diaphragm in f/11 or f/16 (Braga & González-Peña, 2016). One can see in the literature the adoption of BSL using alternative optical imaging in root growth monitoring (Vega & Torres, 2015) an isolated system lights for imaging, a laser pointer 633 nm 10 mw as coherent light source, a diffuser and a laptop for processing video. The equipment enables the acquisition and storage of video, also calculated of different descriptors of statistical analysis (vector global accumulation of activity, activity matrix accumulation, cross-correlation vector, autocorrelation coefficient, matrix Fujji etc. or even in drying paint (Pérez et al., 2018) without a diaphragm. Therefore, we can hypothesize that it is possible to improve the outcomes of digital mini microscopy using a diaphragm to restrict the amount of light, enhancing the quality of the speckle pattern. The reduced size of the equipment allows its portability (Braga, 2017). The use of portable equipment can open the way for dedicated and accessible devices to analyze all types of biological samples, such as seeds, which are among the most commonly used samples in BSL applications (Vieria & Braga, 2020, Cai et al., 2019; Yu et al., 2022; Wu et al., 2024). BSL have been sensible to monitor biological activities in maize seeds (Braga et al., 2001), in soybean seeds (Singh et al., 2022) as well as in coffee seeds (Braga et al., 2020) among others. Seed analysis is at a turning point when novel methodologies have been developed (Xing et al., 2023), and this is the case of hyper-spectral imaging technology (HIS) (Pang et al., 2022; Feng et al., 2021), X-Ray (Medeiros et al., 2021) and the case of biospeckle laser (BSL) technique, supported by Xing et al. (2023) as an emerging test of seed vigor. Therefore, as an emerging technique the DLS must be accessible to users in seed analysis laboratories, and the portability of the system plays a key point. Thus, the portability of DLS system, in this case, means that it can be accessible by final users, such as seed analysts.
This work aimed to test a digital mini microscope with an adapted diaphragm to reduce its size while guaranteeing a reliable speckle pattern in portable version accessible to seed analysis laboratories. The diaphragm inserted after the associated CMOS sensor and the macro was tested with different opening diameters and compared to the traditional setup to monitor drying paint and to provide seed analysis.
Material and Methods
The ability of the digital mini microscopes to acquire speckle images was tested with and without a diaphragm. The apertures of the diaphragm varied from 3 to 4 to 5 mm.
Traditional setup and proposed setup
Traditional setup
The Figure 1 shows the setup with the CCD camera and the macro zoom lens plus the diaphragm. The diaphragm was set in f-16 (Braga & González-Peña, 2016). The He-Ne laser was 632 nm, 10 mW from Coherent, USA, and the CCD was a JAI CV-S3200, 640 × 486 pixels, coupled with a macro zoom lens (Navitar’s ZOOM 7000-2) that had an adjustable iris (diaphragm) embedded. The intensity of the laser beam was adjusted by a variable optical density filter from Edmund Optics. The traditional setup has dimensions of 1140 mm in length, 560 mm in width and 570 mm in height, which results in a volume of approximately 0.36 m3.
Proposed setup using a mini microscope and diaphragm.
The proposed setup had a diode laser with a wavelength of 635 nm and a power of 3.0 mW (LRM-03/635 L, LASERLine, Amparo, Brazil). The beam was expanded using the objective lens of an optical microscope. A neutral filter from the Edmund Optics brand was used (transmission 10%; NIR ND Filter 1.0 OD; diameter 25 mm; thickness 3 mm).
Images were acquired using a mini microscope that has a 5-megapixel sensor and a resolution of 2592 x 1944 pixels (AM7013MZT, Dino-Lite, Torrance, CA, USA). An unbranded adjustable aperture diaphragm was attached to the mini-microscope after the macro zoom lens was integrated. It was positioned vertically over the sample at 48 mm, and the diaphragm inlet was positioned at 36 mm. The laser was fixed at 164 mm from the sample and at an angle of 40º. The proposed setup had dimensions of 210 × 220 × 190 mm (length, width, height), with a volume of 0.009 m3 (Figure 2).
Proposed setup used to acquire speckle patterns with a mini-microscope and diaphragm attached.
Four treatments were carried out with the diaphragm coupled in the mini microscope, and the diameters of the apertures were 3 mm, 4 mm, and 5 mm. Finally, the diaphragm was completely open (that is, without a diaphragm).
The relative angle between the mini microscope and the laser beam was tested, varying from 10o to 50o, monitoring the drying of a paint process by means of the BSL index AVD1.
Drying paint
Commercial nail enamel from the Impala brand, Nude Classic, was used, and the samples were prepared by applying a thin layer of nail enamel to the surface of a glass coverslip with a brush.
Images were acquired at a rate of 10 fps, at a resolution of 640 × 486 pixels and a grayscale of 8 bits pixel-1. After the nail enamel was applied, 128 images were acquired every 5 minutes for 30 minutes.
The experiments with drying paint were carried out with a CCD camera and a mini microscope. In the CCD camera, the adjustment of the diaphragm was f/16, and in the mini microscope, apertures of 3, 4 and 5 mm were tested, as well as the complete absence of an aperture.
Validation of drying paint using a scale
Gravimetric analyses consisted of monitoring the variation in the mass of samples at regular time intervals during a specific period. Thus, several studies have compared this technique with the results obtained with the dynamic laser speckle method (Amalvy et al., 2001; Blotta et al., 2011; Faccia et al., 2009).
Therefore, the variation in the weight of the nail enamel after drying was monitored over a period of 30 minutes; from the moment the nail enamel was first applied, the weight was measured every 5 minutes.
Seed analysis
The advanced tests were performed using biological samples, particularly the bean seed Phaseolus vulgaris L. Two treatments, with 36 seeds each, were tested: one related to high vigor seeds and the other related to high-vigor seeds passing through an early aging process. To achieve premature aging, the seeds were placed in germination box containers with 40% distilled water and then in a BOD incubator at 42°C for 96 hours (Ortiz, Gomes, & Takahashi 2024).
The experiments with seeds were carried out both in the setup with the CCD camera and with the mini microscope. The aperture of the CCD was f/16, and that of the mini microscope was 3 mm.
Image
Analysis of dynamic laser speckle
To obtain a quantitative value of the nail enamel drying process and the activity in seeds, the absolute value of the differences (AVD) was determined as a numerical index of dynamic laser speckle (or BSL) in biological samples. The AVD index that was applied in the sequences of images from the speckle patterns in the time domain is presented in Equation (1) (Braga, Pujaico-Rivera & Moreira, 2016).
where COM is the co-occurrence matrix obtained from the time history of the speckle pattern. The inner values of the COM represent the number of gray values in the pixels of the THSP. Normalization is the total summation of the occurrences in the COM, and i and j are the coordinates of the COM matrix that varies from 0 to 255 gray levels.
The time history of the speckle pattern (THSP) was constructed using random points in a Gaussian distribution. The random points selected in the first image stand still to all the images in time. In Figure 3, the red circles represent the Gaussian distribution of the points in the region of interest. The region of interest (ROI) was defined after the quality test protocol, which tested the embryo area of the bean seed for homogeneity, contrast and level of light in the speckle pattern (Braga, Pujaico-Rivera & Moreira, 2016).
Bean seed with red circles indicating the area from which the points of the THSP were constructed.
The Region of Interest was chosen considering the area of the seed most active during the germination process. And in the case of the bean seed, the region is in the embryo and the radicle. The data extracted from the ROI were related to a cloud of random points in a Gaussian distribution (Figure 3). The selection of the area is carried out by the user is an interactive way when running the application developed in GNU Octave with open access in www.nongnu.com\bsltl and detailed in Braga, Pujaico-Rivera and Moreira (2016).
Results and Discussions
Drying paint with a mini microscope
The monitoring of drying paint using a mini microscope with different diaphragm apertures is shown in Figure 4, where the normalized curves represent the drying process for 30 minutes using the AVD index of the biospeckle laser and the weight of the same drying process on a scale.
Evolution of the drying enamel observed using the proposed setup (AVD values) with a diaphragm of apertures 3 (red), 4 (blue) and 5 (green) mm in diameter, without the diaphragm (pink) and the loss of mass measured by a scale (black).
The profile of the curves presents the behavior of the setup with a 3 mm aperture closer to the weight process than the other treatments (4 mm, 5 mm and without an iris). Figure 5 shows that the dispersion images obtained using the weight as a reference confirm the efficiency of the 3 mm aperture with an R2 of 0.944 when linear fitting is adopted.
Dispersion graphics with the weight of the paint (enamel) compared to the diaphragms (a) 3 mm (b) 4 mm (c) 5 mm in diameter and (d) without an iris (aperture).
The profile of the curves presented by the use of a mini microscope reproduces those presented in the literature when DLS was adopted to monitor the drying paint (Amalvy et al., 2001; Blotta et al., 2011; Faccia et al., 2009), particularly when using a CMOS camera in a portable setup (Pérez et al., 2018).
In Figure 6, the results of the relative angle variation between the mini microscope and the laser are presented. The drying paint was monitored by the BSL numerical index AVD1 and the curves presented the same profile, with the relative angle of 50o presenting a profile not so close as the angles from 10o to 40o. This let we conclude that the set up presented robustness regarding the variation of the angle between the laser beam and the digital camera.
BSL numerical index of drying paint with relative angles between the camera and the laser beam varying from 10 to 50o.
The proposed setup using a range of relative angles between the laser and the camera presented the same evaluation of the traditional setup. The test of the angle proved that the proposed setup is robust and does not change the results when an adjustment of the setup varies. That condition allows the construction of the proposed setup by the users without compromising their results.
Drying paint with CCD and a coupled macro zoom lens
The results of the use of the traditional DLS setup to monitor the drying paint (enamel) can be observed in Figure 7. The adjustment of a linear function presented an R2 of 0.966, which is in close agreement with the mass loss measured by the scale during drying. R2 is close to the best R2 from the mini microscope with a coupled aperture of 3 mm, R2 = 0.944.
Dispersion graphics with the weight of the paint (enamel) compared to the traditional CCD and coupled macro zoom lens with a f/16 aperture.
The results presented by both setups using a well-known phenomenon show the reliability of the proposed setup.
Analysis of seeds
The results of DLS in seeds (high-vigor and early aged) were conducted separately regarding the traditional and the proposed setups. The laser intensity in both cases were different, and thus the final values of DLS numerical outcomes presented an offset. Therefore, two Analysis of Variance were conducted with the treatments been the high-vigor and the early aged condition of the seeds.
The results of the seed analysis using the traditional experimental configuration with CCD and macro zoom lenses can be observed in Table 1, where the analysis of variance (ANOVA) presented the ability to separate the high-vigor seeds from those seeds passed by the early aging process. The significance was set at the 5% level.
Where DF = degrees of freedom; SSQ = sums of squares; MSQ = mean square variance; and F = ratio of two mean square values.
Unfolding the ANOVA, one can see the Tukey test using the seed treatment as the source, where the means showed significant separation at the 5% level (Table 2).
The same approach was used for the proposed portable setup, and the ANOVA results are shown in Table 3.
The results of the Tukey test using the seed treatment as the source are presented in Table 4, where the means were significantly different at the 5% level.
The ability to analyze seeds using the DLS is reinforced by the results, which are consistent with the literature (Singh et al., 2020; Vieira & Braga Jr., 2020; Xing et al., 2023), particularly when sources of aging, such as temperature, compromise germination (Chatterjee, Disawal, R., & Prakash, 2017; Singh et al., 2022).
The proposed setup presents some advantages to seed analysis demands. The viability to use the proposed compact setup offers to seed analysts the adoption of a fast, non-subjective and less time-consuming system that can be placed in seed analysis laboratories. Efficiency in seed analysis was compared to traditional germination tests (Vivas et al., 2017), and the compact system proposed addresses the challenges of industry scaling presented by Zdunek et al. (2014) and Braga (2017).
The proposed setup presents some issues related to the traditional method, and the field of view is the main one. The field of view is reduced by the diaphragm after the macro zoom lens instead of the unlike the case of the integrated diaphragm adopted in the traditional CCD and macro zoom lens. However, despite the restriction caused by that, the application in seeds analysis isn’t compromised, since the Region of Interest (ROI) in the seed could be observed in full by the adapted 3 mm diaphragm.
Finally, one can note that the variety of devices known as mini microscopes demands adjustments in the size of the aperture and in the distance between the device and the sample. However, the results showed that the use of a diaphragm plays a key role in DLS images, mostly related to the change in the depth of field and the reduction in over-exposure to light in the optical CMOS sensor, which enhance the sharpness and contrast of the speckle pattern. The compact system proposed does not affect its use in seed analysis laboratories, since the environmental factors, temperature and humidity, should be the same of optical laboratories. The external light and mechanical vibration must be considered in the compact equipment using a case and an anti-vibration device under the optic table.
Conclusions
This work proved the feasibility to use an adapted digital mini microscope with a 3 mm diaphragm for DLS in seed analysis. The proposed setup presented linear correlation between the DLS activity and the weight of paint drying of R2 = 0.944, while the traditional setup was 0.966. The traditional and proposed setup separated, at the 5% level, high from low vigor seeds. The DLS should be considered as an alternative to traditional germination test of seed vigor analysis.
Acknowledgments
This work was partially supported by CNPq - Brazil (316938/2021-1).
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Editor de seção:
Renato Paiva
