Automatic separation system for marine meiobenthos based on laser-induced fluorescence technology

Automatic separation system for marine meiobenthos based on laser-induced fluorescence technology 1 first-author 1 †E-mail:gshaojia@hotmail.com †E-mail:gshaojia@hotmail.com gshaojia@hotmail.com 1 2 3 School of Life Science, Zhejiang University, Hangzhou 310027, China School of Life Science, Zhejiang University, Hangzhou 310027, China Second Institute of Oceanography, Hangzhou 310027, China Second Institute of Oceanography, Hangzhou 310027, China Department of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China Department of Mechanical Engineering, Zhejiang University, Hangzhou 310027, China An automatic system for marine meiobenthos separation was developed by using laser-induced fluorescence technology. Rose Bengal was used as organism dye and the spectrums of Rose Bengal were measured. Laser-induced fluorescence system was established to detect marine meiobenthos in sediments. Data obtained from experiments were analyzed by using a mathematical model. The results showed that laser-induced fluorescence technology worked well in the system. The system could select the meiobenthos efficiently and precisely. Meiobenthos Automatic separating system Optical sensor Laser-induced fluorescence (LIF) Rose Bengal INTRODUCTION 1 Meiobenthos is defined as marine meiofauna that can pass a screen with 250 μm apertures but cannot pass a screen with 32 μm apertures. It occupies about 80% of the total marine biomass and is of great importance in the marine ecology and the marine mineralogy (Mclntyre, 1969 1969 ). So that, meiobenthos investigation is a very active research field (Kitazato et al., 2003 2003 ; Gwyther, 2004 2004 ). Separating meiobenthos accurately from sediments is crucial in quantificative investigation of meiobenthos, and will influence the reliability and precision of research results. Various separation methods have been developed ever since meiobenthos research began. However most reported methods for separating meiobenthos from sediments are primarily based on the difference of specific mass between meiobenthos and sediment grains, which limit their application (Dillon, 1964 1964 ; Heip et al., 1974 1974 ; De Jonge and Bouwman, 1977 1977 ; Nichols, 1979 1979 ; Schwinghamer, 1981 1981 ; Jensen, 1982 1982 ). There is still no formal report on meiobenthos separation system development. At present, picking up meiobenthos manually under microscope is still ongoing in most Chinese and the foreign labs despite of the manual method’s large consumption of human power and time. In order to promote the efficiency and free scientists from the boring manual work, we developed an automatic separation system by applying laser-induced fluorescence (LIF) technology (Li et al., 1996 1996 ), which disregards the properties of the sediments. The system involves the technologies of biology, optics, electronics and mechanics. In this system, LIF is used to identify the organism, photoelectric transducers were used to detect the fluorescence signal, and an electronically controlled robot hand is used to collect the samples (The system is shown in Fig. 1 1 ). The LIF detecting technology is the main part of the automatic separation system. In this work, the fluorescence detecting system was established by measuring the spectra of the organism dye, Rose Bengal, to help choose the parameters of the optical parts. A mathematical model of the system signals was set up to analyze the experimental data. LIF SYSTEM SETTING 1 Organism dye 2 We chose Rose Bengal as the organism dye for its good chromaticity and high fluorescence quantum yield (Larkin et al., 1999 1999 ). In order to choose the photoelectric component with appropriate parameters, the fluorescence emission spectrum and absorption spectrum of Rose Bengal dissolved in basic ethanol (96% ethanol:water:2 mol/L NaOH=500:475:257) were measured. There were an obvious absorption peak at 550 nm and an emission peak at 580 nm in the spectra (Fig. 2 2 ). System setup 2 The key problem of the automatic separation system is to determine whether there is organism in the flume based on the weak optical output of the photomultiplier tube (PMT). The laser beam is first filtered, and then continuously focussed on the center of the transparent capillary by the lens. When there is dyed meiobenthos in the mixture liquid flowing through the laser beam, it will be induced to fluorescence with far more intense luminance than that of the background optical noise. When the dispersion laser is blocked by the filter, the PMT detects the fluorescence, and then proportionally generates electronic signals that will be sent to the electronic detector and controller. The compressed meiobenthos cause the fluorescence to project in one direction. Two detecting settings were arranged in different directions to catch more fluorescence signals (The optical layout is shown in Fig. 3 3 , Parameters of the optical parts of the setting are shown in Table 1 1 ). Parts Parameters Laser Wavelength λ 0 =532 nm; output power W 0 =5 mW; output aperture Φ =8 mm. Filter1 Narrow band-pass filters; center wavelength λ 0 =532 nm; bandwidth Δ λ =10 nm; transmittance≥50%. Filter2–3 High-pass filters: λ > 560 nm, transmittance≥80%; λ ≤560 nm, blocking≈10 −6 . PMT1–2 Luminous sensitivity (with 2856K light source) ν =2×10 8 V/lm; spectrum response at wavelength of 185 nm~900 nm and the maximum response at 600 nm; dark current of 1 nA; optical window s =7.0×16.0 mm 2 ; linearity of ±0.5%. MODELING ANALYSIS 1 The model of the LIF detecting system for the meiobenthos was set up, and the electronic signals of the PMT were analyzed. The intensity of the fluorescence I is defined by I =a Φ 0 I 0 (1−e − εlc ) where a is the constant of the instrument, Φ 0 is the fluorescence quantum yield, I 0 is the intensity of the laser, ε is the molar extinction coefficient, l is the light length, and c is the concentration. Because the concentration of the sample was very low, 1−e − εlc ≈ εlc , and the Eq.( 1 1 ) could be revised to I =a εlcΦ 0 I 0 With the intensity of the fluorescence I , the luminous flux F is defined by $$F = \int_{{\text{ }}0}^{{\text{ }}\Omega } {I{\text{d}}\Omega }$$ , where Ω is the solid angle. The fluorescence can be divided into many dot light sources. Considering the position of the PMT and the area of its optical window, the luminous flux F of the fluorescence received by the PMT is given by $$F{\text{ = a}}\varepsilon lc\Phi _0 I_0 \int_{{\text{ }}0}^{{\text{ }}s} {\frac{{f_i (r_i ,\Delta s_d ,\theta )}}{{r_i ^2 }}{\text{d}}s}$$ where s is the cross section of the meiobenthos. The position of the dot fluorescence is charactered by f i ( r i ,Δ s d , θ ), where r i is the distance between dot fluorescence source and the PMT, Δ s d is the optical area of the PMT, and θ is the angle between the fluorescence and the optical window of the PMT. $$\int_{{\text{ }}0}^{{\text{ }}s} {\frac{{f_i (r_i ,\Delta s_d ,\theta )}}{{r_i^2 }}} {\text{d}}s$$ is the intensity of all dot fluorescence. Therefore, at luminous sensitivity ν of the PMT, the output voltage of the PMT is given by $$V{\text{ = }}F\nu {\text{ = a}}\varepsilon lc\Phi _0 I_0 \nu \int_{{\text{ }}0}^{{\text{ }}s} {\frac{{f_i (r_i ,\Delta s_d ,\theta )}}{{r_i ^2 }}{\text{d}}s}$$ From the analysis above, we can conclude that the output voltage is mainly influenced by the parameters of f i ( r i ,Δ s d , θ ) and the size of the meiobenthos. RESULTS AND DISCUSSION 1 The electronic output of the PMT must interface well with the amplifier and be able to drive the collecting machine. The maximal velocity of the flow is 1 m/s, the gain of PMT is adjusted to 4×10 6 , the sampling frequency of the A/D transducer is 1024 Hz, data were collected every 2 s. A typical example is shown in Fig. 4a 4a . Four peaks in Fig. 4a 4a are shown in Fig. 4b 4b with expanded scales. The results are discussed in below. 1. As shown in Fig. 4a 4a , there are obvious peaks in the background of the steady noise signals. There is a very high voltage output of 1.5137 V, which almost reaches the A/D sampling output limit of 1.6 V. The peaks indicate high fluorescence intensity, which in turn indicate the appearance of dyed organism. 2. The maximum values of the peaks are different. The two results from the modelling analysis can explain this phenomenon. One of the reasons is the different size of the meiobenthos; the other is the angle θ between the fluorescence of the compressed meiobenthos and the optical window of the PMT. That is to say, the fluorescence intensity is not proportional to the size of the organism. 3. Data around every peak first increases and then decreases, which as shown in Fig. 4b 4b . The variation indicates that the organism flows with the water through the cross section of the laser beam and the change of the angle θ between the fluorescence of the compressed meiobenthos and the optical window of the PMT. 4. According to Fig. 4b 4b , every peak wave occupies about 6 points, indicating that every organism passes through the optical window of the PMT in a similar duration. Therefore, it can be concluded that the appearance of every meiobenthos corresponds to a 6-point wave. 5. The waves are not always so regular and clear as shown in Fig. 4b 4b (curves B and C). Actually, there are some strange conjoined waves. After the analysis, we conclude that if the length of the conjoined waves is less than 6 points, there is only one organism whose random motions in the flow cause the aberration of the wave as shown in Fig. 4b 4b (curve B), while if the length of two conjoined waves is over 6 points, there must be more than two organisms present proximately as shown in Fig. 4b 4b (curve C). According to the discussions above, experiments are successful. Excellent performance in separating meiobenthos is demonstrated by the obtained precision of above 95%. CONCLUSION 1 According to the experiment and the data analysis, the automatic system for separation of marine meiobenthos based on LIF technology can work well. It can select the organisms in the sediments efficiently and precisely. Compared with other conventional detecting methods, LIF technology has the advantages of easy set-up, high sensitivity and resolving ability. Laser induced fluorescence can improve the sensitivity about 2~10 times that of other light sources induced fluorescence. Now, the LIF technology has been developed well, and is widely applied in many fields (Kopp et al., 1997 1997 ; Chuck et al., 2002 2002 ; Michael et al., 2004 2004 ). Because the key part of the automatic separation system is based on LIF detecting technology, a similar technology used in many other biological detecting systems, such as flow cytometry (Lu et al., 2004 2004 ), the success of developing such a set of systems will be of great value in the development of other instruments. References RS Chuck , ME Arnoldussen , A Behrens . Fluorescence-guided laser removal of chemically damaged cornea . Journal of Cataract & Refractive Surgery , 2002 . 28 ( 10 ): 1847 - 1852 . . Fluorescence-guided laser removal of chemically damaged cornea VN De Jonge , LA Bouwman . 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Extraction of living meiofauna from marine sediments by centrifugation in a silica Sol-Sorbitol mixture 10.1631/jzus.2005.B0535 Project (Grant No. DY105-03-01-15) supported by “Tenth Five Year Plan of R & D of International Deep-Sea” of National Ocean Mineral Resources R & D Association, China 2004-09-09 2004-11-26 2005-05-22 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 6B