Non-contact and Non-invasive Photonic Device for Qualitative ... - wseas

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investigation and bio-chemical analysis, a non- ... 9th WSEAS Int. Conf. on MATHEMATICS & COMPUTERS IN BIOLOGY & CHEMISTRY (MCBC '08), Bucharest, Romania, June 24-26, ..... [1] Jagdish Singh, Surya Thakur Ed., Laser Induced.
9th WSEAS Int. Conf. on MATHEMATICS & COMPUTERS IN BIOLOGY & CHEMISTRY (MCBC '08), Bucharest, Romania, June 24-26, 2008

Non-contact and Non-invasive Photonic Device for Qualitative Fungal Contamination Control LAURENTIU ANGHELUTA1, JOAKIM STRIBER1, ROXANA RADVAN1 , IOANA GOMOIU2 ,VIVIAN DRAGOMIR3 1 National Institute of Research and Development for Optoelectronics INOE 2000 1 Atomistilor Str., Magurele MG5 ROMANIA 2

National University of Arts from Bucharest, 19 G-ral Budisteanu, 1 Bucharest ROMANIA 3

Village Museum “Dimitrie Gusti” Sos. Kiseleff nr. 28-30, Bucharest ROMANIA

Abstract: - This paper presents a complex portable photonic setup that exploits LIF advantages for biological contamination control of a surface and a case study that validates the investigation procedure. Based on LIF method the necessary information for microbiological species discrimination is collected from distance in realtime, without mechanical contact and preserving surface reliefs. Key-Words: laser, fluorecence, spectroscopy, microbiology, scanning device high energy levels can decay to lower levels by emitting radiation. This applied method is a molecular emission spectroscopic technique based in the absorption of light emitted by a laser. [1] The LIF spectrum of the each specie provides information directly related to the molecular structure of materials on the irradiated area. Besides the molecules in a molecular substrate, or some impurities or crystal defects into solids, the microbiological species are responsible of specific fluorescence emissions. Pulsed UV laser is succesfully and often used for inducing fluorescence because solid substances have broad absorption bands in the UV region. Due to the laser design field development, pulsed UV laser are now available and portable LIF systems are reachable. Such devices can assure on site high resolution identification of small traces of substances, chemical compounds or, even, incipient biocontaminations. It is useful to add that LIF results can be associated with high-resolutin thermographie, and high-resolution 3D models obtained by laser scanning, obtaining diagnostics of complex structures at a moment. [2] This paper presents a complex portable photonic setup that exploits LIF advantages for biological contamination control of a surface and a case study

1 Introduction As you can see for the title of the paper you must Beyond the naked eye observation, accurate optical investigation and bio-chemical analysis, a noncontact and non-invasive device is the desired mean for surface contamination control. Over all these advantages, a fast and accurate technique is an advanced task. In the last decades a lot of studies in the field of laser interaction with the matter were made. Among the most important challenges in various applications, investigation and analysis techniques of the surfaces based on the laser interactions on materials faced serious requests in biological application. Two investigation methods that are using the laser beam as the surface composition detection processes inductor are the laser induced breakdown spectroscopy (LIBS) and laser induced fluorescence spectroscopy (LIF). Within these two methods, it is applied laser radiation over a surface producing interaction processes that are monitored, analyzed and interpreted by the means of some dedicated devices and software applications. As it is wellknown, every material has its own characteristic electromagnetic absorption and emission spectrum, and LIF method speculates this phenomena. Atoms or molecules that are excited to

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that validates the investigation procedure. There is large range of applications from dermatology, food [4] and pharmaceutical industies to cultural heritage conservation [5].

The irradiation source L is a solid state passive Qswitched diode pumped laser that emits at 266 nm with a maximum repetition rate of 3 KHz at 1,25 μJ of energy per pulse. The L1 lens focuses the laser beam on the scanned surface. For detection it was used a low wavelength high sensitive spectrometer, best suited for fluorescence acquisition, detecting light radiation in the 200-1100 nm bandwidth with a 90% quantum efficiency. To synchronize the irradiation spot’s position changes with the spectra emission acquisition, it was used a device that allows the external software triggering. The TRIG break-out box is the communication link between the spectrometer and the computer or other triggering devices. The redirecting mirror’s rotational ensemble device is constituted by two stage step motors. These motors can proceed at high precision rotations, with a minimum step of 0.0005˚ with a maximum speed of 80˚/s. Both motors are connected to controller CTRL to communicate with the computer through a serial port. A software application designed under the LabView platform ensures the full control of the motors operation. The optical collector L2 is a 25.4 mm diameter collimating lens. This lens transmits the collected data to the spectrometer SP through the optical fiber FO. In front of the collector it is mounted the UV filter F to avoid the acquisition of the incident laser beam reflection. Being design as portable and stable device, the whole ensemble is constructed on a solid platform, all the components being arranged and fixed on it as the figure shows. [3] Critical aspects that must be faced are regarding the acquisition timing synchronization with the laser

2 Portable LIF scanning device setup Within LIF method the necessary information for contamination discrimination will be picked up from distance in real-time, without mechanical contact and preserving surface relief. Point by point interrogation of a surface offers detailed data about that surface quality. Therefore an automated device that could actually scan a defined area of that surface based on a certain algorithm using an acquisition technique that can provide us with the desired data is developed. Laser scanning of the area implies the redirection of the laser beam, emitted by the source, to every spot of that area (respectively with the input resolution) following a predefined algorithm. The laser beam is optically deflected by mirrors system mounted on a two stage motors ensemble, controlled by a computer that allows the high precision two central axes rotation of the mirror (0.0005˚). The laser beam interacting with the surface will induce the fluorescence emission of the molecules that will be propagated in all directions. Partly, the emitted fluorescence falls on the redirecting mirror and is transmitted through an optical ensemble to a detection device. The optical ensemble is constituted by a perforated mirror, which allows the laser beam to propagate through its aperture but reflects the fluorescence emission that comes from the redirecting mirror; another redirecting mirror that reflects the fluorescence emission through-out an UV filter (266 nm) up to the optical collector; optical fiber for information transmission to the spectrometer.

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Fig. 1. Laser induced fluorescence scanning setup

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focus point right on the surface that has to be scanned. To identify each characteristic spectrum of the surface’s contaminated areas – as a restorers request – there have been made LIF analysis at different interogation duration times.

spot repositioning. LIF emission takes place in a short interval after the initial light absorption event, in around 10-15 s. The fluorescence lifetime, the total time spent in the excited electronic state, is of the order of few nanoseconds to microseconds. P1

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Fig. 2. Waiting time condition The acquisition time [Tac] represents the duration of time while the surface is being irradiated for one signal acquisition. The waiting time [Tw] set in the scanning software interface represents the duration of a step cycle, including the triggeration time [Tt], acquisition time [Tac] and the motor repositioning duration time [Tp]. The duration time of a full scanning process is a multiple of Tac. To synchronize the laser spot repositioning with the induced emitted fluorescence radiation, the waiting time before motor rotation up to the new position [Tw] must be equal or greater with the sum of the triggering time [Tt], the motor repositioning duration time [Tp] and the acquisition time [Tac]. (Fig.2 ) The main goal of these mapping interpretations is to detect a specific known fluorescence characteristic of biological attacks over the surface, helping the researcher to have a better knowledge of the objects surface composition and to prevent the further negative effects of the biological attacks.

Fig. 3. a - The analysed icon a.-front side

3 Case study The case study validates the described device efficiency and proper functioning, performing a high resolution scan on an old wood painted Orthodox icon (from 1867) belonging to open air Village Museum „Dimitrie Gusti” from Bucharest, trying to detect and identify the microbial contamination based on different fluorescence emissions. To demonstrate biunique relationship between record fluorescence signals and cultivation methods results, the experiment was developed in both ways. As it can be seen in Fig. 3, the 353x295 mm area of the icon’s back side surface containing degradated and stained areas, dirt deposition, was selected . Using a laser diode that emits in the visibile spectrum, the operator adjusts the optical collector

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Z2 X1 Fig. 3.b The analysed icon b- back side

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To obtain a good spectrum shape the interogation time for the acquisition was set to 500 ms. The acquisition aplication was set to record only the average intensity of several 10 nm bandwidths of the characteristic spectrum, Fig.4-f , (445-455 nm, 495505 nm, 550-555 nm, 595-605 nm, 655-665 nm) to fasten the acquistion process and to ease the interpretation work. For discussion, the attention is focussed on a 10x10 mm area. This scanned area can be seen in Fig. 4-a. The scanning resolution was set at 0.2 mm per step (on horizontal and vertical axes) taking advantage of the high precision laser spot positioning. This means a total 50x50 points to be interogated. The distance between the repositioning mirror and the scanned surface was 1 m. Acquistion time Tac was set at 500 ms and the wating time Tw at 550 ms. After choosing the starting point and with the other scan parameters all set, the scanning process is started and the laser spot is repositioned each time for a new acquisition, following the pattern provided by the programmed algorithm.

Fig.4 c.Intensity distribution map 450 nm bandwidth (Z2 area)

Fluorescence intensity distributions maps - (Z2) versus:

Fig.4d. Intensity distribution map 500 nm bandwidth (Z2 area) Fig. 4 a. Macro Mode Picture (Z2 area)

Fig. 4e. Intensity distribution map 555 nm bandwidth (Z2 area)

Fig. b. RGB scanning representation 4-2-0 (Z2 area)

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From these specific points classical microbiological sample have been collected and processed. The sampling areas were established taking in consideration the macroscopic deterioration of wood and specific morphology of fungal growth on the wood surface. Samples had been taken with a sterilized cotton swab and with a sterile tweezers and introduced in 9ml of distilled water containing 0,2 mg/ml1streptomycine. Then each 1ml of suspension was put in Petri dishes and poured nutrient medium (malt extract-agar). Finally, those 9 Peri dishes had been incubated at 28°C for 14 days. Fungal genera had been identified using pure culture and analytical keys. Using cultivation methods for samples took from X1 area it was isolated Acremonium sp with white mycelium, slender; conidiophore is simple upright bearing conidia hyaline, 1 celled borne singly. Using the same method for samples took from Z2 area were isolated 3 fungal strains: Sclerotium sp., Penicillium sp 1 and Penicillium sp 2. (Fig.6 a) Sclerotium sp has white mycelium and spores lacking; sclerotia are olive to brown to blackglobose or irregular Penicillium sp 1 and Penicillium sp 2 have fluffy mycelium with conidiophores branched near the apex to form a shape like brush; phialides bear conidia in dry chains; conidia are hyaline or brightly colored mostly blue-green, globose or ovoid.

In the Fig.4 there is represented information regarding the selected area: Fig.4 –a - shows the macro mode picture of the scanned area. The scanned area contains fungi mycelia on wooden surface. Fig.4 –b - Represents the RGB visualisation of the scanned data, constituted from three different bandwidth channels: R for 660 nm, G for 555 nm and B for 450 nm. Fig.4 - c. to e. depicts the intensity distribution map of the scanned data for each bandwidth of interest: 450 nm, 500 nm, and 555 nm. This representation uses a three color gradient to illustrate the distribution map of the intensities of each bandwidth of the emitted fluorescence as follows: the brightest areas represents the highest intensities, while the bluer, darker colored areas represents the regions with the lowest fluorescence emittance. Fig.4 -f. represents a full spectra acquisition, depicting the LIF characteristic of the fungus collony. This characteristic is influenced in small measure by the underlying material, the wood. This spectrum will be compared with the spectrum of the cultivated sample. Part of the numerous selected points that responds to the laser stimulation by fluorescence emission having a large enough development have been detected and captured by high magnification optical microscopy (up to 500 x). Fig. 5 emphasized presents hyphae and mycelium (Fig.5 a- from Z2 area, and FIG.5 b- from X1 area).

6a. Sclerotia

Mycelium

Fig. 5a – Z2 area

Penicillium 6c. sp.

Fig. 5b – X1 area

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areas shape, and the possibility to have qualitative information about the existing species. The presented setup is part of the mobile laboratory based on photonic techniques for artwork investigation and diagnosis. Future developments of the spectra databases will increase the diagnosis efficiency. An exhaustive database will lead to significant investigation time winning.

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References: [1] Jagdish Singh, Surya Thakur Ed., Laser Induced Breakdown Spectroscopy, Elsevier ISBN-13: 978-0-444-51734-0, 2007; [2] Simileanu, M., Maracineanu, W., Striber, J., Deciu, C., Ene,D., Angheluta,L., Radvan,R., Savastru, R., Advanced research technology for art and archaeology – ART4ART mobile laboratory -JOAM, vol.10, nr.2, 2008 , pg. 470473 [3] Simileanu, M., Maracineanu, W., Deciu, C., Striber, J., Radvan, R., A complex portable optoelectronic setup for on-site interventions: case studies, SPIE Proc., 2006, VOL 6345, pages 63450U, ISSN-0277-786X [4] F. Morales, C. Romero, and S. Jiménez-Pérez, in Food Chemistry, Vol. 57, 3, 1996

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Wavelength [nm] 6d. Fig.6. a. Cultivation method results; b-. LIF spectrum of Sclerotia., c- LIF spectrum of Penicillinum sp.,d- LIF spectrum of Mycelium . In spite of the evident fluorescence signals that were obtained between 450 and 555 nm from the scanned surface of the object, the punctually LIF spectra on the cultivated samples in laboratory were necessary for biunique relationship between record fluorescence signals. The same experimental condition have been used for the analysed isolated species (Fig.6 a). The significant peaks for each isolated species are found on the same spectral range of 450-550 nm.

4 Conclusions In summary the use of LIF method - exploited on site due to a portable complex device - is an extremely useful tool for microbiological contamination control. As a non-contact and non-invasive method, it must be used especially for fragile, deteriorated, or dangerous surfaces inspection. Over all mentioned advantages and in spite of the limited efficiency in case of the materials with low fluorescence response, the presented case study prooves the detection accuracy of contaminated

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