Laser-induced Fluorescence Imaging System Research Articles

Hyperspectral, Time-Resolved, Fluorescence Imaging System for Large Sample Sizes: Part I. Development of a High-Energy Line-Illumination Source

Abstract. To reduce the risk of foodborne illnesses, produce fields are visually surveyed prior to harvest for signs of fecal contamination. To improve the efficacy of surveys, a hyperspectral, line-scan, laser-induced fluorescence imaging system was developed. The goal is to incorporate the imaging system into a field-deployable apparatus to survey produce fields. The system includes a gated intensified camera, a spectral adapter, a 355 nm pulsed laser, and a Powell lens that is used to expand the laser beam into a line-illumination source. Software was developed to facilitate alignment of the Powell lens with the laser beam and the resulting line-illumination profile with the line-imaging field. Comparisons were made of uniformity and efficiency measures for regions within illumination profiles that corresponded to the camera field of view for the Powell lens and previously developed simple and homogenizing expansion optics. Spatial and temporal uniformity measures were similar for the Powell and homogenizing optics, and both were better than for simple optics; however, total efficiency was better for Powell compared to homogenizing optics at 28.5% and 3.0%, respectively. After spectral calibrations, theoretical and measured spectral peaks of five fluorescent standards were identical. Images of apples and spinach artificially contaminated with dilutions of dairy manure demonstrated high contrast. By selecting appropriate gate timing parameters, it was possible to create images in which responses from contamination sites were still evident but responses from normal surfaces were effectively extinguished. These results demonstrate that the system has potential to be used to detect sites of fecal contamination in produce fields. Keywords: Fecal detection, Fluorescence imaging, Food safety, Machine vision, Powell lens.

Hyperspectral, Time-Resolved, Fluorescence Imaging System for Large Sample Sizes: Part II. Detection of Fecal Contamination on Spinach Leaves

Abstract. To reduce the risk of foodborne illnesses resulting from fecal contamination in produce fields, a hyperspectral, line-scan, laser-induced fluorescence imaging system was developed with the goal of eventually incorporating the imaging system into a pre-harvest detection apparatus for fecal contamination. The imaging system includes an intensified, gated camera, a spectral adapter, a 355 nm pulsed laser, and laser expansion optics that produce a line-illumination profile. To validate and test the system, spinach leaves inoculated with dilutions of bovine fecal material were imaged repeatedly using a predefined set of imaging parameters. These images were used to evaluate methods for detecting fecal contamination. Methods investigated included ratio, edge, threshold, and slope detection. Differences in the magnitude of averaged intensities for the spectral range of 450-500 nm for regions within fecal contamination sites and in nearby uncontaminated surface areas suggested that the 450-500 nm waveband would be a good region for use in detection tests. Validation tests that used threshold or slope detection, the 450-500 nm waveband, and that took advantage of the slower fluorescence decay rates of fecal contamination sites relative to uncontaminated surface areas showed almost 100% detection of 1:2, 1:10, and 1:100 dilution sites and over 70% detection of 1:200 dilution sites with essentially zero false positives. These results suggest that the imaging system has potential for development of a commercially viable apparatus for pre-harvest detection of fecal contamination in produce fields and for detection of fecal contamination of leafy green vegetables in general. Keywords: Fecal detection, Fluorescence imaging, Food safety, Machine vision, Spinach, Time-resolved imaging.

Development and application of a high-speed planar laser-induced fluorescence imaging system to evaluate liquid and vapor phases of sprays from a multi-hole diesel fuel injector

A high-speed imaging system capable of acquiring elastic scattering images and planar laser-induced fluorescence (PLIF) images in a near-simultaneous fashion has been developed. Acquiring both elastic scattering and PLIF images enables the liquid phase to be discriminated from the vapor phase. High-speed imaging allows the temporal evolution of flow structures to be evaluated. Images of sprays from a multi-hole diesel fuel injector operating under engine-like conditions were acquired. The vapor phase images reveal intricate fluid dynamic structures that exhibit a high degree of variability, indicative of a turbulent gas jet.

Development of a laser-induced fluorescence imaging system for root activity and rhizosphere visualisation

Non-invasive methods that enable continuous monitoring of spatial and temporal variations of physical and chemical parameters in the roots and the rhizosphere in situ are required in order to study the complex soil–roots interactions. In this work we present the use of laser-induced fluorescence imaging for visualizing these processes. The system consists of a Nd:YAG Q-switched laser that excites the root and rhizosphere auto-fluorescence, and an intensified gated camera (intensified charged coupled device (ICCD)) that is synchronized with the laser so that it captures the very short auto-fluorescence signal even in broad daylight. By using very short gating times (1 ns) and varying the delay between the laser pulse and the gating operation, time-resolved fluorescence profiles are obtained for each pixel in the image. The potential of the system is illustrated with several examples that show that both fluorescence intensity and temporal evolution profiles provide information about root activity and root–soil interactions.

Characterization of a CH planar laser-induced fluorescence imaging system using a kHz-rate multimode-pumped optical parametric oscillator

The performance characteristics of a new CH planar laser-induced fluorescence (PLIF) imaging system composed of a kHz-rate multimode-pumped optical parametric oscillator (OPO) and high-speed intensified CMOS camera are investigated in laminar and turbulent CH4-H2-air flames. A multi-channel Nd:YAG cluster that produces up to 225 mJ at 355 nm with multiple-pulse spacing of 100 μs (corresponding to 10 kHz) is used to pump an OPO to produce up to 6 mJ at 431 nm for direct excitation of the A-X (0, 0) band of the CH radical. Single-shot signal-to-noise ratios of 82:1 and 7.5:1 are recorded in laminar premixed flames relative to noise in the background and within the flame layer, respectively. The spatial resolution and image quality are sufficient to accurately measure the CH layer thickness of ~0.4 mm while imaging the detailed evolution of turbulent flame structures over a 20 mm span. Background interferences due to polycyclic-aromatic hydrocarbons and Rayleigh scattering are minimized and, along with signal linearity, allow semi-quantitative analysis of CH signals on a shot-to-shot basis. The effects of design features, such as cavity finesse and passive injection seeding, on conversion efficiency, stability, and linewidth of the OPO output are also discussed.

Detection of Fecal Residue on Poultry Carcasses by Laser‐Induced Fluorescence Imaging

Feasibility of fluorescence imaging technique for the detection of diluted fecal matters from various parts of the digestive tract, including colon, ceca, small intestine, and duodenum, on poultry carcasses was investigated. One of the challenges for using fluorescence imaging for inspection of agricultural material is the low fluorescence yield in that fluorescence can be masked by ambient light. A laser-induced fluorescence imaging system (LIFIS) developed by our group allowed acquisition of fluorescence from feces-contaminated poultry carcasses in ambient light. Fluorescence emission images at 630 nm were captured with 415-nm laser excitation. Image processing algorithms including threshold and image erosion were used to identify fecal spots diluted up to 1: 10 by weight with double distilled water. Feces spots on the carcasses, without dilution and up to 1: 5 dilutions, could be detected with 100% accuracy regardless of feces type. Detection accuracy for fecal matters diluted up to 1: 10 was 96.6%. The results demonstrated good potential of the LIFIS for detection of diluted poultry fecal matter, which can harbor pathogens, on poultry carcasses.

Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

Detection of Contaminants on Poultry Processing Plant Equipment Using Laser-Induced Fluorescence Imaging

Fluorescence techniques have demonstrated great potential for detection of the presence of fecal and other biological substances that can harbor pathogens. We used a recently developed laser-induced fluorescence imaging system (LIFIS) to demonstrate the potential use of fluorescence techniques for detection of a range of diluted poultry feces from various sections of the digestive tract, including gizzard, duodenum, small intestine, ceca, and colon, on processing plant equipment. The use of the LIFIS allowed tunable excitation in the visible with selection of emission wavebands for multispectral imaging. Thus, both fluorescence excitation and emission parameters can be optimized (e.g., 415 nm excitation, and 580 and 630 nm emission bands). The results showed that 1:5 and 1:10 diluted feces samples could be detected with 100% detection rates at the 580 and 630 nm emission bands regardless of feces types. Detection rates for 1:50 and 1:100 diluted samples at the 580-nm band were 96.0 and 89.3%, respectively, and those at the 630-nm band were 94.7 and 84.0%, respectively. Even minute amounts of fecal matter on processing plant equipment surfaces, not clearly visible to the human eye, could be detected.

Laser-induced fluorescence imaging of plants using a liquid crystal tunable filter and charge coupled device imaging camera

We developed a laser-induced fluorescence imaging system for plant monitoring use, with which it was possible to make an image at any wavelength between 430 and 750nm. The excitation source for the fluorescence was a cw ultraviolet laser diode with 398nm, and the detector was an image-intensified charge coupled device. A liquid crystal tunable filter was used as the fluorescence wavelength selection device. All of the system performance including the wavelength tuning was electrically controlled, so that it could be operated with no mechanical vibration noise. The fluorescence images of a coffee tree leaf obtained at 440, 530, 685, and 740nm clearly showed a distribution pattern of the fluorescence intensity over the leaf. The pattern reflected the different physiological statuses of the plant. Advantages of the imaging system were experimentally discussed on a point of detection of inhomogeneous physiological activities over a plant leaf.

A Laser‐Induced Fluorescence Imaging System for Isoelectric Focusing*

This paper describes a laser‐induced fluorescence (LIF) imaging system for detecting proteins separated by isoelectric focusing (IEF). IEF was carried out in either microfabricated glass devices or in capillaries. Compared to the single‐point‐detection used in capillary IEF (CIEF), the imaging approach eliminates the need for mobilizing the focused proteins to the detector, thereby reducing proteins' de‐focusing during mobilization and improving separation resolution. The LIF imaging system consists of a laser; its diameter is first increased through a beam expander and subsequently the laser beam is focused into a line using a cylindrical lens. The resultant laser line is used to image an entire channel or capillary in which protein separation take place. The fluorescence emission is collected with a cooled, scientific grade charge‐coupled device (CCD) camera. The detection limit was determined using a series of concentrations of fluorescein solutions. The imaging system was demonstrated by isoelectric focusing of proteins, including Green Fluorescent Protein, B‐phycoerythrin, and R‐phycoerythrin. In addition, we can obtain temporal profiles of protein separation, providing useful information about the dynamic behavior of protein migration during IEF. *Portions of this work were presented in “Lab‐on‐a‐chip: platforms, devices, and applications” at the Optics East conference, Philadelphia, PA, Oct., 2004.

MULTISPECTRAL LASER INDUCED FLUORESCENCE IMAGING TECHNIQUES FOR NONDESTRUCTIVE ASSESSMENT OF POSTHARVEST FOOD QUALITY AND SAFETY

Ns-scale, time dependent fluorescence emission characteristics of apples contaminated with a range of diluted cow feces were acquired using a recently developed multispectral laser-induced fluorescence imaging system. Four spectral bands, F670, F680, F685 and F730, centered near the emission peak wavelengths of the major constituents responsible for the red fluorescence emissions from apples and animal feces were examined. The objective of the study was to investigate ns-scale fluorescence emission characteristics of samples to determine a suitable single red fluorescence band and optimal gate-delay time for detection of fecal contamination on apples. The results based on the ns decay curves show that 670 nm with 10 nm FWHM provides the greatest difference in time-dependent fluorescence responses of feces treated and untreated apples surfaces at a gate-delay of 4 ns from the laser excitation peak. With these detection parameters, fluorescence emissions from the diluted feces spot account for a minimum of 27% of the total emissions.

Uses of Hyperspectral and Multispectral Laser Induced Fluorescence Imaging Techniques for Food Safety Inspection

Spectral imaging including machine vision and multispectral imaging can provide a rapid, nondestructive means to assess agricultural commodities for their quality and safety for human consumption. These nondestructive techniques generally rely on reflectance measurements; the most commonly used spectral regions range through the visible (Vis) to the near infrared (NIR). Another optical sensing technique is based on fluorescence that is generally regarded as more sensitive optical sensing tools compared to reflectance techniques. Recently, we developed two imaging systems for food safety research; a laboratory-based hyperspectral imaging system capable of both reflectance and fluorescence measurements and a multispectral laser induced fluorescence imaging system. Because many contiguous spectral bands are acquired with each hyperspectral image pixel, the resulting data quantity makes uses of hyperspectral imaging spectrometer for on-line food safety inspection impractical. Instead, the spectral and spatial information is used to determine several optimal wavebands that can be implemented to a multispectral imaging system. We present a systematic method for using hyperspectral image data to identify few wavebands to be used in multispectral detection systems. Also presented is a two-band ratio as a multispectral fusion method along with automated detection for animal feces contamination on apples. The fluorescence imaging techniques demonstrated that even minute animal feces, not clearly visible to human eye, could be detected. Practical considerations for development of multispectral fluorescence imaging systems for on-line uses are also discussed.

Fluorescence sensing systems: In vivo detection of biophysical variations in field corn due to nitrogen supply

Leaf and canopy fluorescence properties of field corn ( Zea mays L.) grown under varying levels of nitrogen (N) fertilization were characterized to provide an improved N sensing capability which may assist growers in site-specific N management decisions. In vivo fluorescence emissions can occur in the wavelength region from 300 to 800 nm and are dependent on the wavelength of illumination. These light emissions have been grouped into five primary bands with maxima most frequently received from corn at 320 nm (UV), 450 nm (blue), 530 nm (green), 685 nm (red), and 740 nm (far-red). Two active fluorescence sensing systems have been custom developed; a leaf level Fluorescence Imaging System (FIS), and a canopy level Laser Induced Fluorescence Imaging System (LIFIS). FIS sequentially acquires high-resolution images of fluorescence emission bands under darkened laboratory conditions, while LIFIS simultaneously acquires four band images of plant canopies ≥1 m 2 under ambient sunlit conditions. Fluorescence emissions induced by these systems along with additional biophysical measures of crop condition; namely, chlorophyll content, N/C ratio, leaf area index (LAI), and grain yield, exhibited similar curvilinear responses to levels of supplied N. A number of significant linear correlations were found among band emissions and several band ratios versus measures of crop condition. Significant differences were obtained for several fluorescence band ratios with respect to the level of supplied N. Leaf adaxial versus abaxial surface emissions exhibited opposing trends with respect to the level of supplied N. Evidence supports that this confounding effect could be removed in part by the green/blue and green/red ratio images. The FIS and LIFIS active fluorescence sensor systems yielded results which support the underlying hypothesis that leaf and canopy fluorescence emissions are associated with other biophysical attributes of crop growth and this information could potentially assist in the site-specific management of variable-rate N fertilization programs.