A PHOTON COUNTING TV CAMERA EQUIPPED WITH AN IMAGE GUIDE FOR RAPID DETECTION AND COUNTING OF SINGLE BACTERIA IN A WIDE FIELD

Abstract— We developed a photon counting TV camera system, which uses an image guide consisting of tapered optical fibers, for the rapid detection and counting of microscopic sized luminous particles in a wide field. Using a luminous bacterium as a standard, we compared this image guide-coupled TV camera to a lens-coupled TV camera by determining their light collecting powers and detection abilities for single bacteria on a membrane filter. The image guide shortened the detection time by an order of magnitude, making the photon counting TV camera a practical system for rapid counting of bacteria.     

Application of charge-coupled device technology for measurement of laser light and fluorescence distribution in tumors for photodynamic therapy

Laser and fluorescence light distributions with applications for photodynamic therapy were measured in mouse tumors using a non-invasive electronic optical imaging system. The system consists of a liquid-nitrogen-cooled, charge-coupled-device (CCD) array camera under computer control with 576 x 384 detection elements having dimensions of 23 microns x 23 microns. The available dynamic range of the array is approx. 10(3), and the effective wavelength range is 400-1000 nm. An interstitially placed cylindrical diffusing optical fiber was used to provide tumor illumination. The light distribution pattern from the fiber was determined by immersing the cylindrical diffusing tip in a fluorescing solution and recording the emission image. Fluorescence imaging facilitates an accurate measurement of light intensity distribution while avoiding problems associated with the directional nature of other detection methods used with diffusing fibers. Radiation-induced fibrosarcoma tumors on C3H mice were grown to about 1 cm diameter for in vivo recording of light distribution from the tumor volume and for determination of effective light penetration distance at 18 wavelengths in the range 458-995 nm. Endogenous tumor fluorescence and Photofrin II fluorescence intensity were measured over the wavelength range 585-725 nm to investigate the possible application of CCD imaging technology for drug distribution measurements. Model experiments were begun to evaluate the relative importance of potential distortions of light distribution measurements using this approach.     

ArtTAX - a new mobile spectrometer for energy-dispersive micro X-ray fluorescence spectrometry on art and archaeological objects

A newly developed spectrometer for energy-dispersive micro X-ray fluorescence spectrometry has been designed for the demands of archaeometry. ArtTAX combines the advantages of non-destructive and sensitive multi-elemental analysis at sub-mm resolution with the possibility of working outside the laboratory. The spectrometer consists of an air-cooled, low-power molybdenum tube, new generation polycapillary X-ray optics, a silicon drift detector without the need for liquid-nitrogen cooling, a CCD camera, and three light diodes for sample positioning. The motor-driven measurement head is fixed on a x,y,z-flexible tripod support which can be assembled and dismantled within minutes. The spot size of the primary X-ray beam was determined to be 94 microm for the Cu(Kalpha) energy, the detection limits are in a range of a few tens of microg g(-1) for the medium energy-range in glass. Additional open helium purging in the excitation and detection paths enables the determination of elements down to sodium, thus avoiding vacuum conditions or a size-limiting sample chamber. A selection of qualitative and quantitative results on pigment, metal, glass, and enamel analyses are presented to show the potential of ArtTAX in the field of art and archaeology.     

X-ray fluorescence imaging with polycapillary X-ray optics

X-ray fluorescence spectrometry imaging is a powerful tool to provide information about the chemical composition and elemental distribution of a specimen. X-ray fluorescence spectrometry images were conventionally obtained by using a μ-X-ray fluorescence spectrometry spectrometer, which requires scanning a sample. Faster X-ray fluorescence spectrometry imaging would be achieved by eliminating the process of sample scanning. Thus, we developed an X-ray fluorescence spectrometry imaging instrument without sample scanning by using polycapillary X-ray optics, which had energy filter characteristics caused by the energy dependence of the total reflection phenomenon. In the present paper, we show that two independent straight polycapillary X-ray optics could be used as an energy filter of X-rays for X-ray fluorescence. Only low energy X-rays were detected when the angle between the two optical axes was increased slightly. Energy-selective X-ray fluorescence spectrometry images with projection mode were taken by using an X-ray CCD camera equipped with two polycapillary optics. It was shown that Fe Kα (6.40 keV) and Cu Kα (8.04 keV) could be discriminated for Fe and Cu foils.     

Whole column fluorescence imaging on a microchip by using a programmed organic light emitting diode array as a spatial-scanning light source and a single photomultiplier tube as detector

A novel miniaturized, integrated whole-column imaging detection (WCID) system on a microchip is presented. In this system, a program controlled organic light emitting diode (OLED) array was used as a spatial-scanning light source, to achieve imaging by the time sequence of the excited fluorescence. By this mechanism, a photomultiplier tube (PMT) instead of a charge coupled detector (CCD) can be applied to the imaging. Unlike conventional systems, no lenses, fibers or any mechanical components are required either. The novel flat light source provides uniform excitation light without size limitations and outputs a stronger power by pulse driving. The scanning mode greatly reduced the power consumption of the light source, which is valuable for a portable system. Meanwhile, this novel simplified system has a broader linear range, higher sensitivity and higher efficiency in data collection. Isoelectric focusing of R-phycoerythrin (PE) and monitoring of the overall process with WCID were performed on this system. The limit of detection (LOD) was 38 ng mL(-1) or 3.2 pg at 85 nL per column injection of PE. The system provides a technique for WCID capillary isoelectric focusing (cIEF) on chip and can be used for throughput analysis.     

Lensless CCD-based fluorometer using a micromachined optical Söller collimator

In this paper, we describe a simple charge-coupled device (CCD) based lensless fluorometer with sensitivity in the range of current ELISA plate readers. In our lensfree fluorometer, a multi-wavelength LED light source was used for fluorophore excitation. To collimate the light, we developed a simple optical S?ller collimator based on a "stack of pinholes" (a stack of black PMMA with array of pinholes machined with laser) enabling the light to be collimated from the LED through the filters and the assay's microfluidics directly onto the CCD without a lens. The elimination of the lens that is used in almost all other current CCD based detection systems has four major advantages: (1) It simplifies the device design and fabrication while reducing cost. (2) It reduces the distance between the sample and the measuring device (without a lens the distance needed to focus the image on the CCD is reduced and the fluorometer can be more compact). (3) It couples the CCD and the detected surface by using an optical S?ller Collimator which allows the use of filters for fluorescence detection. (4) It also uncouples the CCD and the microfluidics to enable the use of interchangeable fluidics while protecting the delicate CCD. The lensless CCD-based fluorometer is capable of detecting 16 samples simultaneously, and was used for in vitro detection of botulinum neurotoxin serotype A (BoNT-A) activity with a FRET assay that measures cleavage of a fluorophore-tagged peptide substrate specific for BoNT-A (SNAP-25) by the toxin light chain (LcA). The limit of detection (LOD) of our lensless fluorometer is 1.25 nM, which is similar to the LOD of a modern ELISA plate reader. Combined with microfluidics, this simple low cost point-of-care (POC) medical diagnostic system may be useful for the performance of many other complex medical diagnostic assays without a laboratory and thus potentially enhancing the accessibility and the quality of health care delivery in underserved populations.     

Detection of particulate polycyclic aromatic hydrocarbons by laser-induced time-resolved fluorescence

Summary Laser-induced fluorescence is introduced as an analytical technique for the detection of particle-bound PAHs, which can be found as a result of most combustion processes. A quartz fiber is used to couple the light of a frequency-doubled excimer-pumped dye laser into the sensor head. The fluorescence light is detected using collecting optics, a set of interference filters and a photomultiplier. PAHs in different forms (crystalline, as homogeneous particles and coated on NaCl particles) were investigated. Fluorescence spectra and time-resolved signals, which exhibit characteristic decay times, are presented.     

Slot scintillation detector modeling for digital radiography

A scanning slot X-ray digital detector operating at relatively high photon energies, mainly 70 kVp for dental radiography, has been designed and modeled. In this energy range, further improvements are required in order to attain optimal image quality for the lowest achievable mean absorbed dose to the patient. This paper concerns the investigation of the physics of imaging using a structured screen composed of a scintillating fiber optic (LKH-6/CCD system) and its performance by Monte Carlo (MC) simulation. The A_s factor, X-ray cross-talk, quantum noise effect on resultant values of Detective Quantum Efficiency (DQE) and Modulation Transfer Function (MTF) are discussed. The results of this paper should serve as a useful guide in the engineering design of future structured screen/CCD based systems.     

A simple light-emitted diode-induced fluorescence detector using optical fibers and a charged coupled device for direct and indirect capillary electrophoresis methods

We constructed a simple fluorescence detector for both direct and indirect CE methods using a blue light-emitted diode (470 nm) as excitation source, a bifurcated optical fiber as a waveguide, and a CCD camera as a detector. The connection of all the components is fairly easy even for nonexperts and the use of a CCD camera improves the applicability of this detector compared to the others using PMTs because it permits the recording of 2-D electropherograms or phosphorescence measurements. This detector provides a compact, low cost, and rapid system for the determination of native fluorescence compounds which have high quantum yields by CE with direct fluorescence detection, showing an LOD of 2.6 x 10(-6) M for fluorescein; the determination of fluorescence derivative compounds by CE with direct fluorescence detection, showing an LOD of 1.6 x 10(-7) M for FITC-labeled 1,6-diaminohexane; and nonfluorescence compounds by CE with indirect fluorescence detection with an LOD of 2.7 x 10(-6) M for gallic acid.     

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser‐induced fluorescence detection

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK_a determination of small molecule analytes is demonstrated with the multilane microchip.     

PNA-based probe for quantitative chemiluminescent in situ hybridisation imaging of cellular parvovirus B19 replication kinetics

To allow the ultrasensitive localization and the quantitative detection of parvovirus B19 nucleic acids in single infected cells at various times post-infection, a peptide nucleic acid (PNA)-based in situ hybridisation (ISH) assay with chemiluminescent detection has been developed. The assay is based on the use of a biotin-labelled PNA probe detected by a streptavidin-linked alkaline phosphatase and a chemiluminescent dioxetane phosphate derivative substrate. The luminescent signal was quantified and imaged with an ultrasensitive nitrogen-cooled CCD camera connected to an epifluorescence microscope. The assay was used to analyze the parvovirus B19 infection process in cell cultures and to quantify the amount of viral nucleic acids at different times after infection.The chemiluminescent ISH-PNA assay is characterized by high resolution providing a sharp localization of B19 nucleic acids within single cells, with higher sensitivity with respect to conventional colorimetric ISH detection. Thanks to the high detectability and wide linear range of chemiluminescence detection, an objective evaluation of the percentage of infected cells, which reached its maximum at 24 h after infection, following a B19 virus infectious cycle could be accurately evaluated. Chemiluminescence detection also allowed the quantitative analysis of viral nucleic acids at the single-cell level, showing a continuous increase of the content of viral nucleic acids in infected cells with time after infection.The developed chemiluminescent ISH-PNA assay could thus represent a potent tool for the assessment of viral infections and for the quantitative evaluation of the virus nucleic acid load of infected cells in virus studies and diagnostics.     

Fluorescence detection system for capillary separations utilizing a liquid core waveguide with an optical fibre-coupled compact spectrometer

A fluorescence detection system for capillary liquid separation methods is described. The system is based on a silica capillary coated with a low refractive index fluoropolymer Teflon AF that serves both as a separation channel and as a liquid core waveguide (LCW). A fibre-coupled laser excites separated analytes in a detection point and arising fluorescence is collected at one end of the LCW capillary into the other optical fibre which brings it to a compact charge-coupled device (CCD) array spectrometer installed in a desktop computer. No additional components such as focusing optics or filters are necessary. This system was used for detecting isoelectrically focused fluorescent low-molecular-mass pI (isoelectric point) markers and fluorescein isothiocyanate (FITC) labelled proteins. The ability of the system to acquire fluorescent spectra is also demonstrated.     

Photoelectron microscopy and applications in surface and materials science

We review the recent achievements of photoelectron microscopy (PEM), which is a rapidly developing technique that is significantly advancing the frontiers of surface and materials science. The operation principles of scanning photoelectron microscopes (SPEM), using different photon optic systems to obtain a micro-probe of sub-micrometer dimensions, and of the full-field imaging microscope, using electrostatic lenses for magnification of the irradiated sample area, are presented. The contrast mechanisms, based on photon absorption and photon-induced electron emission, are described and the expected development in the photon and electron optics and detection systems are discussed. Particular attention is paid to the present state-of-art performance of the microscopes collecting photoelectrons (PEs), which carry specific information about the lateral variations in the chemical, magnetic and electronic properties of the material under investigation. Selected results, obtained recently with instruments installed at synchrotron light facilities, are used to illustrate the potential of PEM in characterising micro-phases and dynamic processes with a lateral resolution better than 100 nm.     

使用一维机械模板的高分辨阿达玛变换显微图像探测技术研究

阿达玛变换（HT）是一种类似于傅里叶变换（FT）的光谱调制技术,具有多通道同时检测和多通道成像能力等优点,但两者的数学模型、对光信号的调制方法和调制手段都不一样。由于HT仅涉及四则运算,而FT涉及较为复杂的三角函数和复数运算,所以HT的解码速度快于FT。在成像技术方面,HT具有直接成像的能力,而FT只能对通过其它方式获取的图像进行加工处理。     

Design and development of a field-deployable single-molecule detector (SMD) for the analysis of molecular markers

Single-molecule detection (SMD) has demonstrated some attractive benefits for many types of biomolecular analyses including enhanced processing speed by eliminating processing steps, elimination of ensemble averaging and single-molecule sensitivity. However, it's wide spread use has been hampered by the complex instrumentation required for its implementation when using fluorescence as the readout modality. We report herein a simple and compact fluorescence single-molecule instrument that is straightforward to operate and consisted of fiber optics directly coupled to a microfluidic device. The integrated fiber optics served as waveguides to deliver the laser excitation light to the sample and collecting the resulting emission, simplifying the optical requirements associated with traditional SMD instruments by eliminating the need for optical alignment and simplification of the optical train. Additionally, the use of a vertical cavity surface emitting laser and a single photon avalanche diode serving as the excitation source and photon transducer, respectively, as well as a field programmable gate array (FPGA) integrated into the processing electronics assisted in reducing the instrument footprint. This small footprint SMD platform was tested using fluorescent microspheres and single AlexaFluor 660 molecules to determine the optimal operating parameters and system performance. As a demonstration of the utility of this instrument for biomolecular analyses, molecular beacons (MBs) were designed to probe bacterial cells for the gene encoding Gram-positive species. The ability to monitor biomarkers using this simple and portable instrument will have a number of important applications, such as strain-specific detection of pathogenic bacteria or the molecular diagnosis of diseases requiring rapid turn-around-times directly at the point-of-use.     

A precipitator for the detection of thiophilic metals in aqua

The detection of toxic metals including mercury and lead has become a vital analytical tool for environmental remediation and regulation of food stocks. A prevalent obstacle with the current assessment of metal ion contamination originates from the lack of adequate assay throughput. In this context, a critical concern with current analyses stems from the fact that the majority of these assays are solution-based, and thus the response is highly dependent upon the assay environment. Herein, we describe a fluorescent dye-doped crystalline assay that offers convincing metal selection and provides detection comparable to conventional solution-based ligands used for the spectrofluorometric analysis of thiophilic heavy metal ions. While comparable in analytical performance to known methodologies, the formation of crystalline analytes provides for signal amplification and, consequently, a powerful platform whose analysis is directly amenable to high-throughput video capture systems. This procedure has been tested in a variety of scenarios and shows good performance using readily available equipment, including a commercially available Universal Serial Bus (USB) CCD camera. Furthermore, when developed in a microcapillary format, this assay is capable of screening thousands of samples per day for the presence of subnanomolar concentrations of Hg2+ using a conventional fluorescence microscope.     

A unique charge-coupled device/xenon arc lamp based imaging system for the accurate detection and quantitation of multicolour fluorescence

In recent years the use of fluorescent dyes in biological applications has dramatically increased. The continual improvement in the capabilities of these fluorescent dyes demands increasingly sensitive detection systems that provide accurate quantitation over a wide linear dynamic range. In the field of proteomics, the detection, quantitation and identification of very low abundance proteins are of extreme importance in understanding cellular processes. Therefore, the instrumentation used to acquire an image of such samples, for spot picking and identification by mass spectrometry, must be sensitive enough to be able, not only, to maximise the sensitivity and dynamic range of the staining dyes but, as importantly, adapt to the ever changing portfolio of fluorescent dyes as they become available. Just as the available fluorescent probes are improving and evolving so are the users application requirements. Therefore, the instrumentation chosen must be flexible to address and adapt to those changing needs. As a result, a highly competitive market for the supply and production of such dyes and the instrumentation for their detection and quantitation have emerged. The instrumentation currently available is based on either laser/photomultiplier tube (PMT) scanning or lamp/charge-coupled device (CCD) based mechanisms. This review briefly discusses the advantages and disadvantages of both System types for fluorescence imaging, gives a technical overview of CCD technology and describes in detail a unique xenon/are lamp CCD based instrument, from PerkinElmer Life Sciences. The Wallac-1442 ARTHUR is unique in its ability to scan both large areas at high resolution and give accurate selectable excitation over the whole of the UV/visible range. It operates by filtering both the excitation and emission wavelengths, providing optimal and accurate measurement and quantitation of virtually any available dye and allows excellent spectral resolution between different fluorophores. This flexibility and excitation accuracy is key to multicolour applications and future adaptation of the instrument to address the application requirements and newly emerging dyes.     

A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol

A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene microtiter plates; the sample volume was 20 mul and HRP-labeled CLB activity was immediately measured, using different CL substrates, after 10 min incubation time. Emitted light was recorded using a sensitive back-illuminated, cooled CCD camera or a conventional, photomultiplier-based micrtotiter plate reader. The developed method fulfills all the requirements of precision (CV below 10%) and accuracy (mean recovery from 96 to 110%) with a detection limit of 0.08 ppb in urine matrix. The use of 384-wells microtiter plate allows a 5-fold reduction in reagent quantity and the CL detection improves the detectability of the HRP-labeled tracer, thus reducing analysis time. The developed method is therefore suitable for high-throughput screening of CLB in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays, thanks to the possibility to optimize the system in non-equilibrium immunological conditions and with a very fast chemiluminescence detection of the HRP-label activity.     

A Smartphone-assisted Paper-based Analytical Device for Fluorescence Assay of Hg2+

Rapid, efficiency and portable detection systems in low-resource settings with limited laboratory equipment and technical expertise are urgently needed. Herein, an integrated platform composed of a paper-based analytical device and a smartphone detection system for Hg²⁺ onsite testing was developed. Nitrogen-doped carbon dots(N-CDs) were synthesized by a simple hydrothermal method using citric acid as the carbon source and ethanediamine as the nitrogen source, which gave out bright blue fluorescence under the excitation at 350 nm UV light and the absolute fluorescence quantum yield was 17.1%. The fluorescence of the prepared N-CDs can be effectively quenched by Hg²⁺. In addition, an external attachment of smartphone for illumination and external light interference was designed to trace the fluorescence signals, and a software application of Android system with simple operation program was developed to perform snapshot and image processing. The smartphone-assisted detection system was combined with the N-CDs decorated paper chip to achieve the sensitive detection of Hg²⁺ in water samples. This integrated method for reliable sensing of Hg²⁺ shows a good linear detection range of 10-800 μmol/L(R²=0.9595) with detection limit of 1.07×10^-8 mol/L.     

Genotyping from saliva with a one-step microdevice

This paper presents a disposable microfluidic device for on-chip lysing, PCR, and analysis in one continuous-flow process. Male-female sex determination was performed with human saliva in less than 20 min from spit to finish, and requiring only seconds of manual sample handling. This genetic analysis was based on the amplification and detection of the DYZ1 repeat region unique to the Y-chromosome. The flow-through microfluidic chip consisted of a single serpentine channel designed to guide samples through 42 heating and cooling cycles. Cycling was performed by matching the local channel geometry to a steady-state temperature gradient established across the microfluidic chip. 38 channel segments were designed for rapid low volume PCR, and four were optimized for spatial DNA melting analysis. Fluorescence detection was used to monitor the amplification and to capture the melting signature of the amplicon was performed with a basic 8-bit CCD camera. The microfluidic device itself was fabricated from microscope slides and a double-sided tape. The simplicity of the system and its robust performance combine in an elegant solution for lab-on-a-chip genetic analysis.