A solid-state,portable instrument for measurement of chlorophyll luminescence induction in plants

A newly developed compact instrument is described for the measurement of chlorophyll luminescence induction in plants. The instrument operates with a pulsed light emitting diode (LED) as light source and a photodiode as luminescence detector. A special emitter-detector geometry provides for high irradiance of the sample and efficient collection of luminescence by the detector. With insertion of appropriate filters the same probe is also suited for measuring prompt chlorophyll fluorescence. The instrument shows considerable flexibility with respect to pulse frequency, relative lengths of light/dark intervals and luminescence sampling periods. Due to a selective amplifier system only that part of luminescence is processed which is induced by the individual excitation pulses. By this approach, the problem of slow phase accumulation, encountered with conventional phosphoroscopes, is eliminated. Some examples are given for system operation, demonstrating satisfactory performance in measurements with intact leaves and isolated chloroplasts.     

Hyperfine structure in melting profile of bacteriophage lambda DNA.

A high-resolution plotter of differential melting profiles of DNA, RNA, or related biopolymers with an on-line mini-computer is described. With this device, more than 15 transition steps were identified in the thermal melting profile of DNA from bacteriophage lambda. These fine structures were found to be reproducible, and some of them disappear in the deletion mutant. To Examine the melting profile, computer simulations for several hypothetical polynucleotide sequences were performed, and compared with experimental data. The sharp peaks that appeared in the differential melting profile of λ DNA may come from some homogeneous sequences of 500 bases or longer.     

Automated preparation of DNA sequences for publication.

A computer program which draws DNA sequences is described. A simple method is used which enables the user to highlight or annotate specific parts of a sequence. The sizes of the characters in the sequence to be drawn are specified by the user. In addition, vertical spacing between lines and horizontal spacing between characters can be specified. Sequences can be prepared and high quality output produced on a plotter in a short period of time, making the program advantageous to use over typing, computer printing, or preparation by a graphics department.     

Development of a 3‐dimensional tissue lung phantom of a preterm infant for optical measurements of oxygen—Laser‐detector position considerations

There is a need to further improve the clinical care of our most vulnerable patients—preterm infants. Novel diagnostic and treatment tools facilitate such advances. Here, we evaluate a potential percutaneous optical monitoring tool to assess the oxygen and water vapor content in the lungs of preterm babies. The aim is to prepare for further clinical studies by gaining a detailed understanding of how the measured light intensity and gas absorption signal behave for different possible geometries of light delivery and receiver. Such an experimental evaluation is conducted for the first time utilizing a specially developed 3‐dimensional‐printed optical phantom based on a geometry model obtained from computer tomography images of the thorax (chest) of a 1700‐g premature infant. The measurements yield reliable signals for source–detector distances up to about 50 mm, with stronger gas absorption signals at long separations and positions related to the lower part of the lung, consistent with a larger relative volume of this. The limitations of this study include the omission of scattering tissue within the lungs and that similar optical properties are used for the wavelengths employed for the 2 gases, yielding no indication on the optimal wavelength pair to use.      

On-line green fluorescent protein sensor with LED excitation

We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:921-926, 1997.     

FLIM on a wide field fluorescence microscope

Summary FLIM (Fluorescence Lifetime Imaging Microscopy) is a new tool to detect interaction between proteins. The proteins under investigation are fused with fluorescent donor and acceptor molecules. Interaction between the two proteins is accompanied by direct energy transfer from donor to acceptor (FRET), resulting in a shorter lifetime of the fluorescence emitted by the donor molecule. This change in lifetime is detected by FLIM. Fluorescence lifetime imaging can now be done on a widefield fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an Accousto Optical Modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. Power supplies and signal generator are integrated in one control unit that is connected to the light source, detector and computer. All parameters for image acquisition, processing and viewing are easy accessible in the user interface of the software package that uses a modular structure. Lifetime images showing FRET in MCF7 cells with ErbB1-GFP as donor and Py72/Cy3 as acceptor that were taken at EMBL, Heidelberg are shown.     

C.R.T. spot-follower device for eye-movement measurements

Summary An eye-movement detector, using the reflection of a light beam on the cornea and a properly designed feedback loop to counterbalance the ocular rotation by suitably adjusting the position of the light source, is presented. The displacement of the light source is transduced into an electrical signal whose functional dependence on the eye rotation is linear, to a very good approximation, for a proper choice of the parameters of the system. Both two-dimensional and temporal evolution of the eye-movements in any direction, as well as velocity and acceleration components along two perpendicular axes, can be measured and recorded. The range is of the order of 60°, with a noise level of about 10 and a bandwidth of 100 Hz. Some preliminary results, in particular on the saccadic movements during free observation, are presented.     

Investigation of the source‐detector separation in near infrared spectroscopy for healthy and clinical applications

Understanding near infrared light propagation in tissue is vital for designing next generation optical brain imaging devices. Monte Carlo (MC) simulations provide a controlled mechanism to characterize and evaluate contributions of diverse near infrared spectroscopy (NIRS) sensor configurations and parameters. In this study, we developed a multilayer adult digital head model under both healthy and clinical settings and assessed light‐tissue interaction through MC simulations in terms of partial differential pathlength, mean total optical pathlength, diffuse reflectance, detector light intensity and spatial sensitivity profile of optical measurements. The model incorporated four layers: scalp, skull, cerebrospinal‐fluid and cerebral cortex with and without a customizable lesion for modeling hematoma of different sizes and depths. The effect of source‐detector separation (SDS) on optical measurements' sensitivity to brain tissue was investigated. Results from 1330 separate simulations [(4 lesion volumes × 4 lesion depths for clinical +3 healthy settings) × 7 SDS × 10 simulation = 1330)] each with 100 million photons indicated that selection of SDS is critical to acquire optimal measurements from the brain and recommended SDS to be 25 to 35 mm depending on the wavelengths to obtain optical monitoring of the adult brain function. The findings here can guide the design of future NIRS probes for functional neuroimaging and clinical diagnostic systems.      

Time, a quality-control parameter in flow cytometry

Time has been used as a quality-control parameter in our flow cytometer. This parameter was automatically incorporated into the list-mode data base by hooking the computer clock directly into the acquisition logic. Quality control can then be checked by "replaying" fluorescence or light scatter data vs. time and any instrumental drift can be observed and degraded data can be excised. The technique is illustrated with a number of DNA data sets from a tissue culture cell line in which artificial perturbations were introduced during data acquisition to simulate potential causes of instrumental drift.     

DNA biosensor for detection of Neisseria gonorrhoeae causing sexually transmitted disease

A sequence-specific electrochemical sexually transmitted disease (STD) sensor based on self-assembled monolayer of thiolated DNA probe specific to target opa gene for detection of Gonorrhoea, a sexually transmitted disease has been fabricated. 6-Mercapto-1-hexanol (MCH) has been used as a blocking agent to facilitate oligos "stand" up at the surface, a configuration favoring subsequent DNA hybridization and to repel non-specific adsorption of undesired DNA. The results of differential pulse voltammetric studies of this STD sensor reveal low detection limit (1.0 × 10(-18)M) and a wide dynamic range (from 1.0 × 10(-6)M to 0.5 × 10(-18)M) arising due to the stable hybridization using methylene blue as an electro-active DNA hybridization indicator. The experimental results with genomic DNA, clinical patient sample of Neisseria gonorrhoeae, culture of non-N. gonorrhoeae Neisseria species (NgNS) and gram negative bacteria indicate that the fabricated sensor is specific to this STD.     

Ion chromatographic study of sodium,potassium and ammonium in human body fluids with bulk acoustic wave detection

In the present paper, determination of Na⁺, K⁺ and NH₄⁺ in saliva and serum was carried out using an ion chromatography (IC) method with a bulk acoustic wave (BAW) sensor as detector and 2.0 mmol/l nitric acid as mobile phase. The IC–BAW method is simple, rapid and accurate. Comparison between the BAW detector and the conventional conductivity detector (CDD) was discussed. The IC–BAW showed agreement with the commonly used flame photometric method for Na⁺ and K⁺ and enzymatic method for NH₄⁺, as well as IC–CDD for those three cations.     

On-line data acquisition and evaluation of long-term measurements of circadian rhythms in individual cells of Acetabularia

A multitasking time-sharing computer system was implemented for studies of different circadian rhythms in individual cells of the unicellular green alga, Acetabularia. This fully automatized system allows simultaneous data acquisition and analysis. Graphical presentation of untreated and mathematically treated data is permanently available on three graphic displays and on a digital plotter. The sampling rate for the data acquisition in each of the 60 channels connected to the system is 720/24 h. Provisions have been made to guarantee uninterrupted data uptake for these long-term measurements by including an auto-restart module and by providing extremely reliable software for the experimenter using menu techniques.     

The "Flash-Lag" effect occurs in audition and cross-modally

In 1958 MacKay showed that a rigidly moving object becomes visually fragmented when part of it is continuously visible but the rest is illuminated intermittently. For example, the glowing tip of a lit cigarette moving under stroboscopic illumination appeared to move ahead of the intermittently lit body. Latterly rediscovered as "the flash-lag effect" (FLE), this illusion now is typically demonstrated on a computer monitor showing two spots of light, one translating across the screen and another briefly flashed in vertical alignment with it. Despite being physically aligned, the brief flash is seen to lag behind the moving spot. This effect has recently motivated much fruitful research, prompting a variety of potential explanations, including those based on motion extrapolation, differential latency, attention, postdiction, and temporal integration (for review, see ). With no consensus on which theory is most plausible, we have broadened the scope of enquiry to include audition and have found that the FLE is not confined to vision. Whether the auditory motion stimulus is a frequency sweep or a translating sound source, briefly presented auditory stimuli lag behind auditory movement. In addition, when we used spatial motion, we found that the FLE can occur cross-modally. Together, these findings challenge several FLE theories and point to a discrepancy between internal brain timing and external stimulus timing.     

High-performance confocal system for microscopic or endoscopic applications

We designed a high-performance confocal system that can be easily adapted to an existing light microscope or coupled with an endoscope for remote imaging. The system employs spatially and temporally patterned illumination produced by one of several mechanisms, including a micromirror array video projection device driven by a computer video source or a microlens array scanned by a piezo actuator in the microscope illumination path. A series of subsampled "component" video images are acquired from a solid-state video camera. Confocal images are digitally reconstructed using "virtual pinhole" synthetic aperture techniques applied to the collection of component images. Unlike conventional confocal techniques that raster scan a single detector and illumination point, our system samples multiple locations in parallel, with particular advantages for monitoring fast dynamic processes. We compared methods of patterned illumination and confocal image reconstruction by characterizing the point spread function, contrast, and intensity of imaged objects. Sample 3D reconstructions include a diatom and a Golgi-stained nerve cell collected in transmission.     

Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.     

Microdensitometer system for microradiography

Summary A microdensitometer system for point measurements in areas down to a few square micrometers is described. The detector system is connected to an integrating digital voltmeter, constituting a very sensitive system. The digital output signals and a digital identification are registered on a recorder and on a tape punch, thus facilitating computer analysis.The precision of the equipment varied from 1.1 per cent, for low signal levels, to 0.3 percent for the highest signal levels. Stray light, the only important source of systematic errors, was found to influence the output signal level. Concerning determinations of the densities, however, a variation of the order of 1 per cent was only obtained for small areas with densities differing from that of the background.     

Evidence of luminous bacterial symbionts in the light organs of myctophid and stomiiform fishes

The myctophids and stomiiforms represent two common groups of luminous fishes, but the source of luminescence in these animals has remained undetermined. In this study, labeled luciferase gene fragments from luminous marine bacteria were used to probe DNA isolated from specific fish tissues. A positive signal was obtained from skin DNA in all luminous fishes examined, whereas muscle DNA gave a weaker signal and brain DNA was negative. This observation is consistent with luminous bacteria acting as the light source in myctophids and stomiiforms and argues against the genes necessary for luminescence residing on the fish chromosomes. To confirm the location of this signal, a bacterial probe was hybridized in situ to sections of a stomiiform. A strong signal was generated directly over specific regions of the fish light organs, whereas no signal was found over other internal or epidermal tissues of the fish. Taken together, these data provide the first indication that luminous bacterial symbionts exist in myctophids and stomiiforms and that these symbionts account for luminescence in these fishes.     

On-line data acquisition and evaluation of long-term measurements of circadian rhythms in individual cells ofAcetabularia

A multitasking time-sharing computer system was implemented for studies of different circadian rhythms in individual cells of the unicellular green alga,Acetabularia. This fully automatized system allows simultaneous data acquisition and analysis. Graphical presentation of untreated and mathematically treated data is permanently available on three graphic displays and on a digital plotter. The sampling rate for the data acquisition in each of the 60 channels connected to the system is 720/24 h. Provisions have been made to guarantee uninterrupted data uptake for these long-term measurements by including an auto-restart module and by providing extremely reliable software for the experimenter using menu techniques.     

Ultra-High-Sensitive Sensor Based on Surface Plasmon Resonance Structure Having Si and Graphene Layers for the Detection of Chikungunya Virus

Chikungunya virus has been discovered in about 60 countries of the world. It leads to joint pain, joint swelling, headache, muscle pain, and fatigue of the human body. In this work, a surface plasmon resonance (SPR)based sensor is developed to detect chikungunya virus through normal and infected platelets and plasma blood cells. The proposed SPR-based sensor uses silicon and graphene layers coated over the base of a glass prism sputtered with a silver layer. The graphene layer has the advantage of enhancing the biomolecules adsorption on the metal layer. The silicon layer between silver and graphene enhances the sensor performance. The number of graphene layers along with the thicknesses of silicon and silver layers is optimized to get the highest sensitivity of the detector. To investigate the effect of the light source wavelength, simulations are performed for four different wavelengths. The highest sensitivities exhibited by the SPR-based sensor are 393 and 160 deg/RIU for the platelets and plasma cells, respectively.

     

Phototaxis in the flagellates,Euglena gracilis and Ochromonas danica

Oriented movement with respect to laterally impinging white light of the flagellates Euglena gracilis and Ochromonas danica has been analyzed in an individual cell study with a microvideographic technique. Using the deviation of track segments (in given time intervals of 1 s) from the light direction as raw data allowed a computer based analysis of the direction distribution. A number of statistical methods employed to test the significance of the obtained results demonstrated an obvious phototactic orientation in Ochromonas which was positive (toward the light source) in low illuminance (1.25 lx=5.3×10^-3 Wm^-2) and negative in higher illuminance (>12.5 lx=5.3×10^-2 Wm^-2). Since in this flagellate the threshold for negative phototaxis is much lower than that for the step-up photophobic response, the hypothesis that negative phototaxis may be brought about by repetitive step-up phobic responses can be rejected for at least this organism. In Euglena positive phototaxis was observed in 50 lx (=0.21 Wm^-2), while an illuminance of 500 lx (=2.1 Wm^-2) caused a negative phototaxis.The experiments were carried out in this laboratory