Sensory capacities and the nocturnal habit of owls (Strigiformes)

Behavioural studies show that in the eye of the Tawny Owl Strix aluco both absolute visual sensitivity and maximum spatial resolution at low light levels are close to the theoretical limit dictated principally by the quantal nature of light and the physiological limitations on the structure of vertebrate eyes. However, when the owl's visual sensitivity in relation to naturally occurring ligh levels is analysed, it is concluded that at night there will often be occasions when vision can only be used to control the owl's behaviour with respect to large objects.
Owls are capable of detecting and catching prey by hearing alone. However, absolute auditory sensitivity is not superior to that of mammals (including Man), but does appear to have reached the absolute limit on sensitivity in the aerial environment, which is dictated by the minimum ambient sound level.
An explanation of the owl's ability to be active at night based only upon high sensory sensitivity is thus untenable. Many features of the natural behaviour of the Tawny Owl (e.g., the high degree of territoriality, prey catching technique, dietary spectrum) may be interpreted as reflections of an additional requirement for the nocturnal habit beyond high sensory sensitivity: detailed knowledge of local topography.     

Excitation kinetics during induction of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence

We present a chlorophyll fluorometer module system which adapts the intensity to the individual leaf sample by adjusting the quantum flux density of the excitation light so that the fluorescence signal is kept constant. This is achieved by means of a feedback power adjustment of the fluorescence exciting laser diode. Thus, the intensity of the excitation light is adapted to the actual need of a particular sample for quantum conversion without applying exaggeratedly high quantum flux density. We demonstrate the influence of the initial laser power chosen at the onset of irradiation and kept constant during fluorescence rise transient within the first second. Examples are shown for measuring upper and lower leaf sides, a single leaf with different pre-darkening periods, as well as yellow, light green and dark green leaves. The novel excitation kinetics during the induction of chlorophyll fluorescence can be used to study the yield and regulation of photosynthesis and its related non-photochemical processes for an individual leaf. It allows not only to sense the present state of pre-darkening or pre-irradiation but also the light environment the leaf has experienced during its growth and development. Thus, the individual physiological capacity and plasticity of each leaf sample can be sensed being of high importance for basic and applied ecophysiological research which makes this new methodology both innovative and informative.     

Directly probing rapid membrane protein dynamics with an atomic force microscope: a study of light-induced conformational alterations in bacteriorhodopsin.

This paper demonstrates that an atomic force microscope can be used to directly monitor rapid membrane protein dynamics. For this demonstration the membrane-bound proton pump, bacteriorhodopsin, has been investigated. It has been unequivocally shown that the light-induced dynamic alterations that have been observed do not arise from external artifacts such as heating of the sample by the incident light, but that these changes can be directly linked to the light-induced protein conformational alterations in this membrane. In essence, it has been shown that the light energy absorbed by bacteriorhodopsin is converted not only to chemical energy but also to mechanical energy. In summary a new ultrasensitive tool is described for monitoring the molecular dynamics of materials with wide applicability to fundamental and applied science.     

Interferometric Control of Signal Light Intensity by Anomalous Refraction with Plasmonic Metasurface

We propose a new method of controlling signal light intensity by changing the polarization direction of pump light that is incident on a phase-gradient metasurface. Theoretical analyses and simulations demonstrate that, when the incident angles for signal and pump light sources conform to a certain relationship, the anomalous refraction of the pump light superposes with the signal light. The intensity of the signal light can be fully tuned across a broadband range by varying the polarization direction of the pump light. Our methods will contribute to the development of promising techniques to be used in photonics devices such as amplitude modulators and photoswitches.     

A dilution immunoassay to measure myosin regulatory light chain phosphorylation

We examined the quantitation of myosin regulatory light chain phosphorylation (MRLCP) by Western blot and found both offset and saturation errors. The desirable characteristics of an MRLCP assay are that the dynamic range be 60- to 100-fold and that the detection threshold be known and preferably very small relative to total MRLC concentration. No technique examined provided all these characteristics. However, accurate measurements can be obtained by including serial dilutions of the sample to provide a fractional calibration scale in terms of the dephosphorylated light chain and by using interpolation of the phosphorylated band signal intensity to provide values for the relative phosphorylation ratio. We found that this method offers several advantages over methods that rely on signal ratios from single samples: The dilution ratio method is less subject to errors from differences in protein load, it offers estimates of the error in the individual measurement, and has some redundancy that increases the likelihood of obtaining a valid measurement despite gel or membrane artifacts.     

Using an electro-microchip, a nanogold probe, and silver enhancement in an immunoassay

This paper presents a novel immunoassay that uses an electro-microchip to detect the immuno-reaction signal, gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. This study is based on the direct immunoassay (two-layer format) and the sandwich immunoassay (three-layer format). The ANPs were introduced into the electro-microchip by the specific binding of the antibodies-ANPs conjugates and then were coupled with silver enhancement to produce black spots of silver metal. The silver precipitation constructs a "bridge" between two electrodes of the electro-microchip allowing electrons to pass. The variation of impedance can be easily measured with a commercial LCR meter. Various gap sizes (20, 50, 100, and 200 microm) of the electrodes of electro-microchips were designed for the sensitivity study. The experimental data show that a chip with a 20microm gap has the highest sensitivity. There was a significant difference in impedance between the experiment sample and the negative control after 10 min of reaction time. The proposed method requires less time and fewer steps than the conventional enzyme-linked immunosorbent assay (ELISA). In addition, it shows a high detection sensitivity (10 microg/mL of 1st antibody (IgG) immobilized on slides and 1 ng/mL of antigen (protein A)). There is a clear distinction between the signal intensity and the logarithm of the sample concentration. The proposed new immunoassay method has potential applications in proteomics research and clinical diagnosis.     

Circadian rhythms of finches under steadily changing light intensity: Are self-sustaining circadian rhythms self-excitatory?

Summary Finches (Chloris chloris, Fringilla montifringilla) showed clear freerunning circadian rhythms when exposed to constant dim light. Increasing the light intensity by doubling it each day made them become arrhythmic at a certain threshold intensity of illumination, showing continuous locomotor activity. When the light intensity was decreased steadily at the reversed rate, the finches became rhythmic again. 7 out of 8 finches had a clear start in their rhythms, from one day to the next, at light intensities about 4 times higher than the point where they had become arrhythmic. The last finch started its freerunning circadian rhythm gradually, a few days after the light intensity had reached a constant dim illumination (0.2 lux).The results of all birds are taken as proof of the self-excitatory capacity of the circadian system. This means, it characterizes the dynamics of the system that the clock mechanism is continuously in operation, and not only after a passive reaction to external stimuli exceeds any threshold. Simultaneously, the results of all but one bird allow the evaluation of the contribution of proportional and differential effects of light in the control of circadian rhythmicity. A relative change in light intensity by 100% in the course of one day is nearly equivalent to a change of 100% in the absolute intensity of illumination.     

A method for measuring microquantities of DNA

Quantitative analysis of DNA content represents a critical step when only very small amounts of nucleic acids are available. The DNA content of a small RNA-free sample can be measured in a simple and precise way using a two-dimensional approach. DNA samples are spotted on the surface of an agarose gel containing ethidium bromide (EtBr) and the ultraviolet-induced low-light fluorescence emitted by EtBr molecules intercalated into the DNA is evaluated. The high sensitivity and reproducibility of this quantitative method has been obtained using an advanced analysis system capable of distinguishing low-light fluorescent patterns, as in the case of DNA stained with EtBr, from the background. Use of an internal standard is necessary because the intensity of the signal is due to the aperture of camera diaphragm and to gel conditions. Using this two-dimensional analysis system it is possible to obtain rapid and precise quantitation of as little as 2ng of DNA.     

Swim pacemakers in box jellyfish are modulated by the visual input

A major part of the cubozoan central nervous system is situated in the eye-bearing rhopalia. One of the neuronal output channels from the rhopalia carries a swim pacemaker signal, which has a one-to-one relation with the swim contractions of the bell shaped body. Given the advanced visual system of box jellyfish and that the pacemaker signal originates in the vicinity of these eyes, it seems logical to assume that the pacemakers are modified by the visual input. Here, the firing frequency and distribution of inter-signal intervals (ISIs) of single pacemakers are examined in the Caribbean box jellyfish, Tripedalia cystophora. It is shown that the absolute ambient light intensity, if kept constant, has no influence on the signal, but if the intensity changes, it has a major impact on both frequency and ISIs. If the intensity suddenly drops there is an increase in firing frequency, and the ISIs become more homogeneously distributed. A rise in intensity, on the other hand, produces a steep decline in the frequency and makes the ISIs highly variable. These electrophysiological data are correlated with behavioral observations from the natural habitat of the medusae.     

Noise, sensitivity, and resolution of flow cytometers.

The sensitivity and resolution of flow cytometers are functions of the signal produced by a given particle as well as by the noise in the presence of which the signal is detected. The noise is primarily due to the fact that emission of light as well as its detection by photoelectric devises are stochastic processes. This fact leads to equations describing how resolution and sensitivity are limited by the magnitude of the signal, the background, and the photoelectron quantum yield of the detector. The equations are pointing to a method by which the signal and noise of a flow cytometer can be measured in absolute terms, as well as a way to determine fluorescence sensitivity without having to extrapolate to the noise level. The equations appear to be validated when applied to measuring data obtained with two different flow cytometers.     

Interest of second harmonic generation imaging for diagnosis in thick and opaque tissue

In articular hyaline cartilage, chondrocytes are surrounded by an extracellular matrix which is mainly composed by collagen and proteoglycanes. Pathological specimens show a partial or complete degradation of this matrix. Therefore, it could be interesting to know how mechanical or biochemical constraints applied to cartilage specimens induce modifications of the cartilage network. Multiphoton technology combined to Second Harmonic Generation (SHG) enables to image cartilage specimens in a non-invasive mode with high resolution at deep penetration. By placing a band pass filter in front of the transmitted light detector, SHG signal with frequency doubled can be isolated for a new contrast imaging. SHG (second harmonic generation) is a diffusion process generated from organized structures and does not need any fluorescent staining. Due to their non-centrosymetric structure, collagen fibrilles present a high second-order non-linear susceptibility and thus give rise to a strong SHG signal when exposed to high enough electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton microscopy). As the extracellular matrix of cartilage is in part constituted by collagen fibers, it can be imaged with this contrast tool. The intensity of SHG signals strongly depends on the organization of collagen fibers. Thus a modification of the extracellular matrix in terms of 3D-organization of collagen induced by mechanical stress can be shown with this contrast tool.     

A miniaturized, highly sensitive polarimeter as detector in an implantable glucose probe. II. Opto-electronic amplification and processing of measuring signals]

There is a clinical requirement for an implantable telemetric probe for monitoring glucose levels in humans. This probe can measure the glucose content of the intercellular tissue fluid, which reflects glucose levels in the blood. The lifespan of such an implantable probe should be maximal, so that presumably only physical measuring detectors, but not aging-sensitive bio-sensors can be considered. We are in the process of developing a very sensitive miniaturised detector based on polarimetry, capable of determining the measuring parameter--the spatial orientation of the in-plane vibration of a polarised light beam--with high accuracy. This is necessary for our purpose, since the physiological and pathological glucose levels modify in in-plane vibration by only a tiny angle of rotation. The high level of accuracy is achieved by various specific mechanisms both of the measuring parameters and the electric signal. Two suitable optoelectronic amplification methods are described. The first makes use of the ratio of the signal provided by the intensity of two consecutive beams, derived from the original light beam with the aid of a beam splitter. In this way, the sensitivity of determining the spatial position of the in-plane vibration of the polarised light beam can be increased by up to 50-fold in comparison with a "simple" polarimetry. The second method requires two very closely approximated (quasi united) or actually united beams from two sources, which are both "fixed-phase" time-coupled and quantitatively periodically intensity-modulated in opposite sense. Together with the already-mentioned ratio of the intensity signals of two consecutive beams, a periodically modulated signal generated from the individual signals is derived from this quasi-unified beam that enables the use of a phase sensitive rectifier-amplifier (lock-in amplifier) with its enormous amplification factor and noise elimination in the following circuitry.     

Photoacoustic detection of photosynthetic oxygen evolution from leaves. Quantitative analysis by phase and amplitude measurements

The photoacoustic signal from an intact leaf was analyzed as a vectorial summation of photothermal and photosynthetic oxygen-evolution contributions. A method is outlined to estimate each contribution separately. The amplitude of the oxygen-evolution component relative to that of the photothermal singnal decreases as the modulation frequency increases due to two processes which specifically damp the oxygen-evolution modulation: (1) diffusion of oxygen from the chloroplasts to the cell boundary, and (2) electron-transfer reactions occurring between the photochemical act and oxygen evolution. The effects of the two processes are well separated and are observed over different ranges of modulation frequency. Analysis of the data leads to a consistent estimation of the oxygen diffusion coefficient and also to a preliminary idea on the limiting time constant on the donor side of Photosystem II. The dependence of the photoacoustic oxygen-evolution signal on the intensity of added nonmodulated background light is used to construct the light saturation curve of (gross) Photsynthesis, with an estimation of the ratio maximal rate / maximal quantum yield. The photoacoustic method is distinguished by its sensitivity and rapidity (a single measurement takes approx. 1 s), far better than any other method to measure gross photosynthesis. The only disadvantage is in the fact that the quantum yield of oxygen evolution is determined in a relative basis only. Attempts to calibrate the photoacoustic measurements in an absolute sense are underway.     

Intensity Correlation-Based Calibration of FRET

Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determining proximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolute fluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenching and sensitized emission. A crucial point is the determination of the scaling factor α responsible for balancing the different sensitivities of the donor and acceptor signal channels. The determination of α is not simple, requiring preparation of special samples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation (least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of α and the absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the second moments (variances and covariances) of the detected intensity distributions. A quadratic equation for α is formulated with the intensity fluctuations, which is proved sufficiently robust to give accurate α-values on a cell-by-cell basis in a wide system of conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly new approach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines, FT and LS174T. The figures show that whereas the common way of α determination fails at large dye-per-protein labeling ratios of mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer, the new approach can also be straightforwardly used with fluorescence microscopes.     

In intact mammalian photoreceptors, Ca2+-dependent modulation of cGMP-gated ion channels is detectable in cones but not in rods

In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP-gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is approximately 2.3 microM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of approximately 480 ms time constant at 20-21 degrees C and 138 ms at 32 degrees C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors.     

Analysis of cell surface light chain immunoglobulin expression by flow cytometry in normal controls: a new mathematical approach

It has been observed in flow cytometric studies that in normal individuals there are proportionally more kappa+ than lambda+ bearing light chain B-cells. Overt predominance of one type of light chain bearing cell over the other is a characteristic of B-cell neoplasias, a phenomenon called clonal excess (CE). A mathematical model using the Weibull distribution is proposed for studying such an excess. The new approach is desirable for two reasons: First, it is parametric and hence offers a more sensitive and versatile analysis than its nonparametric counterparts. Second, it utilizes only the relevant information from the upper tails of the distributions of the fluorescence intensity of the kappa+ and lambda+ cells. Two measures of CE based on the Weibull model are proposed, and a normal range of variability was determined for each measure using a random sample of 48 normal controls. Such normal ranges are particularly useful in detecting cancer patients with minimal B-cell neoplasias. A comparative study of the new measures, Ault's maximum difference measure, and a measure based on Ligler's method showed that the parametric approach provides much more sensitivity than both the nonparametric ones.     

Adaptation of the visual system

We recorded the total pulse response of the optic nerve in frogs to varying degrees of increase and decrease of light from the original adapting level. On the basis of these data, we plotted curves of dependence of the magnitude of response on the logarithm of relative value of increase and decrease of light (the amplitude characteristic — AC). The AC is steepest in the zone of adapting background and sloped on either side of it. It follows that under stationary conditions of illumination, the eye is capable of finely differentiating light intensity only within a narrow range (one logarithmic unit). After adaptation to a new level of illumination, the AC shifts along the scale of light intensity in such a way that the steepest portion corresponds to the adapting brightness. Increase in steepness of the AC occurs precisely during the process of adaptation. The contrast sensitivity of the human visual system is greatest near the adapting level and declines on either side of it. It follows that in man steepness of the visual system AC is greatest in the zone of the adapting background. Both increase and decrease of intensity of the adapting background are accompanied by a decline of contrast sensitivity, which rises again during the process of adaptation to a new level. Thanks to adaptive shift of the steep portion of the AC along the scale of light intensity, a visual system having a high contrast sensitivity only within a narrow "working" range is capable of finely differentiating light intensity in significantly changing conditions of illumination.Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 1, No. 1, pp. 81–89, July–August, 1969.     

Modulation of synaptic transfer between retinal cones and horizontal cells by spatial contrast

We studied the influence of steady annular light on the kinetics and sensitivity of horizontal cell (HC) responses to modulation of the intensity of small concentric spots in the turtle retina. As shown by previous investigators, when the intensity of the annulus was equal to the mean spot intensity, spot response kinetics were the same as those for the modulation of spatially uniform light. Turning off the annulus attenuated dramatically high-frequency flicker sensitivity and enhanced somewhat low-frequency sensitivity. This phenomenon reflects a modulation of synaptic transfer between cones and second-order neurons that is mediated by cones, and it will be referred to as cone-mediated surround enhancement (CMSE). Our main results are as follows: (a) The change in test-spot response sensitivity and kinetics upon dimming a steady surrounding annulus is a consequence of the change in spatial contrast rather than change in overall light level. (b) Introduction of moderate contrast between the mean spot intensity and steady surrounding light intensity causes a marked change in spot response kinetics. (c) The dependence of spot response kinetics on surrounding light can be described by a phenomenological model in which the steady state gain and the time constant of one or two single-stage, low-pass filters increase with decreasing annular light intensity (d) The effect of surrounding light on spot responses of a given HC is not determined by change in the steady component of the membrane potential of that cell. (e) Light outside the receptive field of an HC can affect that cell's spot response kinetics. (f) In an expanding annulus experiment, the distance over which steady annular light affects spot response kinetics varies among HCs and can be quite different even between two cells with closely matched receptive field sizes. (g) The degree of CMSE is correlated with HC receptive field size. This correlation suggests that part of the enhancement mechanism is located in the HC. Taken together, our results suggest the involvement of the inner retina in CMSE.     

Directional surface plasmon-coupled emission: application for an immunoassay in whole blood

We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. This approach, which is based on surface plasmon-coupled emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal was studied. It was demonstrated that the kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately two- and threefold, respectively, as compared with buffer, resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood with no need for washing steps.     

Simulation of the effect of local adaption with nonlinear spatial filters

The visual system of vertebrates is capable of processing pattern signals over a wide range of intensity reaching from nearly absolute darkness to very bright sunlight. Typically the visual system of humans extracts fine contours of patterns of sufficiently high intensity or at high background intensity level, showing signal processing properties which can be explained by a bandpass system. Conversely, at very low intensity levels that system shows low-pass response: only coarse contours of patterns are recognized, however, the amplification of the signals has increased. The effect is called local adaption. A model is shown on the basis of a one-stage nonlinear spatial filter which, controlled by the local distribution of pattern intensity, can alter its frequency characteristic between low-pass response and bandpass response. Results are stated for computer-modelled filters. The investigation is restricted to one-dimensional filters, however, the results can be used to explain the function of two-dimensional filters qualitatively.