The two-wavelength method of microspectrophotometry. III. An extension based on photographic color transparencies

The two-wavelength method of microspectrophotometry corrects for distributional error and measures the amount of absorbing material by taking advantage of certain spectral characteristics of the specimen. Under certain circumstances, such as the absorption of nucleic acids in the ultraviolet and of black or multiple stains in the visible, the spectral characteristics are not suitable for the application of the method. To circumvent this, a photomicrograph of the object is taken with monochromatic light of a suitable wavelength. A second plate is exposed as a contact print of the photomicrograph and is developed in the presence of a coupling agent. After bleaching and fixation, the positive appears as a monochromatic color transparency. Two-wavelength analysis of such a transparency can be made in terms of the new color. The measurements will be free of distributional error and can be equated to the original object. The necessary formulae are derived, and a method which has proven suitable for color development is given. The photographic and the direct two-wavelength method were found to give equivalent results when both were used on the same series of liver nuclei. The application of the photographic method to ultraviolet absorption has been demonstrated. The new method is potentially applicable to other types of photographic densitometry involving heterogeneous images.     

THE TWO-WAVELENGTH METHOD OF MICROSPECTROPHOTOMETRY : IV. A New Solution

In connection with the potential development of automatic two-wavelength microspectrophotometry, a new version of the two-wavelength method has been formulated. Unlike its predecessors, the Ornstein and Patau versions, the new method varies the area of the photometric field seeking to maximize a relationship between distributional errors at the two wavelengths. Stating this distributional error relationship in conventional photometric terms, the conditions at the maximum are defined by taking the first derivative with respect to field size and setting it equal to zero. This operation supplies two equations; one relates the transmittances at the two wavelengths, and a second states the relative amount of chromophore in the field in terms of transmittance at one wavelength. With the first equation to drive a servomechanism which sets the appropriate field size, the desired answer can then be obtained directly and continuously from the second equation. The result is identical in theory with those of the earlier methods, but the technique is more suitable for electronic computing.     

PHOTOGRAPHIC VISUALIZATION OF FLORAL COLORS AS PERCEIVED BY HONEYBEE POLLINATORS

An inexpensive photographic technique for visualizing the ultraviolet and visible wavelengths perceived by honeybees is described. Using a standard daylight-balanced color slide film and illumination from blacklight and filtered daylight fluorescent lamps, a recording balance was achieved which approximates the spectral sensitivity of the honeybee eye. The technique was used to illustrate floral features among Rudbeckia species and among color morphs of Phlox. The Rudbeckia have inflorescences that are similar in visible coloration but distinctive in ultraviolet patterning and Phlox color morphs are distinctive in visible coloration but similar in ultraviolet patterning. The efficacy of the technique was judged from comparison with in vivo reflectance spectra of the floral subjects. Generally, photographic visualizations of entomophilous flowering plants portray only the ultraviolet or the visible components of floral coloration. This technique emphasizes the importance of considering the entire spectrum of floral colors relevant to most insect pollinators.     

The two-wavelength method of microspectrophotometry. I. A microspectrophotometer and tests on model systems

In spite of a number of examples of the apparently successful application of the two-wavelength method of microspectrophotometry to biological materials, a demonstration of the effective transition from the theoretical to the practical aspects of the method has never been given. The following tests were carried out on model systems in order to confirm the practical validity of the method: (1) Spherical droplets of Sudan blue or Sudan IV (varying in volume by as much as 400 times) were measured with the surrounding blank field included and were found to have amounts of chromophore which conformed to the estimated droplet volume to within 3 to 6 per cent. (2) Repeated determinations on the same isolated nucleus gave results which did not vary in spite of wide variations in the area of the photometric field. (3) The results of repeated determinations on the same droplet of dye were unaffected by gross changes in the focus of the objective. The microspectrophotometer on which these measurements were made has been briefly described; it was designed specifically for the two-wavelength method. Some of the details of instrumentation have been discussed, in particular, the effects of non-uniform illumination and the contrasting demands of the two integral parts of this type of photometry. Although it has certain limitations, the two-wavelength method conforms to its theoretical promise and is a practical method of measuring light absorption in complex objects.     

The Two-Wavelength Method of Microspectrophotometry : I. A Microspectrophotometer and Tests on Model Systems

In spite of a number of examples of the apparently successful application of the two-wavelength method of microspectrophotometry to biological materials, a demonstration of the effective transition from the theoretical to the practical aspects of the method has never been given. The following tests were carried out on model systems in order to confirm the practical validity of the method: (1) Spherical droplets of Sudan blue or Sudan IV (varying in volume by as much as 400 times) were measured with the surrounding blank field included and were found to have amounts of chromophore which conformed to the estimated droplet volume to within 3 to 6 per cent. (2) Repeated determinations on the same isolated nucleus gave results which did not vary in spite of wide variations in the area of the photometric field. (3) The results of repeated determinations on the same droplet of dye were unaffected by gross changes in the focus of the objective. The microspectrophotometer on which these measurements were made has been briefly described; it was designed specifically for the two-wavelength method. Some of the details of instrumentation have been discussed, in particular, the effects of non-uniform illumination and the contrasting demands of the two integral parts of this type of photometry. Although it has certain limitations, the two-wavelength method conforms to its theoretical promise and is a practical method of measuring light absorption in complex objects.     

Discovery of signaling effect of UV-B/C light in the extended UV-A/blue-type action spectra for step-down and step-up photophobic responses in the unicellular flagellate algaEuglena gracilis

Summary Cultures of unicellular algal flagellateEuglena gracilis grown in different conditions were subjected to action spectroscopy for step-down and step-up photophobic responses, respectively. The spectral region was extended into the UV-B/C as well as in the UV-A and visible regions with the Okazaki Large Spectrograph as the monochromatic light source. The photophobic responses of the cells were measured with an individual-cell assay method with the aid of a computerized video motion analyzer. In the UV-A and visible regions, the shapes of the action spectra were the so-called UV-A/blue type. In the newly studied UV-B/C region, new action peaks were found at 270 nm for the step-down response and at 280 nm for the step-up one. The absorption spectrum of flavin adenine dinucleotide (FAD) appeared to fit the action spectrum for the step-up response, whereas the shape of the step-down action spectrum, which has a UV-A peak (at 370 nm) higher than the blue peak (at 450 nm), appeared to be mimicked by the absorption spectrum of a mixed solution of 6-biopterin and FAD. These observations might also account for the fact that the UV-B/C peak wavelength at 270 nm of the action spectrum for the step-down response is shorter by 10 nm than the action spectrum for the step-up response at 280 nm.Abbreviations FAD flavin adenine dinucleotide - FWHM spectral full width at half maximum - NIBB National Institute for Basic Biology - OLS Okazaki Large Spectrograph - PFB paraflagellar body - UV-A ultraviolet light of spectral region between 320 and 400 nm - UV-B/C ultraviolet light of spectral region between 190 and 320 nm     

Wavelength-shifting molecular beacons

We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.     

Spectral responses of the tsetse fly, Glossina morsitans morsitans

Spectral responses of male Glossina morsitans morsitans Westw, were investigated using behavioural and electrophysiological techniques. Phototactic responses to monochromatic lights of different wavelengths but equivalent intensities (measured either in energy or quantal units) were tested in an apparatus which permitted the simultaneous presentation of pairs of lights to groups of flies. Near ultraviolet light was the most attractive, followed by blue and red, with green and far red light giving no significant responses above control levels. The spectral sensitivity of the compound eye was also determined from the electroretinogram (ERG) recorded from electrodes implanted in the eye which was stimulated with flashes of monochromatic light. Three areas of maximum sensitivity were found by this method in the UV, blue/green and red. The relationship between the behavioural and electrophysiological sensitivity characteristics is explained with reference to visual systems of other Diptera which have previously been described.     

Electrophysiology and anatomy of medulla interneurons in the optic lobe of the cockroach,Periplaneta americana

1. Medulla interneurons of the optic lobe of P. americana were studied to determine their spectral properties. These neurons exhibited tonic firing which changed with monochromatic broadfield illumination of the ipsilateral eye. The response patterns of these neurons were analyzed by inferring their relation to the ultraviolet (UV) and green (G) photoreceptor groups of the eye. Their anatomy was described after injection of Lucifer yellow. 2. Broadband neurons received either excitatory or inhibitory input from both UV and G receptors. These neurons were not strictly sensitive to luminosity levels and had large cell bodies in the central rind of the medulla and wide dendritic arbors in the medulla neuropil. 3. Narrow band neurons received input from predominantly one receptor type. Their spectral sensitivity curves were more finely tuned than those of the primary receptors presumably due to neural interactions within the optic lobe. 4. Color opponent neurons were inhibited by UV and excited by G inputs in their sustained response. Under certain conditions, some of these neurons also showed G inhibition. These neurons suggested the presence of a subsystem involved in color vision. 5. Broadband, narrow band and color opponent properties were seen in some single neurons when tested over a 5-6 log unit range of intensity. The responses of some of these neurons changed when stimulus duration was increased. These findings indicated that functional classification for these neurons was dependent on stimulus intensity and duration. 6. Polarizational sensitivity was tested in preliminary experiments. Two neurons responded to the movement and direction of polarized light.     

The Nature, Significance, and Evaluation of the Schwarzschild-Villiger (SV) Effect in Photometric Procedures

The Schwarzschild-Villiger effect has been experimentally demonstrated with the optical system used in this laboratory. Using a photographic mosaic specimen as a model, it has been shown that the conclusions of Naora are substantiated and that the SV effect, in large or small magnitude, is always present in optical systems. The theoretical transmission error arising from the presence of the SV effect has been derived for various optical conditions of measurement. The results have been experimentally confirmed. The SV contribution of the substage optics of microspectrophotometers has also been considered. A simple method of evaluating a flare function f(A) is advanced which provides a measure of the SV error present in a system. It is demonstrated that measurements of specimens of optical density less than unity can be made with less than 1 per cent error, when using illuminating beam diameter/specimen diameter ratios of unity and uncoated optical surfaces. For denser specimens it is shown that care must be taken to reduce the illuminating beam/specimen diameter ratio to a value dictated by the magnitude of a flare function f(A), evaluated for a particular optical system, in order to avoid excessive transmission error. It is emphasized that observed densities (transmissions) are not necessarily true densities (transmissions) because of the possibility of SV error. The ambiguity associated with an estimation of stray-light error by means of an opaque object has also been demonstrated. The errors illustrated are not necessarily restricted to microspectrophotometry but may possibly be found in such fields as spectral analysis, the interpretation of x-ray diffraction patterns, the determination of ionizing particle tracks and particle densities in photographic emulsions, and in many other types of photometric analysis.     

不同光波及光强度下棉铃虫(Helicoverpa armigera)成虫的行为反应

运用室内行为实验方法研究了不同波长单色光和白光刺激刺激的棉铃虫「Ｈｅｌｉｃｏｖｅｒｐａ ａｒｍｉｇｅｒａ（Ｈｕｂｎｅｒ）」成虫趋光、避光反应及其与光强度之间的相互关系,结果显示：（１）一定时间暗适应的棉铃虫蛾对单光和白光刺激均具有趋光行为反应,依其趋光反应率大小,其光谱反应曲线在３４０－６０５ｎｍ波谱范围内,表现为大小较复杂上峰,其中两个峰分别在蓝光区４８３ｎｍ、紫外３４０ｎｍ,另外近紫外的４０ｎ     

Connecting the navigational clock to sun compass input in monarch butterfly brain

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.     

Fitting Age Related Reference Intervals to Ocular Axial Length in Children Using a Four-Parameter Curve Family

There exist a number of methods to determine age dependent reference intervals. Some of those are based on standard parametric classes of distributions like normal or lognormal and standard parametric classes of age functions like linear or polynomial of some order. Others are based on more flexible distribution classes like Box-Cox transformation of the normal distribution, which allows for skewness. There exist also purely nonparametric methods, where the bounds of the reference intervals are only assumed to be nondecreasing and they are directly estimated by a suitable error function without any distributional assumption. In this paper we propose a flexible four-parameter age function class for the reference interval bounds and a method to estimate those. The four parameters in the class have concrete meanings; starting value at age 0, asymptotic value at increasing age, time scale and shape. The function class satisfies some desirable properties, which are discussed. The estimation of the parameters in the model uses the same type of error function as in the purely nonparametric methods. With our method we also get an estimate of the distributional position of an observation for a new individual given its age. The method is illustrated by an application example, where a 90% reference interval for ocular axis length of children up to age 18 years are determined.     

Evidence for a two pigment visual system in the fiddler crab, Uca thayeri

Intraocular recordings were made from the eyestalks of dark-adapted fiddler crabs (Uca thayeri) during presentation of monochromatic light flashes of different wavelengths and intensities. Two types of signals were recorded in different experiments: slow potentials (electroretinogram) and fast potentials (spikes). The latter were also recorded in the presence of a continuous green or red adapting light. The resulting visual spectral-sensitivity curves, when fitted to rhodopsin-based visual pigment absorption spectra (from Dartnall nomograms), indicated the presence of two visual pigments, one with an absorption maximum near 430 nm, and the other with a peak absorption between 500 nm and 540 nm. The data also provided evidence for some differential bleaching of the pigments in the presence of a colored adapting light, but most of the adaptation effect was probably due to changes in screening pigment and neural desensitization or inhibition. These two observations suggest that an adequate substrate for color vision may exist in this and other species of fiddler crabs. The electroretinogram and spike-recording methods produced similar visual-sensitivity data, suggesting that latter technique, a much more efficient way of collecting data that is physiologically relevant, may be the method of choice for determining spectral sensitivity in crustaceans.     

Noninvasive diagnosis of melanoma with tensor decomposition-based feature extraction from clinical color image

We propose a method for feature extraction from clinical color images, with application in classification of skin lesions. Proposed feature extraction method is based on tensor decomposition of the clinical color image of skin lesion. Since color image is naturally represented as a three-way tensor, it is reasonable to use multi-way techniques to capture the underlying information contained in the image. Extracted features are elements of the core tensor in the corresponding multi-way decomposition, and represent spatial-spectral profile of the lesion. In contrast to common methods that exploit either texture or spectral diversity of the tumor only, the proposed approach simultaneously captures spatial and spectral characteristics. The procedure is tested on a problem of noninvasive diagnosis of melanoma from the clinical color images of skin lesions, with overall sensitivity 82.1% and specificity 86.9%. Our method compares favorably with the state of the art results reported in the literature and provides an interesting alternative to the existing approaches.     

Determination of lansoprazole and five metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of lansoprazole, a new proton-pump inhibitor, and five of its metabolites in human plasma is described. Lansoprazole, its metabolites, and internal standard (omeprazole) were extracted into diethyl ether-methylene chloride and separation was obtained using a reversed-phase column under isocratic conditions. The method features monochromatic ultraviolet detection at 285 nm, and single extraction, single evaporation sample handling. The lower limit of quantitation, based on standards with acceptable coefficients of variation, was 10 ng/ml for all compounds. No endogenous compounds were found to interfere. This method has been demonstrated to be suitable for pharmacokinetic studies in humans.     

Absorption cytophotometry: evaluation of three methods for the determination of DNA in Feulgen stained nuclei.

Three methods for the evaluation of the relative amount of DNA per nucleus by absorption cytophotometry are compared. A combination of the two-wavelength method (Patau, 1952; Ornstein, 1952), the one-wavelength two-area method (Garcia, 1965) and the determination of transmission through nuclear plugs is proposed in order to estimate systematic errors made by absorption cytophotometry.     

Evidence for a sensitizing pigment in the ocellar photoreceptors of the fly (Musca, Calliphora)

Summary A method is described that allows the spectral sensitivity of photoreceptors to be measured with high spectral resolution. It is shown that the sensitivity of the ocelli ofMusca andCalliphora has a vibrational fine structure in the ultraviolet, strongly indicative of a sensitizing pigment. The visual pigment has its absorption maximum close to 425 nm.     

铜绿丽金龟对不同光谱的行为反应

【目的】旨在获得铜绿丽金龟 Anomala corpulenta Motschulsky敏感的波谱范围及性别差异。【方法】本研究利用室内行为学的方法,测试了在波长为340~610 nm的14个单色光刺激下铜绿丽金龟的趋、避光行为反应,并计算趋、避光反应率曲线。【结果】观测显示,各单色光均能引起铜绿丽金龟产生一定的趋、避光反应,其雌雄虫的趋光敏感光区位于紫外光（405 nm）、蓝光（460 nm）和绿光区（505和570 nm）,性别对趋光行为有一定的影响,但是仅存在于趋光反应率曲线波峰的大小,而对波峰的位置没有影响。铜绿丽金龟的避光行为反应,在雌雄之间无明显的性别分化,其避光敏感光谱为紫外光（380 nm）、蓝光（440 nm）和绿光区（492和505 nm）。【结论】铜绿丽金龟成虫对不同波长光的趋性存在差异,性别对其光谱行为反应有一定的影响。该研究结果为铜绿丽金龟光视觉的深入研究奠定必要的理论基础,也为利用其趋光性对该金龟甲进行综合治理提供科学依据。     

Absorption cytophotometry: Evaluation of three methods for the determination of DNA in feulgen stained nuclei

Summary Three methods for the evaluation of the relative amount of DNA per nucleus by absorption cytophotometry are compared. A combination of the two-wavelength method (Patau, 1952; Ornstein, 1952), the one-wavelength two-area method (Garcia, 1965) and the determination of transmission through nuclear plugs, is proposed in order to estimate systematic errors made by absorption cytophotometry.