The Two-Wavelength Method of Microspectrophotometry : II. A Set of Tables to Facilitate the Calculations

The calculations required for two-wavelength measurements are time consuming and laborious. In order to circumvent this limitation of the method, a set of tables which combined four operations into one has been designed and is reproduced within. The tables are based on Patau's formulae. The two transmission readings obtained according to the photometric method provide the coordinates which lead directly to a value for the relative absorbance. The product of this absorbance and the area of the photometric field gives the relative amount of chromophore in the field. The range of transmission values covered in the table corresponds to the effective range of the two-wavelength method.     

The two-wavelength method of microspectrophotometry. II. A set of tables to facilitate the calculations

The calculations required for two-wavelength measurements are time consuming and laborious. In order to circumvent this limitation of the method, a set of tables which combined four operations into one has been designed and is reproduced within. The tables are based on Patau's formulae. The two transmission readings obtained according to the photometric method provide the coordinates which lead directly to a value for the relative absorbance. The product of this absorbance and the area of the photometric field gives the relative amount of chromophore in the field. The range of transmission values covered in the table corresponds to the effective range of the two-wavelength method.     

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 : 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. 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.     

Two-wavelength cytophotometry: the choice of the wavelengths from a practical point of view.

Methods in use for the choice of the two-wavelengths and the effect of a faulty choice of the two wavelengths in the two-wavelength cytophotometric method of Patau and Ornstein are studied by means of a computer simulation and in practice upon pararosaniline-Schiff stained nuclei. A simplification of the measuring strategy is proposed without affecting the accuracy.     

Factors affecting the estimation of the relative amount of chromophore and chromophore area by the two-wavelength method of patau and ornstein

Summary Factors influencing the calculation of the relative amount of chromophore and the chromophore area by the two-wavelength method are examined. The study was carried out with the help of models and further tested on Feulgen stained preparations. Except for certain restrictions the difference between the chromophore area as calculated from the two transmissions measurements and the chromophore area obtained by planimetry can be used as a guide for determining the proper measuring conditions, including the choise of the two wavelengths.     

Factors affecting the estimation of the relative amount of chromophore and chromophore area by the two-wavelength method of Patau and Ornstein.

Factors influencing the calculation of the relative amount of chromophore and the chromophore area by the two-wavelength method are examined. The study was carried out with the help of models and further tested on Feulgen stained preparations. Except for certain restrictions the difference between the chromophore area as calculated from the two transmissions measurements and the chromophore area obtained by planimetry can be used as a guide for determining the proper measuring conditions, including the choise of the two wavelengths.     

Is There a Third Photoreceptor Involved in the Control of Chloroplast Movements in Mougeotia?

The photometric method was used to test a possibility proposed recently that a new photoreceptor with maximum activity at 620 nm is involved in mediating chloroplast rotation in Mougeotia (Z Lechowski, J Bialczyk [1988] Plant Physiol 88: 189-193). The hypothesis was tested under conditions of continuous dichromatic unilateral or mutually perpendicular irradiation with red light of wavelengths 620 or 660 (680) nanometers and far-red. When the red light was polarized parallel to the long cell axis, chloroplast response could be monitored by changing the direction of far-red irradiation. The level of the response obtained with red and far-red applied from the same direction depended on far-red intensity: at higher fluence rates the maximum response was shifted to longer wavelengths of red light. A high fluence rate of far-red inhibited the response. The absorption coefficients of Mougeotia chloroplasts were measured for the studied wave-lengths using the microphotometric method. Possible impact of absorption by the chloroplast on photoreception has been discussed. Current and previous results can be interpreted in terms of phytochrome action and do not support the involvement of the hypothetical 620 nanometer photoreceptor.     

Localization of Light with Two Metallic Wedges: A Step Toward Surface Plasmon Sources

A new technique for localization of the light in subwavelength scale is proposed by using two adjacent metallic wedges, which are made by different metals, i.e., Al and Ag. Green’s function surface integral equation method is exploited to numerically calculate magnetic field at different points. The results illustrate that by controlling the phase difference between two surface plasmon polariton waves propagating on the sides of the first wedge and the distance between two wedges the amplitude and the phase of SPP waves generated on the sides of the second wedge can be adjusted.     

Intrinsic guild structure: determination from competition experiments

A new approach is introduced for determining the intrinsic guild classification of a group of species. Previous delimitations of intrinsic guilds have used evidence of spatial distributions (i.e. species co-occurrences), but this is rather indirect evidence. The new method is based on the results of species pairwise competition experiments, and thus uses direct data on species interactions. As with the spatial-distribution intrinsic guild approach, no prior assumptions are made about the classification, nor about which characters are related to guild membership.
The method is applied to the results of two published experiments. For one, little independent evidence is available to judge the classification. There is no correlation between the guild classification obtained and gross morphology, but there is no reason to expect any such correlation. For the second experiment, intrinsic guild classifications had previously been obtained from distributional data, and the experimentally-based intrinsic classifications was identical to a distributionally-based one.
We suggest that combining evidence from field distributions with experimental evidence offers a rigorous way to determine the true guild structure of communities, offering convincing conclusions when the two lines of evidence converge.     

Craniofacial variation of the Chinese macaques explored with Morphologika

In order to analyze, separately and dynamically, the variation in cranial size and shape of Chinese macaques, a new method, Morphologika, was used to illustrate 3D profiles based on the coordinates of 26 landmarks on the skull. Striking image variation between the two sexes was detected on the facial region: males exhibited a larger and more protrusive facial structure. Males also displayed a bigger cranium than females. The two sexes also showed quite different images in skull shape. However, they expressed the same allometric pattern with regard to the relationship between size and shape, which was significantly positively associated with each other along the first axis. The same relationship was negatively displayed along the second axis when the two sexes were analyzed together. However, only the relationship for females reached a significant level when the two sexes were studied separately. This was considered to be related to their differentiation in growth trajectory. This study also tested the concept of size revealed by the second axis of principal components analysis based on traditional morphometric methods on the same taxa.     

Nonlinear optical effects in oxygen-binding reactions of hemoglobin A0

Optical spectra have been taken in the Soret band (440-400 nm) under different oxygen partial pressures for hemoglobin (Hb) A0 at pH 7.0, 15 degrees C, 2-3 mM heme, 30 mM inositol hexaphosphate, 0.1 Hepes and 0.1 M NaCl. Application of the matrix method of singular value decomposition (SVD) to the difference spectra for different oxygen pressures shows the presence of at least two distinct optical transitions. From this result one concludes that the optical response to oxygen binding is nonlinear in the Soret band. The degree of nonlinearity has been determined by fitting the data at different wavelengths to the four-step reaction Adair equation with the inclusion of optical parameters that describe the intermediate oxygenated species. It is found that the data are well-represented by two optical parameters at each wavelengths, one which represents the optical change for the addition of the first and second oxygen molecules and the other which corresponds to the change for the addition of the third and fourth oxygen molecules. The ratio of these optical parameters depends only moderately upon wavelength with an average value of 0.8 over the Soret band. Thus, there is an approx. 20% smaller optical response for the first two ligated species than that for the last two ligated species. The overall Adair equilibrium constants are evaluated as follows: beta 1 = 0.081 +/- 0.003 Torr-1, beta 2 = 2.53 x 10(-3) +/- 2.4 x 10(-4) Torr-2, beta 3 = 1.25 x 10(-5) +/- 1.0 x 10(-6) Torr-3, beta 4 = 1.77 x 10(-6) +/- 1.5 x 10(-7) Torr-4.     

A new method for nonhomogeneous time course expression analysis

Time course expression analysis constitutes a large portion of applications of microarray experiments. One primary goal of such experiments is to detect genes with the temporal changes over a period of time or at some interested time points. Difficulties arising from data with small number of replicates over only a few unaligned time points in multiple groups pose challenges for efficient statistical analysis. Some known methods are limited by the unverifiable assumptions or by the scope of applications for only two groups. We present a new method for detecting differentially expressed genes under nonhomogeneous time course experiments in multiple groups. The new method first models the time course curve of one gene by a Gaussian process to align the nonhomogeneous time course data and to compute the gradient of the time course curve as well, the latter of which is used as directional information to enhance the sensitivity of detection for temporal changes. Second, we adopt a nonparametric method to test a surrogate hypothesis based on the augmented data from the Gaussian process model. The proposed method is robust in terms of model fitting and testing. It does not require any distributional assumption for the observations or the test statistic and the method works for the case with as few as triplicate samples over four or five time points under multiple groups. We show the effectiveness and superiority of the new method in comparison with some existing methods using simulated models and two real data sets.     

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.     

Nest-site size as a short-term constraint on the reproductive success of paternal fishes

Synopsis Because female signal blennies, Emblemaria hypacanthus (Pisces: Chaenopsidae), exhibited a preference for the larger of two males in laboratory mate-choice experiments, male reproductive success was predicted to be positively correlated with male size in field populations. This prediction was met at one field site, but not a second where shelter size, rather than male size, was the primary correlate of male reproductive success. This finding appeared to be the consequence of the relatively small size of shelters (vacant gastropod shells) at the second site. At this site, significantly more males had their shelters filled with eggs and variance in male reproductive success was lower than at the first site. Limited area for deposition of eggs is potentially an important constraint on the short-term reproductive success of paternal males of shelter- or crevice-spawning fishes.     

An optical method for the determination of platelet count in platelet samples contaminated with red blood cells.

We present a simple photometric method to determine the total concentration of platelets present in a sample independently of red blood cell concentration. Standard optical density curves for platelet samples ranging in concentration from 0 to 1.5 x 10(9) cells/ml and contaminated with red blood cells ranging in concentration from 0 to 0.03 x 10(9) cells/ml are determined. A study of the absorbance spectra of red blood cells and platelets suggests that by calculating the absorbance difference between two wavelengths, an estimate of red blood cell concentration can be made. Then, in the second step of this two-step method, the individual absorbance measurements at the two wavelengths are matched to the standard values determined previously to derive an estimate of platelet concentration. In a trial of 62 unknown platelet samples contaminated with red blood cells, the standard deviation for the error in platelet count was 0.16 x 10(9) cells/ml with a mean difference of 0.011 x 10(9) platelets/ml. We conclude that our method may be useful in laboratories not equipped with electronic cell counters as well as in applications such as the development of noninvasive measurements of platelet concentration in platelet transfusion packs.     

Algorithmic Optimisation Method for Improving Use Case Points Estimation

This paper presents a new size estimation method that can be used to estimate size level for software engineering projects. The Algorithmic Optimisation Method is based on Use Case Points and on Multiple Least Square Regression. The method is derived into three phases. The first phase deals with calculation Use Case Points and correction coefficients values. Correction coefficients are obtained by using Multiple Least Square Regression. New project is estimated in the second and third phase. In the second phase Use Case Points parameters for new estimation are set up and in the third phase project estimation is performed. Final estimation is obtained by using newly developed estimation equation, which used two correction coefficients. The Algorithmic Optimisation Method performs approximately 43% better than the Use Case Points method, based on their magnitude of relative error score. All results were evaluated by standard approach: visual inspection, goodness of fit measure and statistical significance.     

The relationship between the transition matrix model and the diffusion model

The diffusion equation model and the Lefkovitch matrix model have been employed independently in plant population ecology in order to analyze the dynamics of growth and size structure. The two models describe the dynamics of size structure in biological populations, and thus there must be some relationship between them. In the present paper, we examine the theoretical relationship between these two models. We demonstrate, on a certain assumption, that the one-step Lefkovitch matrix model corresponds to a difference equation of the diffusion equation and that the two- and three-step Lefkovitch matrix model correspond to difference equations of the 4th- and 6th-order Kramers-Moyal expansions, respectively. It is also shown that 2n moments (the first to the 2n-th moments) of growth rate are necessary and sufficient to rewrite uniquely the n-step Lefkovitch matrix model in terms of the linear combination of the moments. We finally discuss the relationship between the species characteristics of census data and the appropriate types of the Lefkovitch matrix.