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 : 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.     

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.     

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.     

Sensitive Detection of the Leaching-out from Membrane-surrounded Compartments: a New Approach Explored with Cells and Tissues of Nicotiana tabacum L.

In search of a simple method for testing the very early events of ozone damage to susceptible plants as well as complete destruction after threshold-exceeding treatments an over-all measurement of cell ingredients by their optical density in the UVB and UVC range was investigated. The cell particles were liberated after membrane permeabilization or after cell bursting. Uncontrolled results could be excluded. Furthermore, the results of the developed spectrophotometrical test could be, in the case of tissue samples (leaf discs), very well reproduced with an osmometrical measurement. The latter was less sensitive and not suitable for cellular samples because the protoplasts must be dissolved in a nearly isotonic medium which caused too large a background for this method but not for the UVS test. Contrary to the osmometric measurement, the photometric one cannot be used for determining the absolute amount of cell ingredients but only for relative measurement between samples in a given range of concentrations. Oxidative changes of the liberated ingredients do not influence their detection, which was demonstrated with ascorbate. The developed leaching test was also useful for determining the membrane damage caused by the detergent Triton X-100, although this was known to have UV absorbance by itself. It was noted that the far UV maximum is not only caused by absorbance and scattering is discussed as an additional explanation.     

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.     

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.     

Spectrophotometric determination of the critical micellar concentration of bile salts using bilirubin monoglucuronide as a micellar probe. Utility of derivative spectroscopy.

We have developed a simple biologically non-invasive method for determining the critical micellar concentration (CMC) of bile salts using pure naturally occurring bilirubin IX alpha monoglucuronide (BMG), an important bile pigment present in virtually all mammalian biles. This methodology employs visible absorbance spectroscopy of BMG in bile salts over a range of bile salt concentrations that include the reported CMC. Using 100 microM-BMG in 0.4 M-imidazole buffer at pH 7.8, we calculated that the CMC for sodium taurochenodeoxycholate is between 2.5 and 3.0 mM based on: (1) an abrupt change in lambda max. in this concentration range, (2) a precipitous decrease in the amplitude of the absorbance shoulder at 450 nm, (3) a sudden decrease in the second derivative absorbance of BMG at 400 nm and an increase in absorbance at 470 nm, (4) a sharp change in the 4th derivative absorbance at 375 and 395 nm. In contrast, sodium taurocholate, a bile salt that reportedly does not have a CMC but continuously self-associates over a wide concentration range, exhibited none of these changes. The use of derivative spectroscopy enhances the ability to detect the CMC changes and also indicates the number of BMG species in solution and their relative energy states.     

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.     

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.     

A simple spectrophotometric streptavidin-biotin binding assay utilizing biotin-4-fluorescein

A new assay for biotin binding capacity of Streptavidin (SA) is presented in this work. The assay is based on the large decrease in the extinction coefficient at 493 nm that accompanies binding of biotin-4-fluorescein (B4F) to SA. This decrease is attributed to formation of a charge transfer complex between the B4F-donor and one or more SA residues. We show that one may observe the stoichiometric binding via monitoring the absorbance at 493 nm using either SA or B4F as the titrant. The sensitivity of the assay is at the lower end of similar fluorimetric and photometric assays. Though the sensitivity is not substantially lower than other comparable techniques, this assay allows one added flexibility in working range and instrumentation, since the same stock solutions may be used for this new photometric assay or the fluorescence assay for which this ligand was first developed.     

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.     

Modification of a quantitative laser-spectrographic method of determination of cations contained in tissue slices.

A quantitative method of determination of elements in histological slices by means of laser microanalysis has been modified. The modification consists essentially in such methodological approach that allows expressing the results by figures which are directly proportional to the relative content of the element in question. The final result, i.e. the relative quantity of the element to be determined is obtained by subtracting the photometric value yielded by the spectrographic line of the Ag standard from that yielded by the spectrographic line of the element under investigation (J = JX-JAG). To avoid negative figures the Ag standard has to be prepared in such a way, as to yield the lowest possibly photometric values still lying on the straight line of the standard curve.     

利用双波长方法对非均匀地形人工林蒸散的测量

千米级地表蒸散通常利用遥感影像并结合经验或半经验模型进行估算,且估算结果还需结合地表实测数据进行验证,但实际中很难获得与遥感像元尺度匹配的实测数据.本研究在黄河小浪底森林生态系统定位研究站人工林的生长期(2016年7月1日-10月19日),基于近红外与微波联合观测技术进行千米级地表蒸散的观测分析.结果表明:近红外与微波联合技术在不同天气条件下均能表现出良好的日变化特征,且整体能量闭合率可达0.87.该技术对空气湿度的变化敏感,土壤湿度的变化对该项技术的结果影响较小.不同波文比条件和大气稳定度均会使该技术的观测产生不同程度的误差,与涡动相关方法相比,误差在合理范围.采用双波长方法研究得出,该区域的最大日蒸散量为3.5 mm,总蒸散量为162.4 mm.     

Asymptotic Relative Efficiency of Some Jackknife Estimators of a Common Odds Ratio

In the situation of several 2 × 2 tables the asymptotic relative efficiencies of certain jackknife estimators of a common odds ratio are investigated in the case that the number of tables is fixed while the sample sizes within each table tend to infinity. The estimators show very good results over a wide range of parameters. Some situations in which the estimators have low asymptotic relative efficiency are pointed out:.     

Quantitative surface studies of protein adsorption by infrared spectroscopy. I. Correction for bulk concentrations

A method for correcting attenuated total reflectance (ATR) infrared spectra of surface-adsorbed proteins for the contribution of proteins in the bulk solution has been demonstrated. This procedure estimates the soluble protein contribution to the ir spectra from parameters determined from transmission experiments, and uses the absorbance of the water band in ATR as an internal reference. Based on this procedure, the soluble protein contribution to the total spectrum is estimated to range from approximately 3% for IgG or albumin in solution at 1 mg/ml, to approximately 35% when whole blood (from sheep) is adsorbed onto a 45 degrees germanium ATR crystal.     

A microassay for proteases using succinylcasein as a substrate.

A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is demonstrated.     

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.     

A quantitative correlation between the kinetics of solutes and water translocation in Liver Mitochondria

Summary A procedure is described for the calculation of solute fluxes in mitochondria from absorbance measurements. The procedure assumes that mitochondria behave as osmometers and that they are always at osmotic equilibrium.The rates and amounts of K⁺ translocation have been calculated simultaneously, with the photometric procedure and electrometrically, during passive, K⁺ efflux coupled to Ca⁺⁺ uptake and during active K⁺ uptake and passive K⁺ release coupled with anion translocation. Good agreement has been found between the two sets of measurements. The data are compatible with the concept that the energy-linked, ion translocation-coupled, mitochondrial swelling is osmotic in nature. It is concluded that the changes of absorbance are quantitatively related to changes in the inner volume and therefore the photometric procedure can be used to calculate, ion fluxes of osmotically active species under various circumstances.