Random signal variation as a cause of systematic microdensitometric error

A high noise/signal ratio in microdensitometry, owing, for example, to the use of a very small measuring spot or light at an extreme of the spectrum, decreases the precision (increases the variability) of results. This can be compensated for by making more measurements, but under some circumstances random signal variations can also cause systematic errors that are not so easily corrected. The present article explains how such errors can arise when measuring either very pale or very dense objects and suggests methods to detect and avoid them. The principles described are applicable to many types of microdensitometer, not only the Vickers flying-spot instruments used in this investigation.     

Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells.     

Demonstration of a highly-sensitive portable double-flash kinetic spectrophotometer for measurement of electron transfer reactions in intact plants

A highly sensitive, portable spectrophotometer for use in measuring flash-induced absorbance changes in intact leaves is demonstrated. The design of the instrument is modified for whole plant use from that suggested by Joliot and Joliot (Biochim. Biophys. Acta 765, 210–218). The spectrophotometer uses trifurcated light guides to deliver measuring and actinic beams to two comparable areas of the leaf. The measuring beam is provided by a series of short, relatively intense light pulses from a xenon flashlamp in place of the constant weak measuring beam used in conventional machines. The use of a flash measuring beam and differential detection allows for a high signal-to-noise ratio (noise levels of 10^-5A) without significant actinic effects. The time resolution of the instrument is 2 sec and the noise level is independent of the experimental time range. The instrument is battery or mains powered, computer operated, and has a liquid crystal display for computer-user interface and dialogue, and to show the kinetic traces graphically. Wavelength selection is provided by interchangeable interference filters. The instrument can communicate with a laboratory-based computer, receiving programming information and sending experimental data to be processed and plotted. The instrument is demonstrated by following the kinetics of the electrochromic shift, the change in redox states of cytochrome f and the b cytochromes in an intact cucumber leaf, and in the same leaf after infiltration with DCMU.     

Minimizing Cognitive Errors in Site-Specific Causal Assessments

Interest in causal investigations in aquatic systems has been a natural outgrowth of the increased use of biological monitoring to characterize the condition of resources. Although biological monitoring approaches are critical tools for detecting whether effects are occurring, they do not identify the cause of the observed effects. Formal approaches to causal evaluation can provide a mechanism to build on expert knowledge, increasing the likelihood that remedial efforts will achieve the desired environmental improvement. This paper examines how formal approaches to causal investigations minimize common errors. We reviewed common cognitive errors reported in the literature, and compared them with considerations suggested for strength-of-evidence approaches. Many of the causal considerations are directed toward distinguishing spurious correlations from true causal relationships. However, this is only one type of error; others include hypothesis dependence, confirmation bias, hypothesis tenacity and anchoring. We suggest three general principles for minimizing error in site-specific investigations: (1) Conduct the causal evaluation as a fair, transparent comparison among alternatives; (2) Carefully describe and quantify the conjunction of cause and effect; and (3) Consider that conjunction between cause and effect is spurious, or that a real conjunction was masked.     

Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity

Synopsis Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigatingin vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.     

A user''s guide for avoiding errors in absorbance image cytometry: a review with original experimental observations

Summary The sources of errors which may occur when cytophotometric analysis is performed with video microscopy using a charged-coupled device (CCD) camera and image analysis are reviewed. The importance of these errors in practice has been tested, and ways of minimizing or avoiding them are described. Many of these sources of error are known from scanning and integrating cytophotometry; they include the use of white instead of monochromatic light, the distribution error, glare, diffraction, shading distortion, and inadequate depth of field. Sources of errors specifically linked with video microscopy or image analysis are highlighted as well; these errors include blooming, limited dynamic range of grey levels, non-linear responses of the camera, contrast transfer, photon noise, dark current, read-out noise, fixed scene noise and spatial calibration. Glare, contrast transfer, fixed scene noise, depth of field and spatial calibration seem to be the most serious sources of errors when measurements are not carried out correctly. We include a table summarizing all the errors discussed in this review and procedures for avoiding them. It can be concluded that if accurate calibration steps are performed and proper guidelines followed, image cytometry can be applied safely for quantifying amounts of chromophore per cell or per unit volume of tissue in sections, even when relatively simple and inexpensive instrumentation is being used.     

Are geometric morphometric analyses replicable? Evaluating landmark measurement error and its impact on extant and fossil Microtus classification

Geometric morphometric analyses are frequently employed to quantify biological shape and shape variation. Despite the popularity of this technique, quantification of measurement error in geometric morphometric datasets and its impact on statistical results is seldom assessed in the literature. Here, we evaluate error on 2D landmark coordinate configurations of the lower first molar of five North American Microtus (vole) species. We acquired data from the same specimens several times to quantify error from four data acquisition sources: specimen presentation, imaging devices, interobserver variation, and intraobserver variation. We then evaluated the impact of those errors on linear discriminant analysis‐based classifications of the five species using recent specimens of known species affinity and fossil specimens of unknown species affinity. Results indicate that data acquisition error can be substantial, sometimes explaining >30% of the total variation among datasets. Comparisons of datasets digitized by different individuals exhibit the greatest discrepancies in landmark precision, and comparison of datasets photographed from different presentation angles yields the greatest discrepancies in species classification results. All error sources impact statistical classification to some extent. For example, no two landmark dataset replicates exhibit the same predicted group memberships of recent or fossil specimens. Our findings emphasize the need to mitigate error as much as possible during geometric morphometric data collection. Though the impact of measurement error on statistical fidelity is likely analysis‐specific, we recommend that all geometric morphometric studies standardize specimen imaging equipment, specimen presentations (if analyses are 2D), and landmark digitizers to reduce error and subsequent analytical misinterpretations.     

A diffused-optics flash kinetic spectrophotometer (DOFS) for measurements of absorbance changes in intact plants in the steady-state

Measurements of steady-state light-induced absorbance changes in intact plants are often hindered by interference from large changes in the light-scattering properties of the chloroplasts. In this work we present a new instrument, the diffused-optics flash spectrophotometer (DOFS), which reduces the magnitude of light scattering interference to manageable levels. In this spectrophotometer, the conventional light path is replaced with a set of light-scrambling chambers formed from a highly light-scattering plastic. The main scrambling chamber acts both to homogeneously diffuse as well as to split the measuring beam into sample and reference channels. Since the measuring beam has no defined incident angle, it is essentially 'pre-scattered', and further scattering changes that occur in the sample have minimal effect on the apparent absorbance changes. The combination of a pulsed probe light and differential optics and electronics provides a high signal-to-noise ratio, stable baseline and high time resolution. We also introduce a technique to account for residual scattering changes. Sets of measurements are made with the instrument in optical configurations that are differentially sensitive to light-scattering changes but yield nearly identical absorbance changes. The difference in apparent absorbance spectra taken with the two configurations reveals the spectral shape of the scattering changes without interference from absorbance signals. Spectra of the scattering contributions are then used to eliminate residual scattering interference from kinetic traces. We suggest that DOFS is ideally suited for study of steady-state electron transfer reactions in intact plants.     

New multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam for photosynthesis research

A multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam and differential optics has been constructed for measurements of light-induced absorbance and fluorescence yield changes in isolated chlorophyll-proteins, thylakoids and intact cells including algae and photosynthetic bacteria. The measuring beam, provided by a short (2 μs) pulse from a xenon flash lamp, is divided into a sample and reference channel by a broad band beam splitter. The spectrum in each channel is analyzed separately by a photodiode array. The use of flash measuring beam and differential detection yields high signal-to-noise ratio (noise level of 2 × 10⁻⁴ in absorbance units per single flash) with negligible actinic effect. The instrument covers a spectral range between 300 and 1050 nm with a spectral resolution of 2.1, 6.4 or 12.8 nm dependent on the type of grating used. The optical design of the instrument enables measuring of the difference spectra during an actinic irradiation of samples with continuous light and/or saturation flashes. The time resolution of the spectrophotometer is limited by the length of Xe flash lamp pulses to 2 μs.     

Interference by pigment in the estimation of microalgal biomass concentration by optical density

Optical density is used as a convenient indirect measurement of biomass concentration in microbial cell suspensions. Absorbance of light by a suspension can be related directly to cell density using a suitable standard curve. However, inaccuracies can be introduced when the pigment content of the cells changes. Under the culture conditions used, pigment content of the microalga Chlorella vulgaris varied between 0.5 and 5.5% of dry weight with age and culture conditions. This led to significant errors in biomass quantification over the course of a growth cycle, due to the change in absorbance. Using a standard curve generated at a single time point in the growth cycle to calculate dry weight (dw) from optical density led to average relative errors across the growth cycle, relative to actual dw, of between 9 and 18% at 680 nm and 5 and 13% at 750 nm. When a standard curve generated under low pigment conditions was used to estimate biomass under normal pigment conditions, average relative errors in biomass estimation relative to actual dw across the growth cycle were 52% at 680 nm and 25% at 750 nm. Similar results were found with Scenedesmus, Spirulina and Nannochloropsis. Suggested strategies to minimise error include selection of a wavelength that minimises absorbance by the pigment, e.g. 750 nm where chlorophyll is the dominant pigment, and generation of a standard curve towards the middle, or across the entire, growth cycle.     

Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence imaging

A new phytotoxicity bioassay based on chlorophyll fluorescence imaging of algae suspensions in multiwell plates is introduced. Phytotoxicity is quantified via inhibition of photosystem II quantum yield, Y(II), assessed with the saturation pulse method. The basics of this approach as well as the factors enhancing and limiting its performance are outlined. Compared to other established techniques the new system allows exceptionally rapid and accurate measurements of phytotoxicity using pulse-amplitude-modulation (PAM) fluorometry. While instrument related errors are negligibly small, optimal performance depends on appropriate choice of algae and illumination conditions. Illustrative examples for the response of Phaeodactylum tricornutum to diuron are presented. The standard deviation involved in the Y(II) determination of a single well amounts to the equivalent of 44 ng/L diuron. A decisive role is played by the light (measuring light, saturation pulses, actinic light) to which samples are exposed during the bioassay: (1) the inhibitor response is enhanced at high measuring light intensity. (2) Saturation pulses may be considered non-invasive only, if applied at low frequency and as long as physiologically healthy algae cultures are used. (3) Continuous actinic light may be problematic, as it induces complex physiological reactions that limit the performance of the approach; it is not required for assessment of diuron-type inhibitors at high measuring light intensity.     

Selection of optimal transmittances with the cytophotometric two-wavelength method

With the help of a computer program using Newton's iterative method, it is possible to obtain a practically instantaneous solution for the two-wavelenght method equation even for values different from 2 for the ratio k of the extinction coefficients. The influence of the extinction ratio and of the mean transmittance on the correction of the distributional error and on the propagation of stochastic and systematic errors into the computed chromophore amount has been studied. After due precautions are taken to minimize chromatic and non-specific light loss errors, the selection of the optimal wavelenght couple will basically depend only on the compromise between correction of the distributional error and the least propagation of stochastic errors into the computed chromophore amount. In many instances, the use of interference filters will be convenient, since their use will eliminate the uncertainties of monochromator readjustment and will allow evenly illuminated field to be obtained more easily, particularly in the case of large measuring fields.     

Apparent anomalies in nuclear feulgen-DNA contents. Role of systematic microdensitometric errors

The Feulgen-DNA contents of human leukocytes, sperm, and oral squames were investigated by scanning and integrating microdensitometry, both with and without correction for residual distribution error and glare. Maximally stained sperm had absorbances which at lambdamax exceeded the measuring range of the Vickers M86 microdensitometer; this potential source of error could be avoided either by using shorter hydrolysis times or by measuring at an off-peak wavelength. Small but statistically significant apparent differences between leukocyte types were found in uncorrected but not fully corrected measurements, and some apparent differences disappeared when only one of the residual instrumental errors was eliminated. In uncorrected measurements, the apparent Feulgen-DNA content of maximally stained polymorphs measured at lambdamax was significantly lower than that of squames, while in all experimental series uncorrected measurements showed apparent diploid:haploid ratios significantly greater than two. In fully corrected measurements no significant differences were found between leukocytes and squames, and in four independent estimations the lowest diploid:haploid ratio found was 1.99 +/- 0.05, and the highest 2.03 +/- 0.05. Discrepancies found in uncorrected measurements could be correlated with morphology of the nuclei concerned. Glare particularly affected measurements of relatively compact nuclei such as those of sperm, polymorphs and lymphocytes, while residual distribution error was especially marked with nuclei having a high perimeter:area ratio (e.g. sperm and polymorphs). Uncorrected instrumental errors, especially residual distribution error and glare, probably account for at least some of the previously reported apparent differences between the Feulgen-DNA contents of different cell types. On the basis of our experimental evidence, and a consideration of the published work of others, it appears that within the rather narrow limits of random experimental error there seems little or no reason to postulate either genuine differences in the amounts of DNA present in the cells studied, or nonstoichiometry of a correctly performed Feulgen reaction.     

Asymmetry of Early Paleozoic trilobites

Asymmetry in fossils can arise through a variety of biological and geological mechanisms. If geological sources of asymmetry can be minimized or factored out, it might be possible to assess biological sources of asymmetry. Fluctuating asymmetry (FA), a general measure of developmental precision, is documented for nine species of lower Paleozoic trilobites. Taphonomic analyses suggest that the populations studied for each taxon span relatively short time intervals that are approximately equal in duration. Tectonic deformation may have affected the specimens studied, since deviations from normal distributions are common. Several measures of FA were applied to 3–5 homologous measures in each taxon. Measurement error was assessed by the analysis of variance (ANOVA) for repeated measurements of individual specimens and by analysis of the statistical moments of the distributions of asymmetry measures. Measurement error was significantly smaller than the difference between measures taken on each side of a specimen. However, the distribution of differences between sides often deviated from a mean of zero, or was skewed or kurtosic. Regression of levels of FA against geologic age revealed no statistically significant changes in levels of asymmetry through time. Geological and taphonomic effects make it difficult to identify asymmetry due to biological factors. Although fluctuating asymmetry is a function of both intrinsic and extrinsic factors, the results suggest that early Cambrian trilobites possessed genetic or developmental mechanisms used to maintain developmental stability comparable to those of younger trilobites. Although the measures are biased by time averaging and deviations from the normal distribution, these data do not lend strong support to 'genomic' hypotheses that have been suggested to control the tempo of the Cambrian radiation.     

Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.     

Reappraisal of nuclear DNA content of rodlet cells compared to several cell types from some freshwater teleosts, using two methods of microdensitometry

Nuclear DNA contents of rodlet cells from Catostomus commersoni, Semotilus atromaculatus and Cyprinus carpio were compared with nuclear DNA of erythrocytes and larger cells of the same species, using scanning microdensitometry and averaging microdensitometry. This study reappraises the work of Barber & Westermann (1983), which employed averaging microdensitometry only, and compared rodlet cell nuclear DNA only with erythrocyte DNA. In addition, this work considers sources of error in both methods of microdensitometry, and comments upon the use of microdensitometry of either method as a mechanism for making distinctions among the DNA contents of cells of different types. The results of the present work consistently indiate no significant differences within species between nuclear DNA content of rodlet cells and larger teleost cells, using either method of microdensitometry. Because of the lack of statistically significant difference in DNA content between nuclei of rodlet cells and those of known teleost cells, it has been concluded that the rodlet cell itself is probably of teleost origin. However, the method indicates nothing about the origin of the rodlets, which have also been shown to contain DNA, but are Feulgen-negative.     

Effects of oxidation and reduction on the spectral properties of the egg capsules of Raja erinacea Mitchill

At oviposition, egg capsules of Raja erinacea Mitchill were a deep greenish-brown. The dorsal wall was translucent while the ventral wall appeared transparent. Spectral analysis of capsulai material was performed by placing capsular specimens in microcuvettes in a scanning spectrophotometer and measuring absorbance at wavelengths in the visible spectrum between 350 and 900 nm. The capsule wall completely absorbed light between 350- and 460-nm wavelengths. At higher wavelengths, absorbance decreased while the amount of transmitted light increased. The dorsal wall absorbed more light > 460 nm than did the ventral wall because the dorsal wall was thicker and contained higher catechol concentrations. Reduction of capsule specimens with NaBH₄ decreased absorbance of light but did not alter the wall thickness or catechol concentration. Reduced specimens appeared transparent light green. Oxidation of capsule with FeCl₃ effectively increased absorbance of light > 460 nm so that little light of any wavelength passed through the specimen. In reflected light, FeCl₃-treated specimens appeared dark brownish-black, mimicking the natural darkening of capsules which occurs during incubation. These results indicate that R. erinacea egg capsules remain chemically reactive following oviposition. Capsules possess inherent redox potential at oviposition and contain catechols which can be oxidized to quinones forming dark pigments. These observations suggest that catechol oxidation plays a role in the change in color and transparency of R. erinacea egg capsules during incubation.     

A digital registration system for net photosynthesis and transpiration measurements in the field and an associated analysis of errors

Summary A digital registration system used with temperature- and humidity-controlled cuvettes for net photosynthesis and transpiration measurements in the field is described. The associated errors of the measured parameters and calculated data are estimated. The digitalization is based on an analogue registration which is of primary importance in the control of experimental conditions in the cuvettes. The digital system is connected to the analogue registration in series. The error associated with digitalization is 0.1% across 70% of the scale. This error increases to 0.2% between 3 and 30% on the scale due to a minor lack of linearity. The reproducibility of the digitalization is ±0.024%.The error associated with data transfer in the digitalization and the errors of the analogue registration are estimated for temperature and humidity measurements (error of air and leaf temperature is ±0.1° C; error of the dew point temperature is ±1.1° C dew point). The effect of these errors on the calculation of relative humidity and the water vapour difference between the leaf and the air is determined using the progressive error law. At 30° C and 50% relative humidity, the error in relative humidity is ±7.4%, the error for the water vapour difference is ±6.6%. The dependence of these errors on temperature and humidity is shown.The instrument error of the net photosynthesis measurement is calculated to be ±4.2%. Transpiration measurements have an average inaccuracy of ±8.3%. The total diffusion resistance which is calculated from values of transpiration and the water vapour difference has an average error of ±10.9%. The sizeable influence of errors in humidity and temperature measurements on the calculated diffusion resistance is demonstrated. The additional influence of biological errors associated with field measurements is discussed.     

Interpreting the evolutionary regression: the interplay between observational and biological errors in phylogenetic comparative studies

Regressions of biological variables across species are rarely perfect. Usually, there are residual deviations from the estimated model relationship, and such deviations commonly show a pattern of phylogenetic correlations indicating that they have biological causes. We discuss the origins and effects of phylogenetically correlated biological variation in regression studies. In particular, we discuss the interplay of biological deviations with deviations due to observational or measurement errors, which are also important in comparative studies based on estimated species means. We show how bias in estimated evolutionary regressions can arise from several sources, including phylogenetic inertia and either observational or biological error in the predictor variables. We show how all these biases can be estimated and corrected for in the presence of phylogenetic correlations. We present general formulas for incorporating measurement error in linear models with correlated data. We also show how alternative regression models, such as major axis and reduced major axis regression, which are often recommended when there is error in predictor variables, are strongly biased when there is biological variation in any part of the model. We argue that such methods should never be used to estimate evolutionary or allometric regression slopes.     

Non-contacting electrode system for the measurement of strain generated potentials in bone

Current methods employing contact electrodes for the measurement of the electromechanical properties of bone produce errors in the measurement due to the effects of polarization at the bone-electrode interface, and the flow of electric charges in the bone measuring circuit. In addition, signal artefacts may result from the movement of an electrode in contact with a specimen undergoing mechanical deformation. The principles for a non-contacting method, based on charge induction on a conductive plate placed in the field of a charged body (bone), and the resulting non-contacting electrode system are presented in this paper. The new electrode enabled measurement of strain generated potentials (SGP) in bone with minimal effect from the measuring circuit and provided new results previously masked by contacting measurement methods. Furthermore, the new electrode is a potential tool for further investigation of the in vitro electromechanical behaviour of bone, particularly in partially hydrated specimens and in vivo, thereby avoiding invasive methods or use of ionizing radiation.