From Biochemistry to Physiology: The Calorimetry Connection

This article provides guidelines for selecting optimal calorimetric instrumentation for applications in biochemistry and biophysics. Applications include determining thermodynamics of interactions in non-covalently bonded structures, and determining function through measurements of enzyme kinetics and metabolic rates. Specific examples illustrating current capabilities and methods in biological calorimetry are provided. Commercially available calorimeters are categorized by application and by instrument characteristics (isothermal or temperature-scanning, reaction vessel volume, heat rate detection limit, fixed or removable reaction vessels, etc.). Advantages and limitations of commercially available calorimeters are listed for each application in biochemistry, biophysics, and physiology.     

Test and calibration processes for microcalorimeters, with special reference to heat conduction instruments used with aqueous systems

Calorimeters are normally calibrated by use of electrical heaters, but the calibration values derived may not always be representative for the chemical or biological process investigated. It is therefore important to have available chemical test processes by which the electrical calibration values can be controlled. In some cases, electrical calibration should be replaced by chemical calibrations. With particular reference to needs in work with thermophile heat conduction microcalorimeters, a number of test and calibration processes are presented. Some calibration problems for various types of reaction vessel are discussed.     

The development of a continuous isothermal titration calorimetric method for equilibrium studies

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12-20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60-180 min to generate 20-30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba(2+) to 18-crown-6 and the binding of cytidine 2'-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.     

Construction of a Promoter-cloning Vector in Pseudomonas aeruginosa

Computer simulations were employed to reconstruct the growth thermograms that are observable for microbial cultures in batch calorimeters having a culture vessel in the calorimetric unit. Differential equations were derived to characterize the heat evolution process on the basis of Monod’s growth equation with some modification. Theoretical growth thermograms were compared with actually observed calorimetric recordings and it was shown that the heat effect due to the microbial cells can be well reproduced for both non-growing and growing cultures of bakers’ yeast. It is concluded that the method used here gives a reasonable characterization of the thermograms observed for microbial cultures in a batch calorimeter, indicating the possibility of determining the appropriate kinetic parameters from calorimeter recordings by a parameter-fitting method.     

The potential use of microcalorimetry in predictive tests of the action of antineoplastic drugs on mammalian cells

The effect of methotrexate (MTX) on cultured T-lymphoma cells in a growing suspension was measured by microcalorimetry. The effect of the drug could be observed within 2 h after its injection into the calorimetric vessel. A dose-response curve was constructed from the calorimetric results obtained with final concentrations of MTX in the range of 0.02-2.00 microM. In another series of experiments calorimetric values were determined after 8 h of MTX-cell interaction. These data were correlated with values for cell counts during an 18 h period following the removal of the drug. A clear correlation was found between the calorimetric and cytotoxicity data. Calorimetric results obtained within 8 h of MTX-cell interaction appear to be of value in predicting the antineoplastic effect of the drug.     

Design and demonstration of a dynamometric horseshoe for measuring ground reaction loads of horses during racing conditions

Because musculoskeletal injuries to racehorses are common, instrumentation for the study of factors (e.g. track surface), which affect the ground reaction loads in horses during racing conditions, would be useful. The objectives of the work reported by this paper were to (1) design and construct a novel dynamometric horseshoe that is capable of measuring the complete ground reaction loading during racing conditions, (2) characterize static and dynamic measurement errors, and (3) demonstrate the usefulness of the instrument by collecting example data during the walk, trot, canter, and gallop for a single subject. Using electrical resistance strain gages, a dynamometric horseshoe was designed and constructed to measure the complete ground reaction force and moment vectors and the center of pressure. To mimic the load transfer surface of the hoof, the shape of the surface contacting the ground was similar to that of the solar surface of the hoof. Following static calibration, the measurement accuracy was determined. The root mean squared errors (RMSE) were 3% of full scale for the force component normal to the hoof and 9% for force components in the plane of the hoof. The dynamic calibration determined that the natural frequency with the full weight of a typical horse was 1744 Hz. Example data were collected during walking on a ground surface and during trotting, cantering, and galloping on a treadmill. The instrument successfully measured the complete ground reaction load during all four gaits. Consequently the dynamometric horseshoe is useful for studying factors, which affect ground reaction loads during racing conditions.     

Exact analysis of competition ligand binding by displacement isothermal titration calorimetry

A rigorous method for the least-squares nonlinear regression analysis of displacement isothermal titration calorimetric data is presented. The method can fit the binding isotherm of a ligand which is competitively inhibited in its binding by another bound ligand to a molecule with n identical and independent binding sites. There are no other assumptions for the method and no approximations. Analysis of previously published data of the strong binding of acarbose to glucoamylase is presented as an example. The regression equations have been programmed for the Origin software supplied with the widely used titration calorimeters from Microcal, Inc., and an Origin Function Definition File with instructions is freely available from the author upon e-mail request.     

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.     

Calibration of position and angular data from a magnetic tracking device

This paper describes a method for calibrating data from a magnetic tracking device. Position and orientation data were collected in a 1. 6x0.8x1.4m(3) volume using a Polhemus Fastrak((R)) in conjunction with both a long-range and standard transmitter. Position and orientation data were calibrated using a locally linear model based on the position of the measurement. After calibration, the average position and angular errors were less than 1.8cm and 1.2 degrees up to 1.8m from the transmitter for the long-range transmitter. For the standard transmitter, even after calibration, errors increased sharply when the sensor was more than 1.2m from the transmitter. Up to that distance, post-calibration errors were less than 1.2cm and 1. 2 degrees, while up to 1.8m they were below 5cm and 4 degrees. These errors could be further reduced by noise filtering. However, use of the standard transmitter is not recommended at distance greater than 1.2m due to orientation-based effects. It was concluded that for the volume investigated, tracking devices could provide similar three-dimensional accuracy to video systems.     

Estimations on the degradability of ores and bacterial leaching activity using short-time microcalorimetric tests

Abstract: This work is based on calorimetric measurements on batch cultures of ferrous iron-oxidizing thiobacilli and leaching cultures degrading solid substrates in the titration vessel under aerobic conditions. It has been demonstrated that heat-flow levels are a direct measure for oxidation rates of known reactions under non-limiting conditions. The total loss of heat-energy per unit of ferrous iron oxidized is identical under similar incubation conditions with different Thiobacillus ferrooxidans strains and different cell-densities in culture suspension. Under limiting conditions a reduced heat-loss occurred. This may have been due to either a more effective use of the substrate or to a shift to different redox reactions. Oxygen was proved to be a limiting factor in dense bacterial cultures with high substrate concentrations in the titration vessel. The experimental duration decreased significantly with the use of increasing cell densities. Therefore cell densities in degradation experiments with solid substrates were adjusted to between 5X10⁹ and 1x10¹⁰ cells/ml in short-term experiments. T. ferrooxidans is the most important species for short-term leaching experiments. Calorimetric measurements allow the best strains for a degradation of unknown substrates to be found. The calculated heat energy is a measure of the amount of converted substrate. High heat-flow only occurs if catabolic reactions take place and maintenance reactions and phenomena like adhesion of cells to surface do not cause significant loss of heat.     

Specific features of thermocouple calorimeter application for measurements of pulsed X-ray emission from plasma

It is shown that the accuracy of time-integrated measurements of pulsed X-ray emission from hot plasma with calibrated thermocouple calorimeters is mainly determined by two factors. The first and the most important factor is heating of the filter by the absorbed X-rays; as a result, the calorimeter measures the thermal radiation of the filter, which causes appreciable distortion of the temporal profile and amplitude of the recorded signal. The second factor is the dependence of the effective depth of X-ray absorption in the dielectric that covers the entrance window of the calorimeter on the energy of X-ray photons, i.e., on the recorded radiation spectrum. The results of model calculations of the calorimeter signal are compared with the experimental data.     

Calibration of pneumotachographs using a calibrated syringe.

Pneumotachograph require frequent calibration. Constant-flow methods allow polynomial calibration curves to be derived but are time consuming. The iterative syringe stroke technique is moderately efficient but results in discontinuous conductance arrays. This study investigated the derivation of first-, second-, and third-order polynomial calibration curves from 6 to 50 strokes of a calibration syringe. We used multiple linear regression to derive first-, second-, and third-order polynomial coefficients from two sets of 6-50 syringe strokes. In part A, peak flows did not exceed the specified linear range of the pneumotachograph, whereas flows in part B peaked at 160% of the maximum linear range. Conductance arrays were derived from the same data sets by using a published algorithm. Volume errors of the calibration strokes and of separate sets of 70 validation strokes (part A) and 140 validation strokes (part B) were calculated by using the polynomials and conductance arrays. Second- and third-order polynomials derived from 10 calibration strokes achieved volume variability equal to or better than conductance arrays derived from 50 strokes. We found that evaluation of conductance arrays using the calibration syringe strokes yields falsely low volume variances. We conclude that accurate polynomial curves can be derived from as few as 10 syringe strokes, and the new polynomial calibration method is substantially more time efficient than previously published conductance methods.     

Validation of oxygen consumption measurements during artificial ventilation

We describe a system for the absolute calibration of indirect calorimeters, under the conditions of artificial ventilation and increased inspired O2 concentration, in which butane, at a measured flow rate, is burned downstream of an artificial lung. One milliliter of butane requires 6.4 ml O2 for its combustion, and the respiratory quotient is 0.615. With the closed-circuit O2-replenishment method there was no significant systematic error in the measurement of either O2 consumption or CO2 output and a random error with a SD of 8.3 ml/min for O2 consumption and 6.3 ml/min for CO2 output. There were no significant differences in the errors with inspired O2 concentrations between 23.8 and 59.5% and O2 consumptions between 89 and 366 ml/min.     

Methods for measuring acoustic power of an ultrasonic neurosurgical device

Measurement of the acoustic power in high-energy ultrasonic devices is complex due to occurrence of the strong cavitation in front of the sonotrode tip. In our research we used three methods for characterization of our new ultrasonic probe for neuroendoscopic procedures. The first method is based on the electromechanical characterization of the device measuring the displacement of the sonotrode tip and input electrical impedance around excitation frequency with different amounts of the applied electrical power The second method is based on measuring the spatial pressure magnitude distribution of an ultrasound surgical device produced in an anechoic tank. The acoustic reciprocity principle is used to determinate the derived acoustic power of equivalent ultrasound sources at frequency components present in the spectrum of radiated ultrasonic waves. The third method is based on measuring the total absorbed acoustic power in the restricted volume of water using the calorimetric method. In the electromechanical characterization, calculated electroacoustic efficiency factor from equivalent electrical circuits is between 40-60%, the same as one obtained measuring the derived acoustic power in an anechoic tank when there is no cavitation. When cavitation activity is present in the front of the sonotrode tip the bubble cloud has a significant influence on the derived acoustic power and decreases electroacoustic efficiency. The measured output acoustic power using calorimetric method is greater then derived acoustic power, due to a large amount of heat energy released in the cavitation process.     

Simultaneous determination of equilibrium constants and enthalpy changes by titration calorimetry: Methods, instruments, and uncertainties

Calorimetric methods have been used to determine equilibrium constants since 1937, but no comprehensive review of the various calorimeters and methods has been done previously. This article reports methods for quantitative comparison of the capabilities of calorimeters for simultaneous determination of equilibrium constants and enthalpy changes, for determining optimal experimental conditions, and for assessing the effects of systematic and random errors on the accuracy and precision of equilibrium constants and enthalpy changes determined by this method.     

Cell Growth and Division: V. Error Analysis of the Collins-Richmond Equation

Error propagation in the Collins-Richmond equation is analyzed in order to obtain the ratio of the fractional error in rate of cell volume increase to the fractional error in each experimental variable. Typical data are analyzed numerically for the total errors resulting from counting statistics, from spectral broadening, and from volume calibration shift. The measurement of 10⁴ cells can give a precision of better than 10% in the volume growth rate with a volume resolution of 3%.     

Forest above-ground volume assessments with terrestrial laser scanning: a ground-truth validation experiment in temperate,managed forests

Background and AimsQuantifying the Earth’s forest above-ground biomass (AGB) is indispensable for effective climate action and developing forest policy. Yet, current allometric scaling models (ASMs) to estimate AGB suffer several drawbacks related to model selection and uncertainties about calibration data traceability. Terrestrial laser scanning (TLS) offers a promising non-destructive alternative. Tree volume is reconstructed from TLS point clouds with quantitative structure models (QSMs) and converted to AGB with wood basic density. Earlier studies have found overall TLS-derived forest volume estimates to be accurate, but highlighted problems for reconstructing finer branches. Our objective was to evaluate TLS for estimating tree volumes by comparison with reference volumes and volumes from ASMs.MethodsWe quantified the woody volume of 65 trees in Belgium (from 77 to 2800 L; Pinus sylvestris, Fagus sylvatica, Larix decidua, and Fraxinus excelsior) with QSMs and destructive reference measurements. We tested a volume expansion factor (VEF) approach by multiplying the solid and merchantable volume from QSMs by literature VEF values.Key ResultsStem volume was reliably estimated with TLS. Total volume was overestimated by +21 % using original QSMs, by +9 % and –12 % using two sets of VEF-augmented QSMs, and by –7.3 % using best-available ASMs. The most accurate method differed per site, and the prediction errors for each method varied considerably between sites.ConclusionsVEF-augmented QSMs were only slightly better than original QSMs for estimating tree volume for common species in temperate forests. Despite satisfying estimates with ASMs, the model choice was a large source of uncertainty, and species-specific models did not always exist. Therefore, we advocate for further improving tree volume reconstructions with QSMs, especially for fine branches, instead of collecting more ground-truth data to calibrate VEF and allometric models. Promising developments such as improved co-registration and smarter filtering approaches are ongoing to further constrain volumetric errors in TLS-derived estimates.     

Potential errors in the quantitative evaluation of biogas production in anaerobic digestion processes

Errors that are commonly made in the quantification of biogas from anaerobic digestion experiments were investigated.For liquid displacement gasometers where a barrier solution separates the biogas and the atmosphere, inaccuracy due to gas diffusion was examined experimentally. Acidified saturated saline solution was the most suitable barrier solution, as biogas characteristics changed least with time. Using acidified or tap water caused considerable biogas losses and should therefore be avoided where biogas is stored before measurement.Errors associated with volume calculation from three common liquid displacement gasometer types were investigated theoretically. Corrections that must be made to obtain gas volumes at standard temperature and pressure when using this equipment are discussed. Regarding experimental errors, gasometer designs where displaced liquid is weighed to determine the volume are the most versatile since errors depend mainly upon balance sensitivity. Using liquid heights to calculate volume requires appropriate sizing of the gasometer relative to the volume of gas measured.The calibration of a low flow gas meter was investigated and an approximately linear variation with flow rate was found; hence in situ calibration is advised for this type of instrument. Correction for atmospheric conditions should be performed in real time to reduce errors.     

Calcium signal communication in the central nervous system

The communication of calcium signals between cells is known to be operative between neurons where these signals integrate intimately with electrical and chemical signal communication at synapses. Recently, it has become clear that glial cells also exchange calcium signals between each other in cultures and in brain slices. This communication pathway has received utmost attention since it is known that astrocytic calcium signals can be induced by neuronal stimulation and can be communicated back to the neurons to modulate synaptic transmission. In addition to this, cells that are generally not considered as brain cells become progressively incorporated in the picture, as astrocytic calcium signals are reported to be communicated to endothelial cells of the vessel wall and can affect smooth muscle cell tone to influence the vessel diameter and thus blood flow. We review the available evidence for calcium signal communication in the central nervous system, taking into account a basic functional unit -the brain cell tripartite- consisting of neurons, glial cells and vascular cells and with emphasis on glial-vascular calcium signaling aspects.