A flow calorimeter for assay of hormone- and metabolite-induced changes in steady-state heat production by tissue

A compact, heat conduction, flow calorimeter for use in monitoring tissue response to metabolic regulators has been designed and constructed. The instrument operates as a perfusion apparatus. Heat production by tissue can be measured continuously, and nutrient and oxygen concentrations can be kept at optimum, constant levels. Introduction of substances of interest is made without production of attendant thermal mixing artifacts. The resolution time of events is less than 10 s. The thermal stability of the instrument is maintained by dynamic methods. The sensitivity of the instrument, 7.2 μW/μV, allows observation of changes in heat production as small as 3 × 10^?4 to 5 × 10^?4 cal/min, the equivalent of 3–5% of the heat production of a representative 1-g quantity of fresh tissue. The calorimeter was used to monitor changes in the steady-state heat production of 250-mg samples of corn coleoptile tissue in response to the plant growth substance, indoleacetic acid.     

Fast titration experiments using heat conduction microcalorimeters.

Thermopile heat conduction calorimeters normally have high time constants. Multistep titration experiments involving fast processes may then require several hours to perform. It is demonstrated that such experiments can be conducted about 10 times faster, without loss of accuracy, by use of a "dynamic correction method". For a new small vessel thermopile conduction calorimeter, injections can be made at 1.5-min intervals.     

An optimized differential heat conduction solution microcalorimeter for thermal kinetic measurements

Heat conduction calorimeters are widely used in biological sciences, but baseline instability, low resolution, electrical noise and motion artifacts have limited their utility. Two main sources of noise, baseline fluctuation or drift and a motion artifact, were traced to amplifier drift, a small (0.015°C) gradient within the constant temperature cylinder, and the method of installing the thermopiles. The addition of heaters to the top and bottom of the cylinder reduced the gradient to approximately 0.003°C and greatly reduced the slow component of the motion artifact. The drift error was reduced by proper mounting of the amplifier and its external components and the enclosure of the calorimeter in a temperature-controlled box.An R-C model of the heat flow in the calorimeter was developed which was employed to discover several means of increasing sensitivity without increasing the rise-time of the calorimeter. Analysis, also based on the model, showed that variations in the air gap between the cell holder can be a major source of error when the calorimeter is used to investigate the kinetics of a chemical reaction. This analysis also showed that the time for the heat to flow through the solution through the solution in the cell can be the dominant factor in determining the rise-time of the instrument.The heat conduction calorimeter described here has improved characterics: a baseline stability of 200 nJ · s^?1 (peak-to-peak) over a 48 h period; a resolution of 200 nJ · s^?1; a sensitivity of 6.504 ± 0.045 J · V^?1 · s^?1 referred to the sensor output; and a rise-time of 122 s for the 10–90% response.     

A differential scanning calorimeter for ice nucleation distribution studies--application to bacterial nucleators

A differential scanning calorimeter has been developed for the automatic detection and measurement of dropwise freezing within a sample of 100-200 water drops. A typical drop size of 1 microliter is employed. The sample is distributed on flat, square (4-cm) thermoelectric sensors and the temperature is scanned downward by conductive cooling to a liquid nitrogen bath. The rate of cooling, typically 1 degree C/min, is set by the choice of a heat conduction rod between the calorimeter and the liquid nitrogen bath. The voltages from the thermopiles along with a system temperature-measuring thermocouple are continuously monitored by digital voltmeters and recorded every half-second in a computer memory. A freezing event in a drop is detected by a characteristic voltage signal whose integral with time is proportional to the size of the drop and its heat of fusion. The half-life of a freezing event signal is 10 s for a 1-microliter drop. The integrated signal produced from multiple freezing events is shown to provide a direct measure of the number of drops frozen at a given temperature. A distribution curve and its smoothed derivative can be constructed directly from these measurements. The instrument, which is termed an "ice nucleometer," is illustrated in determining the ice nucleation distribution in a population of Escherichia coli harboring cloned ice nucleation genes.     

Application of the finite element simulation method to the adiabatic and potentiometric corrections of calorimetric titration data

A general numerical analysis procedure is described which has been applied to an automated differential pH-thermal titration apparatus operated isoperibolically to obtain thermal corrections for heat loss. It is based on the Direct Byte (D-B) finite element computer simulation technique (FEST) applied to the heat conduction behavior of the instrument with time. Thermal constants of the numerical model are determined, and the results of the correction for titration data obtained from acid-base runs show that a constant upper baseline is achieved using this technique for both fast and slow reactions to an accuracy of 2%. The method is equally valid for endothermic and exothermic reactions.     

A twin titration microcalorimeter for the study of biochemical reactions

A small-volume (200 microliter) titration calorimeter of high sensitivity (1 mu cal ) has been developed for the purpose of studying biochemical reactions where the amounts of material are limited to a few nanomoles. High sensitivity is achieved by calorimetric twining , use of glass cells, elimination of vapor space, effective low-energy stirring, and reduction of measurement time. The calorimeter operates using the heat conduction principal with computer-controlled electrical compensation, which reduces the measurement time of each point from 10 to 3 min. This reduction in time is accompanied by a corresponding increase in the precision of measurement. The use of the calorimeter is demonstrated by a measurement of the heat of oxygenation of hemocyanin.     

Design of a Calorimeter for the Continuous Study of Heat Production during Anaerobic Microbial Growth

A conduction type calorimeter has been designed to chase microbial growth in batch system. The calorimeter is of a twin structure having thermopile plates as a temperature sensor, The heat evolution during the microbial growth at a required temperature can be observed as an output-voltage generated at thermopile terminals with a sensitivity of 58.5 mV K^?1

A stainless steel cell with a volume of 300 cm³ serves as a culture cell which is capable of being autoclaved prior to the initiation of calorimetric run, taking out from the calorimeter body.

Because of the twin structure, the apparatus works with sufficient stability in detecting small heat evolution for long duration. Its operation has been demonstrated with the growth of Sacch. cerevisiae grown on liquid synthetic medium under anaerobic condition.     

Estimation of muscle fiber conduction velocity from two-dimensional surface EMG recordings in dynamic tasks

The aims of this study are (1) to demonstrate that multi-channel surface electromyographic (EMG) signals can be detected with negligible artifacts during fast dynamic movements with an adhesive two-dimensional (2D) grid of 64 electrodes and (2) to propose a new method for the estimation of muscle fiber conduction velocity from short epochs of 2D EMG recordings during dynamic tasks. Surface EMG signals were collected from the biceps brachii muscle of four subjects with a grid of 13 × 5 electrodes during horizontal elbow flexion/extension movements (range 120–170°) at the maximum speed, repeated cyclically for 2 min. Action potentials propagating between the innervation zone and tendon regions could be detected during the dynamic task. A maximum likelihood method for conduction velocity estimation from the 2D grid using short time intervals was developed and applied to the experimental signals. The accuracy of conduction velocity estimation, assessed from the standard deviation of the residual of the regression line with respect to time, decreased from (range) 0.20–0.33 m/s using one column to 0.02–0.15 m/s when combining five columns of the electrode grid. This novel method for estimation of muscle fiber conduction velocity from 2D EMG recordings provides an estimate which is global in space and local in time, thus representative of the entire muscle yet able to track fast changes over the execution of a task, as is required for assessing muscle properties during fast movements.     

A New Ultrasensitive Scanning Calorimeter

A new ultrasensitive differential scanning calorimeter is described, having a number of novel features arising from integration between hardware and software. It is capable of high performance in either a scanning or isothermal mode of operation. Upscanning is carried out adiabatically while downscanning is nonadiabatic. By using software-controlled signals sent continuously to appropriate hardware devices, it is possible to improve adiabaticity and constancy of scan rate through use of empirical prerun information stored in memory rather than by using feedback systems which respond in real time and generate thermal noise. Also, instrument response time is software-selectable, maximizing performance for both slow- and fast-transient systems. While these and other sophisticated functionalities have been introduced into the instrument to improve performance and data analysis, they are virtually invisible and add no additional complexities into operation of the instrument. Noise and baseline repeatability are an order of magnitude better than published raw data from other instruments so that high-quality results can be obtained on protein solutions, for example, using as little as 50 μg of protein in the sample cell.     

Dynamic analysis of differential scanning calorimetry data

The apparent heat capacity function measured by high-sensitivity differential scanning calorimetry contains dynamic components of two different origins: (1) an intrinsic component arising from the finite instrument time response; and (2) a sample component arising from the kinetics of the thermal transition under study. The intrinsic instrumental component is always present and its effect on the shape of the experimental curve depends on the magnitude of the calorimeter response time. Usually, high-sensitivity instruments exhibit characteristic time constants varying from 10 to 100 s. This slow response introduces distortions in the shape of the heat capacity function especially at fast scanning rates. In addition to this instrumental component, dynamic effects due to sample relaxation processes also contribute to the shape of the experimental heat capacity profile. Since the nature and magnitude of these effects are a function of the kinetic parameters of the transition, they can be used to obtain kinetic information. This communication presents a dynamic deconvolution technique directed to remove artificial distortions in the shape of the heat capacity function measured at any scanning rate, and to obtain a kinetic characterization of a thermally induced transition. The kinetic characterization obtained by this method allows the researcher to obtain transition relaxation times as a continuous function of temperature. This technique has been applied to the thermal unfolding of ribonuclease A and the pretransition of dipalmitoylphosphatidylcholine (DPPC). In both systems the transition relaxation times are temperature dependent. For the protein system the relaxation time is very slow below the transition temperature (approximately 30 s) and very fast above Tm (less than 1 s) in agreement with direct kinetic measurements. For the pretransition of DPPC, the relaxation time is maximal at the transition midpoint and of the order of approx. 40 s.     

Potentials and limitations of miniaturized calorimeters for bioprocess monitoring

In theory, heat production rates are very well suited for analysing and controlling bioprocesses on different scales from a few nanolitres up to many cubic metres. Any bioconversion is accompanied by a production (exothermic) or consumption (endothermic) of heat. The heat is tightly connected with the stoichiometry of the bioprocess via the law of Hess, and its rate is connected to the kinetics of the process. Heat signals provide real-time information of bioprocesses. The combination of heat measurements with respirometry is theoretically suited for the quantification of the coupling between catabolic and anabolic reactions. Heat measurements have also practical advantages. Unlike most other biochemical sensors, thermal transducers can be mounted in a protected way that prevents fouling, thereby minimizing response drifts. Finally, calorimetry works in optically opaque solutions and does not require labelling or reactants. It is surprising to see that despite all these advantages, calorimetry has rarely been applied to monitor and control bioprocesses with intact cells in the laboratory, industrial bioreactors or ecosystems. This review article analyses the reasons for this omission, discusses the additional information calorimetry can provide in comparison with respirometry and presents miniaturization as a potential way to overcome some inherent weaknesses of conventional calorimetry. It will be discussed for which sample types and scientific question miniaturized calorimeter can be advantageously applied. A few examples from different fields of microbiological and biotechnological research will illustrate the potentials and limitations of chip calorimetry. Finally, the future of chip calorimetry is addressed in an outlook.     

The application of a novel heat flux calorimeter for studying growth of Escherichia coli W in aerobic batch culture

The modification and principle of a novel heat flux calorimeter for the in situ, on-line measurement of the heat generated during microbial growth is described. Data concerning the physical characterization of the calorimeter as a fermentor, including stability and sensitivity of the heat signal, are presented. The calorimeter has been successfully applied to the study of the aerobic batch culture of Escherichia coli W on glucose under carbon and nitrogen limitation. A direct correlation between growth and heat evolution was obtained. Quantitative analysis of the data suggests that the new calorimetric technique could be used for monitoring growth and specific metabolic events, for convenient medium optimization, and as a basis for a novel fermentation process control system.     

Power and heat measurement by direct calorimetry of individual insect response to allelo- and toxic compounds

A microcalorimeter was constructed for the individual insect in order to measure the insect's power output as a function of time (the thermogram). The instrument has a figure of merit of 7 μW/μV. It includes a Peltier pumped thermoelectric control loop which protects from intruding ambient thermals, and holds baseline drift to less than 2 μV/day. The design is a conventional twin, or differential heat conduction calorimeter, with two insect-holding vessels of glass. The vessels are connected by a stopcock, to give the insect the option of crawling from the sample chamber to the reference chamber. Continuous, metered air flow is provided. A small pulse of compound may be born in, as vapor with the air flow, for the study of attractants, toxic compounds, anesthetics and allelochemicals. The insect's reaction to such compounds, appears in the thermogram. Cabbage looper larvae were examined in their irritative exothermic reaction to microgram amounts of benzene, and in their neutral behavior toward similar amounts of aliphatic hydrocarbons delivered as a single pulse of vapor. The male northern corn rootworm was monitored in his attraction to female rootworm extract (‘pheromone’) and his tendency to move upwind, or up airflow, toward the pheromone. The instrument enables discrimination of the transient metabolism of muscle use, from that of the resting state power which is probably the true basal metabolic rate power.     

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.     

Estimation of surface heat flux and temperature distributions in a multilayer tissue based on the hyperbolic model of heat conduction

In this study, an inverse algorithm based on the conjugate gradient method and the discrepancy principle is applied to solve the inverse hyperbolic heat conduction problem in estimating the unknown time-dependent surface heat flux in a skin tissue, which is stratified into epidermis, dermis, and subcutaneous layers, from the temperature measurements taken within the medium. Subsequently, the temperature distributions in the tissue can be calculated as well. The concept of finite heat propagation velocity is applied to the modeling of the bioheat transfer problem. The inverse solutions will be justified based on the numerical experiments in which two different heat flux distributions are to be determined. The temperature data obtained from the direct problem are used to simulate the temperature measurements. The influence of measurement errors on the precision of the estimated results is also investigated. Results show that an excellent estimation on the time-dependent surface heat flux can be obtained for the test cases considered in this study.     

A sensitive direct calorimeter for small mammals

A sensitive direct calorimeter for small animals is presented. Its principle is based on the measurement of the heat transfer from the animal chamber to a heat sink. The system gives repetitive measurements with a high efficiency and allows a detailed time-related measurement of the heat production by the whole animal. Its low response time can be advantageously used for the study of post-prandial heat generation and diet-induced thermogenesis. Data on the heat production by Wistar and lean and obese Zucker rats is also included.     

Transient thermal behavior in biological systems

The Peaceman-Rachford finite difference method is applied to cylindrically symmetric, transient heat conduction problems in biological media. Inhomogeneous media and internal sources which vary in both space and time are permitted. Boundary conditions are satisfied without sacrificing high local resolution by means of an exponentially stretched grid. Computation time on a Philco 2000/210 computer is approximately 5 msec per grid point per time step.     

The linear electrode array: a useful tool with many applications.

In this review we describe the basic principles of operation of linear electrode arrays for the detection of surface EMG signals, together with their most relevant current applications. A linear array of electrodes is a system which detects surface EMG signals in a number of points located along a line. A spatial filter is usually placed in each point for signal detection, so that the recording of EMG signals with linear arrays corresponds to the sampling in one spatial direction of a spatially filtered version of the potential distribution over the skin. Linear arrays provide indications on motor unit (MU) anatomical properties, such as the locations of the innervation zones and tendons, and the fiber length. Such systems allow the investigation of the properties of the volume conductor and its effect on surface detected signals. Moreover, linear arrays allow to estimate muscle fiber conduction velocity with a very low standard deviation of estimation (of the order of 0.1-0.2 m/s), thus providing reliable indications on muscle fiber membrane properties and their changes in time (for example with fatigue or during treatment). Conduction velocity can be estimated from a signal epoch (global estimate) or at the single MU level. In the latter case, MU action potentials are identified from the interference EMG signals and conduction velocity is estimated for each detected potential. In this way it is possible, in certain conditions, to investigate single MU control and conduction properties with a completely non-invasive approach. Linear arrays provide valuable information on the neuromuscular system properties and appear to be promising tools for applied studies and clinical research.     

Control of ecdysis by heat in Manduca sexta

Under constant-light conditions, adult Manduca sexta emerge approximately 5 hr after the middle of a 3 hr 3°C heat pulse. The insects can be entrained to a heat cycle in 3 days. Entrainment will decrease at approximately the same rate. Photic signals can modify the precision of the response but, when the insects are presented with conflicting photic and thermal signals, they respond to the thermal signal. During the 3 days of entrainment, the insects appear to hasten the time of eclosion as much as 6 hr to meet the thermal cycle, but the degeneration of the intersegmental muscles is not similarly hastened. The technique described can be applied to various physiological and behavioural studies.