Isothermal microcalorimetry (IMC) is a laboratory method for real-time monitoring and dynamic analysis of chemical, physical and biological processes. Over a period of hours or days, IMC determines the onset, rate, extent and energetics of such processes for specimens in small ampoules (e.g. 3–20 ml) at a constant set temperature (c. 15 °C–150 °C).
IMC accomplishes this dynamic analysis by measuring and recording vs. elapsed time the net rate of heat flow (μJ/s = μW) to or from the specimen ampoule, and the cumulative amount of heat (J) consumed or produced.
IMC is a powerful and versatile analytical tool for four closely related reasons: All chemical and physical processes are either exothermic or endothermic—produce or consume heat. The rate of heat flow is proportional to the rate of the process taking place. IMC is sensitive enough to detect and follow either slow processes (reactions proceeding at a few % per year) in a few grams of material, or processes which generate minuscule amounts of heat (e.g. metabolism of a few thousand living cells). IMC instruments generally have a huge dynamic range—heat flows as low as 1 μW and as high as 50,000 μW can be measured by the same instrument.
The IMC method of studying rates of processes is thus broadly applicable, provides real-time continuous data, and is sensitive. The measurement is simple to make, takes place unattended and is non-interfering (e.g. no fluorescent or radioactive markers are needed).
However, there are two main caveats that must be heeded in use of IMC: Missed data: If externally prepared specimen ampoules are used, it takes about 40 minutes to slowly introduce an ampoule into the instrument without significant disturbance of the set temperature in the measurement module. Thus any processes taking place during this time are not monitored. Extraneous data: IMC records the aggregate net heat flow produced or consumed by all processes taking place within an ampoule. Therefore, in order to be sure what process or processes are producing the measured heat flow, great care must be taken in both experimental design and in the initial use of related chemical, physical and biologic assays.
In general, possible applications of IMC are only limited by the imagination of the person who chooses to employ it as an analytical tool and the physical constraints of the method. Besides the two general limitations (main caveats) described above, these constraints include specimen and ampoule size, and the temperatures at which measurements can be made. IMC is generally best suited to evaluating processes which take place over hours or days. IMC has been used in an extremely wide range of applications, and many examples are discussed in this article, supported by references to published literature. Applications discussed range from measurement of slow oxidative degradation of polymers and instability of hazardous industrial chemicals to detection of bacteria in urine and evaluation of the effects of drugs on parasitic worms. The present emphasis in this article is applications of the latter type—biology and medicine.
Overview
Definition, purpose, and scope
Calorimetry is the science of measuring the heat of chemical reactions or physical changes. Calorimetry is performed with a calorimeter.
Isothermal microcalorimetry (IMC) is a laboratory method for real-time, continuous measurement of the heat flow rate (μJ/s = μW) and cumulative amount of heat (J) consumed or produced at essentially constant temperature by a specimen placed in an IMC instrument. Such heat is due to chemical or physical changes taking place in the specimen. The heat flow is proportional to the aggregate rate of changes taking place at a given time. The aggregate heat produced during a given time interval is proportional to the cumulative amount of aggregate changes which have taken place.
IMC is thus a means for dynamic, quantitative evaluation of the rates and energetics of a broad range of rate processes, including biological processes. A rate process is defined here as a physical and/or chemical change whose progress over time can be described either empirically or by a mathematical model (Bibliography: Glasstone, et al. 1941 and Johnson, et al. 1974 and rate equation).
The simplest use of IMC is detecting that one or more rate processes are taking place in a specimen because heat is being produced or consumed at a rate that is greater than the detection limit of the instrument used. This can be a useful, for example, as a general indicator that a solid or liquid material is not inert but instead is changing at a given temperature. In biological specimens containing a growth medium, appearance over time of a detectable and rising heat flow signal is a simple general indicator of the presence of some type of replicating cells.
However, for most applications it is paramount to know, by some means, what process or processes are being measured by monitoring heat flow. In general this entails first having detailed physical, chemical and biological knowledge of the items placed in an IMC ampoule before it is placed in an IMC instrument for evaluation of heat flow over time. It is also then necessary to analyze the ampoule contents after IMC measurements of heat flow have been made for one or more periods of time. Also, logic-based variations in ampoule contents can be used to identify the specific source or sources of heat flow. When rate process and heat flow relationships have been established, it is then possible to rely directly on the IMC data.
What IMC can measure in practice depends in part on specimen dimensions, and they are necessarily constrained by instrument design. A given commercial instrument typically accepts specimens of up to a fixed diameter and height. Instruments accepting specimens with dimensions of up to 1 or 2 cm in diameter by 5 cm in height are typical. In a given instrument larger specimens of a given type usually produce greater heat flow signals, and this can augment detection and precision.
Frequently, specimens are simple 3 to 20 ml cylindrical ampoules (Fig. 1) containing materials whose rate processes are of interest—e.g. solids, liquids, cultured cells—or any combination of these or other items expected to result in production or consumption of heat. Many useful IMC measurements can be carried out using simple sealed ampoules, and glass ampoules are common since glass is not prone to undergoing heat-producing chemical or physical changes. However, metal or polymeric ampoules are sometimes employed. Also, instrument/ampoule systems are available which allow injection or controlled through-flow of gasses or liquids and/or provide specimen mechanical stirring.
Commercial IMC instruments allow heat flow measurements at temperatures ranging from 15 °C to 150 °C. The range for a given instrument may be somewhat different.
IMC is extremely sensitive – e.g. heat from slow chemical reactions in specimens weighing a few grams, taking place at reactant consumption rates of a few percent per year, can be detected and quantified in a matter of days. Examples include gradual oxidation of polymeric implant materials and shelf life studies of solid pharmaceutical drug formulations (Applications: Solid materials).
Also the rate of metabolic heat production of e.g. a few thousand living cells, microorganisms or protozoa in culture in an IMC ampoule can be measured. The amount of such metabolic heat can be correlated (through experimentation) with the number of cells or organisms present. Thus, IMC data can be used to monitor in real time the number of cells or organisms present and the net rate of growth or decline in this number (Applications: Biology and medicine).
Although some non-biological applications of IMC are discussed (Applications: Solid materials) the present emphasis in this article is on the use of IMC in connection with biological processes (Applications: Biology and medicine).
Data obtained
A graphic display of a common type of IMC data is shown in Fig. 2. At the top is a plot of recorded heat flow (μJ/s = μW) vs. time from a specimen in a sealed ampoule, due to an exothermic rate process which begins, accelerates, reaches a peak heat flow and then subsides. Such data are directly useful (e.g. detection of a process and its duration under fixed conditions) but the data are also easily assessed mathematically to determine process parameters. For example, Fig. 2 also shows an integration of the heat flow data, giving accumulated heat (J) vs. time. As shown, parameters such as the maximum growth (heat generation) rate of the process, and the duration time of the lag phase before the process reaches maximum heat can be calculated from the integrated data. Calculations using heat flow rate data stored as computer files are easily automated. Analyzing IMC data in this manner to determine growth parameters has important applications the life sciences (Applications: Biology and medicine). Also, heat flow rates obtained at a series of temperatures can be used to obtain the activation energy of the process being evaluated (Hardison et al. 2003).
Development history Lavoisier and Laplace are credited with creating and using the first isothermal calorimeter around 1780 (Bibliography: Lavoisier A & Laplace PS 1780). Their instrument employed ice to produce a relatively constant temperature in a confined space. They realized that when they placed a heat-producing specimen on the ice (e.g. a live animal), the mass of liquid water produced by the melting ice was directly proportional to the heat produced by the specimen.
Many modern IMC instrument designs stem from work done in Sweden in the late 1960s and early 1970s (Wadsö 1968, Suurkuusk & Wadsö 1974). This work took advantage of the parallel development of solid-state electronic devices—particularly commercial availability of small thermoelectric effect (Peltier-Seebeck) devices for converting heat flow into voltage—and vice versa.
In the 1980s, multi-channel designs emerged (Suurkuusk 1982), which allow parallel evaluation of multiple specimens. This greatly increased the power and usefulness of IMC and led to efforts to fine-tune the method (Thorén et al. 1989). Much of the further design and development done in the 1990s was also accomplished in Sweden by Wadsö and Suurkuusk and their colleagues. This work took advantage of the parallel development of personal computer technology which greatly augmented the ability to easily store, process and interpret heat flow vs. time data.
Instrument development work since the 1990s has taken further advantage of the continued development of solid-state electronics and personal computer technology. This has created IMC instruments of increasing sensitivity and stability, numbers of parallel channels, and even greater ability to conveniently record, store and rapidly process IMC data. In connection with wider use, substantial attention has been paid to creating standards for describing the performance of IMC instruments (e.g. precision, accuracy, sensitivity) and for methods of calibration (Wadsö and Goldberg 2001).
Instruments and measurement principles
Instrument configurations