Temperature Modulated Differential Scanning Calorimetry - a tool for evaluation of plant glass transition at low temperatures
Zamecnik, Jiri; Bilavcik, Alois; Faltus, Milos (2008)
Agrifood Research Working papersMTT:n selvityksiä
Myynti MTT, Tietopalvelut 31600 Jokioinen
Myynti MTT, Tietopalvelut 31600 Jokioinen
There are two main cryopreservation groups of methods for plant meristematic tissue. The first group of cryopreservation methods is based on water freezing inside the tissue (mostly outside cells) after slow cooling rates. The second group of the methods is based on vitrification. Low water content and high cooling rates leads to the glassy state in plant tissue. The differential scanning calorimeter (DSC) is an appropriate instrument for both types of the methods to give exact information for controlling and/or improving the cryoprotocol. Water together in liquid, solid and vapour forms play a crucial role for survival of shoot tips after cryopreservation. The DSC measurement can help us to measure the amount of frozen water within cryopreservation protocol. In addition to these characteristics, the DSC determination of ice nucleation temperature is of importance too. For the first method supposing that plants are able to tolerate frozen water outside the cells, the information about ice nucleation temperature is crucial. If the ice nucleation temperature occurs at too low temperatures the burst freezing after deep supercooling inside the cells causes the lethal injury and death. The DSC is important also for the second method vitrification because of the glass transition temperature. It should be close to higher temperatures, close to zero, to decrease the probability of water crystallization during samples exposition to low temperatures. The measurement of ice nucleation is important for vitrification cryoprotocols as well. Vitrification is a method avoiding the ice nucleation, mainly by high rate of cooling and warming. In conclusion, there are four important thermal characteristics which can be obtained by DSC measurements: ice nucleation temperature, melting temperature, amount of frozen water and glass transition temperature. According to our experience the glass transition temperatures of plant samples were very close to their thawing temperatures. In some cases these two thermal events are so close that it is too difficult or in some cases completely impossible to differentiate them. At these cases the conventional DSC technique is unable to separate the thermal events. Recently, the differential scanning calorimetry with modulated temperature gave good tool for measurement of such samples. Since 1992 a new DSC method based on sinusoidal temperature modulation was introduced by Reading and co-workers. By temperature modulated differential scanning calorimetry (TMDSC) it is possible to obtain more information about sample in comparison with conventional DSC using linear change of temperature. TMDSC heat flow signal is composed of two parts: a) reversing heat flow - heat capacity component, heating rate dependent responsible for glass transition and some melting and b) nonreversing heat flow - kinetic component, time dependent responsible for crystallization, some melting and enthalpy relaxation. Conventional DSC can only measure the sum of these two components. Heat capacity (Cp) is generally calculated from the difference in heat flow between blank run and sample run under identical conditions including cooling/heating rate. In TMDSC, Cp is determined by dividing the modulated heat flow amplitude by the modulated heat rate amplitude. The modulation type is specific for each instrument. Sinusoidal and jig-saw type of temperature modulation controlling the sample temperature is mostly used and is typical for each instrument of each producer. A new method stochastic modulated temperature was recently published /2/. Quasi-isothermal temperature modulated DSC (QITMDSC) method is based on analysis of sample response to modulated temperature around the constant temperature. After constant output of heat capacity of the sample the temperature abruptly changes to new modulated temperature. Discrete Fourier transformation is used mostly for data evaluation of TMDSC and QITMDSC methods. The evaluation is a complex of difficult mathematical system of equations but modern software of the instrument is able to evaluate the measured data. TMDSC has several significant practical advantages. For example, in glass transitions studies the limit of detection and resolution increases without loss of sensitivity, that makes the correct assignment more certain and quantification of amorphous phases is more accurate /1/. It is advantage to know the temperature range of glass transition before application of temperature modulated DSC. On the base of preliminary measurement by convenient DSC it is possible to decide which parameters of thermal analysis methods are appropriate for measuring TMDSC or QITMDSC. Three parameters can be chosen independently for each method with modulated temperature - modulated amplitude, modulated period and rate of cooling or heating. Typical modulated amplitudes are between 0.1 1 °C with modulated period 60 - 100s (0.017 0.1Hz). Typical cooling/warming rate used is 10 °C/min. Larger amplitude leads to higher sensitivity, smaller amplitude leads to higher resolution. So far, it has not been possible to change the type of modulated signals in an instrument. This option is influenced by setting of particular instrument by individual producer. Usually, the measurement by temperature modulated DSC technique is performed and the data for analysis are collected during sample warming because stochastic event of ice nucleation is avoided. A proper choice of amplitude and modulation allows keeping plant sample at permanent thawing or at both thawing and freezing events during modulation of the sample. For melting/crystallization studies heat-only" amplitude should be used. There is an exact calculation of the maximum temperature amplitude (Tamp) for heat-only modulation: Tamp=Hr P/2ð, where: Hr is average heating rate, P is period of modulation.
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