302 7.7  Characterizing Physical Properties of Biological Samples in Bulk

7.7  CHARACTERIZING PHYSICAL PROPERTIES OF BIOLOGICAL

SAMPLES IN BULK

There are several methods that enable experimental measurements on relatively macro­

scopic volumes of biological material that use, at least in part, biophysical techniques but

whose mainstream applications are in other areas of the biosciences, for example, test

tube length scale level experiments to measure the temperature changes due to biochem­

ical reactions. Also though, bulk samples of biological tissue can be probed to generate

ensemble average data from hundreds or thousands of cells of the same type in that tissue,

but also encapsulating the effect from potentially several other cell types as well as from

extracellular material. This may therefore seem like a crude approach compared to the high

spatial precision methods utilizing optical techniques discussed earlier in this chapter; how­

ever, what these methods lack in being able to dissect out some of the finer details of het­

erogeneous tissue features they make up for in generating often very stable signals with low

levels of noise.

7.7.1  CALORIMETRY

One of the most basic biophysical techniques involves measuring heat transfer in bio­

logical processes in vitro, which ultimately may involve the absorption and/​or emission of

IR photons. The fact of calorimetry being a very established method takes nothing away

from its scientific utility; in fact, it demonstrates a measure of its robustness. Changes in

thermodynamic potentials, or state variables, such as enthalpy (H), may be measured dir­

ectly experimentally. Other thermodynamic potentials that are more challenging to measure

directly such as entropy (S), or the Gibbs free energy (G) that depends on entropy, need to be

inferred indirectly from more easily measurable parameters, with subsequent analysis util­

izing the first-​order Maxwell’s relations of thermal physics to relate the different thermo­

dynamic potentials.

The most quantifiable parameter is sample temperature, which can be measured using spe­

cifically calibrated chambers of precise internal volumes, which typically include an integrated

stirring device with chamber walls maximally insulated against heat flow to generate an adia­

batic measuring system. Time-​resolved temperature changes inside the chamber can easily

be monitored with an electrical thermometer using a thermistor or thermocouple. Inside, a

biological sample might undergo chemical and/​or physical transitions of interest that may be

exothermic or endothermic, depending on whether or not they generate or absorb heat, and

enthalpic change can be very simply calculated from the change in temperature and know­

ledge of the specific heat capacity of the reactant mixture.

Isothermal titration calorimetry (ITC) is often used as an alternative. Here an adiabatic

jacket made from a thermally highly conductive alloy is used to surround the sample cell,

while an identical reference cell close enough to transfer heat very efficiently just to the

sample cell contains a reference heater whose output is adjusted dynamically so as to main­

tain a constant measured temperature in the sample chamber. ITC has been used to study the

kinetics and stoichiometry of reactants and products through monitoring estimated changes

in thermodynamic potentials as a function of the titration of ligands injected into the sample

cell that contains a suitable reactant, for example, of ligand molecules binding to proteins or

DNA in the sample solution.

The heat transfer processes measured in biology are most usually due to a biochemical

reaction, but potentially also involve phase transitions. For example, different mixtures of

lipids may undergo temperature-​dependent phase transition behavior that gives insight

into the architecture of cell membranes. The general technique used to detect such phase

transitions operates using similar isothermal conditions as for ITC and is referred to as dif­

ferential scanning calorimetry.