The backreflection optics are a module for assessing protein aggregation and precipitation by measuring light intensity loss due to scattering. Data recorded by backreflection optics are therefore labeled "Turbidity" in Prometheus Panta software. This article explains how the backreflection optics work. 

The backreflection optics emit a light beam which passes through the capillary and is reflected by the surface of the thermal element, passing back through the capillary again and detected by the same optical system. Generally speaking, if aggregates are present in the sample, they scatter some of the light, which is therefore not backreflected (see image). The difference between the light issued and the backreflected light that reaches the detector is used for quantification.

The amount of light loss, also called attenuation, depends on multiple parameters such as particle size and concentration. While allowing for a rapid and precise detection of aggregation onset temperatures, the backreflection optics do not allow for a precise determination of particle size or number. The overall degree of aggregation is expressed in Attenuation Units - AU, or as depicted in the software, milli-AU (mAU), and calculated according to the Beer-Lambert law:

Empty slots on the tray show a value of ~ 0 mAU. A water filled capillary yields values in the range of 70 - 100 mAU. Samples with maximal turbidity display values of up to 600 mAU.

The backreflection optics record turbidity data in parallel to a thermal unfolding experiment and on the same capillaries, providing an extremely fast and convenient way of analyzing protein aggregation. For an in-depth analysis of protein aggregation and particle sizes, Prometheus Panta allows acquisition of DLS data in parallel to backreflection and fluorescence measurements on the same capillaries during a single thermal unfolding experiment. The most important of the turbidity output parameters is the onset of turbidity (Tturbidity, see image below), which is the temperature at which a considerable amount of aggregation starts to appear in the sample. This can be a useful parameter for comparing protein colloidal stability, and to determine the maximum temperature to keep your sample below. In general, the onset of aggregation correlates with the unfolding of the protein as shown in the image below. For proteins showing more than one unfolding event, aggregation may happen as a consequence of the first unfolding event or of a later one. 

Particles in the sample with a diameter of around 25 nm and larger will show a turbidity signal. Because the scattering behavior changes with the diameter of the particle, conclusions about the exact size or the number of particles in the sample are not possible with the backreflection optics. Particle sizes can be determined with the DLS optics, which are also sensitive to smaller particles below the backreflection threshold. The backreflection optics allow for analysis of heavy aggregation down to precipitation, while the DLS optics are suited to detect monomeric proteins, the distribution of particle sizes in the sample, and trace amounts of aggregates.