Backreflection.
Backreflection monitors light attenuation due to scattering to assess aggregation and measure sample turbidity.
When to use backreflection.
The technique measures a sample’s turbidity, or cloudiness. As proteins aggregate, they scatter light away from the detection path, reducing the backreflected signal. This provides a sensitive readout of aggregate formation during thermal or chemical stress.
Backreflection is especially useful for proteins that form large, visible aggregates during thermal unfolding. It’s less sensitive to small oligomers or subtle changes in particle size, where DLS excels.
Backreflection is ideal when you need to:
When to use backreflection.
The technique measures a sample’s turbidity, or cloudiness. As proteins aggregate, they scatter light away from the detection path, reducing the backreflected signal. This provides a sensitive readout of aggregate formation during thermal or chemical stress.
Backreflection is ideal when you need to:
- Detect aggregation during thermal stress
- Identify conditions that minimize aggregation
- Distinguish between unfolding and aggregation (when combined with nanoDSF)
- Monitor large aggregate formation that might not be visible by DLS
- Screen formulations for colloidal stability
Backreflection is especially useful for proteins that form large, visible aggregates during thermal unfolding. It’s less sensitive to small oligomers or subtle changes in particle size, where DLS excels.
The science behind backreflection.
Aggregates scatter light and reduce backreflected signal
In a backreflection measurement, light is directed into the sample and reflects off a mirror positioned behind the sample volume. The reflected light passes back through the sample and returns to the detector.
When the sample contains well-folded, monomeric protein, most of the light passes through with minimal scattering. The backreflected signal remains high.
As aggregates form—whether from thermal stress, chemical denaturants, or prolonged incubation—they grow large compared to the folded protein. Large aggregates scatter light at wide angles, deflecting it away from the detection path. This reduces the intensity of light reaching the detector.
The decrease in backreflected light intensity is a direct measure of turbidity—the cloudiness of the solution.
Turbidity increases with aggregate size and concentration
Turbidity depends on both the size and number of aggregates. Small aggregates scatter less light than large ones. As aggregation progresses, both the size and number of aggregates increase, causing turbidity to rise.
Backreflection is particularly sensitive to large, amorphous aggregates that scatter light efficiently. This makes it complementary to DLS, which is better suited for detecting smaller aggregates and measuring their size distribution.
Parameters you measure with backreflection.
Parameters you measure with backreflection.
Onset of aggregation (Tagg or Tturb)
The temperature at which the backreflected signal begins to decrease, indicating the onset of aggregation. This parameter identifies when the sample starts to lose colloidal stability.
Aggregation profile
The shape and magnitude of the turbidity signal over the course of the experiment. This reveals whether aggregation is rapid or gradual, and whether it correlates with protein unfolding.
Better show than tell. See how DLS generates information about your sample.
How backreflection complements other techniques.
Backreflection measures aggregation-induced turbidity, which is distinct from the size and distribution information provided by DLS. When used together, backreflection and DLS give you a complete picture of colloidal stability:
- DLS detects small aggregates and measures their size distribution
- Backreflection detects large aggregates and tracks turbidity changes
Using backreflection, you can characterize colloidal stability even when aggregation is so heavy that DLS struggles. Combining Tonset (from nanoDSF), Tsize from DLS, and Tturbidity (from backreflection), you can determine whether aggregation occurs during unfolding, after unfolding, or independently of unfolding. This insight helps you understand the mechanism of instability and design better formulations.
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Frequently Asked Questions
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