TRIC - Temperature-Related Intensity Change.
TRIC measures changes in fluorescence intensity that occur when a localized temperature increase is applied to a sample.
When to use TRIC.
The technique exploits the temperature sensitivity of fluorophores. When a fluorescently labeled target binds to a ligand, the fluorophore’s local environment changes, altering how it responds to temperature. This change in temperature response provides a readout of the binding event.
TRIC is ideal when you need to:
The science behind TRIC.
Fluorescence is temperature-sensitive
Most fluorophores exhibit temperature-dependent fluorescence. As temperature increases, fluorescence intensity typically decreases. This happens because higher temperatures increase the rate of non-radiative decay—energy dissipates as heat rather than light.
The magnitude of this temperature effect depends on the fluorophore’s chemical environment. A fluorophore buried in a hydrophobic protein pocket responds differently to temperature than one exposed to aqueous solution.
The chemical environment around the fluorophore changes upon binding
This can happen in several ways:
• Shielding from solvent: If binding buries the fluorophore in a more hydrophobic environment, it becomes less exposed to water.
• Conformational changes: Binding may cause the protein to fold or unfold, moving the fluorophore to a different location.
• Proximity to the ligand: The ligand itself may interact with or quench the fluorophore.
An infrared laser creates a rapid temperature jump
In a TRIC experiment, an infrared laser creates a localized temperature increase in the sample—typically a few degrees Celsius. This happens rapidly, within milliseconds.
The fluorescence intensity is measured before and after the temperature jump. The change in fluorescence is quantified as Fnorm (normalized fluorescence), calculated as the ratio of fluorescence after heating to fluorescence before heating.
Environmental changes alter how the fluorophore responds to the temperature increase. The Fnorm value shifts when the target is bound compared to when it’s unbound.
How it works.
How it works.
TRIC reveals ligand-induced aggregation
Like MST, TRIC can detect ligand-induced aggregation. Aggregation produces characteristic changes in the TRIC signal—often a sharp, non-sigmoidal change at higher ligand concentrations.
This aggregation detection is particularly valuable in fragment screening and small molecule discovery, where hydrophobic compounds frequently cause non-specific aggregation. TRIC identifies these problematic compounds, preventing false positives from advancing.
When TRIC is used as an orthogonal method to confirm hits from other assays, its aggregation detection capability often explains why some compounds showed activity in biochemical assays but aren't true binders—they're aggregators.
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