Violet 3.0 & QF-Pro® Technology

Conventional spatial biology reveals where proteins are; Violet 3.0 and QF-Pro® technology reveal what they are doing. Most current proteomic assays are limited to measuring molecular expression levels, overlooking whether proteins are actually activated or interacting with one another. In critical fields such as immuno-oncology, neurobiology and drug development, the relationship between expression and the actual function of proteins is often uncoupled, greatly limiting the predictive value of conventional biomarkers and leading to the failure of numerous therapeutic strategies.

Developed by Hawk Biosystems, the patented QF-Pro® (Quantifying Functions in Proteins) technology is the first and only platform capable of spatially quantifying protein function in situ with nanoscale resolution below 10 nm, directly in complex biological samples such as formalin-fixed, paraffin-embedded (FFPE) tissues and cell cultures.

Through the all-in-one Violet 3.0 bioimaging system and its optimised reagent kits, QF-Pro® translates static expression profiles into dynamic maps of molecular function. By measuring protein-protein interactions (PPIs), post-translational modifications (PTMs) such as phosphorylation, and receptor activation states at the nanoscale, this solution provides a layer of functional information hidden from other omics technologies.

How does it work?

QF-Pro® technology is based on the combination of a two-site antibody binding assay, amplified Förster resonance energy transfer (FRET), and fluorescence lifetime imaging microscopy (FLIM).

FRET-FLIM Operating Principle:

1

Specific two-site labelling: Two primary antibodies from different species are used against two epitopes of interest, whether they are two interacting proteins or a protein and its post-translational modification site.

2

QF-Pro® secondary detection: Specific secondary antibodies coupled to a donor chromophore fluorophore (green) and an amplified chemical acceptor (red) are applied.

3

FRET Energy Transfer: If the epitopes are in genuine physical contact, at a distance of between 1 and 10 nm, non-radiative energy transfer occurs from the excited donor to the acceptor.

4

Fluorescence Lifetime Measurement (FLIM) in Violet 3.0: The instrument measures the decay of the donor fluorescence lifetime with picosecond precision. When FRET occurs, the donor lifetime is significantly reduced. Violet 3.0 scans the sample pixel by pixel to record this reduction.

5

Generation of the QF-Pro® Score: The proprietary software automatically calculates FRET efficiency in each region of interest (ROI), translating it into a continuous numerical value (Score) and a high-resolution spatial functional map without requiring manual curve adjustments or subjective interpretation.