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QF-Pro Primer

Spatial Functional Proteomics:
From Expression to Function

A 42-minute deep dive into FLIM-FRET technology, clinical validation, and the QF-Pro reagent & Violet 3.0 FLIM workflow — with an interactive chapter guide linked to the glossary.

Presented by Russell Ulbrich & James Miles, HAWK Biosystems

Click any chapter below to begin
40 chapters 42 glossary terms 44 min

Chapter Guide

Click a timestamp to jump in the video. Click a glossary term to explore the science.

▶ ACT 1
The Problem — Why Expression Biomarkers Fail
Russell Ulbrich · 4 chapters
0:00 – 4:10
▶ 0:00
Introduction & Framing
The expression-vs-function paradigm; the building/corridor/room analogy; 12% immunotherapy response rates; PD-L1 expression failure as predictive biomarker.
Functional vs Expression PD-1/PD-L1 ICI
▶ 1:11
HAWK Biosystems & QF-Pro Overview
Company origin (Francis Crick Institute, CRUK); patented amplified FRET-FLIM technology; 1–10 nm working range vs PLA (40 nm) and colocalization (70–100 nm).
QF-Pro FRET FLIM PLA Colocalization
▶ 2:28
Overtreated & Undertreated Patients
Patient subgroups misclassified by PD-L1 expression; potential 280% efficacy increase; triple response rates; double overall survival in NSCLC.
Patient Stratification Overall Survival NSCLC
▶ 3:39
The Functional Biomarker Challenge
2.13% FRET efficiency threshold; continuous quantitative measurements; precision medicine future depends on measuring what proteins are doing.
FRET Efficiency Clinical Cutpoint Precision Medicine
▶ ACT 2
The Technology — How FRET-FLIM Works
James Miles · 8 chapters
4:11 – 12:17
▶ 4:11
Who Is HAWK Biosystems?
Bilbao-based biotech spun from Crick Institute / CRUK; mission to study protein functional states in fixed cell/tissue samples.
QF-Pro
▶ 5:40
What Is QF-Pro?
Quantifying Functions in Proteins; enhanced FRET-FLIM bioimaging; two-site immunofluorescence labeling; ATTO488 donor + Alexa594 acceptor.
QF-Pro FRET FLIM Donor-Acceptor Pair Two-Site Assay
▶ 6:53
QF-Pro Working Range: 1–10 nm
Energy transfer only within 1–10 nm; TIGIT/CD155 interaction example; differentiating co-expression from genuine interaction.
FRET TIGIT/CD155 Colocalization PPI
▶ 8:00
FRET Physics Explained
Förster resonance energy transfer; spectral overlap requirement; donor excitation transfers energy to acceptor within 10 nm; classical technology from 1947.
FRET Spectral Overlap Förster Radius
▶ 8:58
Why FLIM Over Intensity-Based FRET?
Intensity measurements are rudimentary; fluorescence lifetime is concentration-independent; specific to the fluorophore; donor-only lifetime ~3.2 ns.
FLIM Fluorescence Lifetime Autofluorescence
▶ 10:00
FLIM Measurement: Donor vs Donor+Acceptor
Donor lifetime decreases to ~2.8 ns with acceptor present; FLIM is reproducible, quantitative, and independent of protein concentration.
FLIM Fluorescence Lifetime FRET Efficiency
▶ 11:12
Amplified FRET: Overcoming Autofluorescence
FRET signals typically masked by autofluorescence in tissue; acceptor amplification increases FRET ~4×; clears signal-to-noise barrier; enables FRET in clinical FFPE samples.
aFRET TSA Autofluorescence FFPE
▶ 12:18
Technology Comparison: Coloc vs PLA vs QF-Pro
Colocalization = same building (70–100 nm); PLA = same corridor (14–40 nm); QF-Pro = same room having a meeting (1–10 nm).
Colocalization PLA QF-Pro
▶ ACT 3
The Product — Violet 3.0 & QF-Pro Workflow
James Miles · 6 chapters
13:20 – 18:33
▶ 13:20
QF-Pro Workflow: Accessibility by Design
Standard immunofluorescence workflow; three products: QF-Pro Reagent Kits (FRET), Violet 3.0 (FLIM), QF-Pro Software (analysis); FFPE and fixed cell compatibility.
QF-Pro FFPE Violet 3.0
▶ 14:17
QF-Pro Reagent Kit
Open kit design for screening any mouse/rabbit antibody pair; donor/acceptor fluorophore pair; amplification reagents; up to 50 experiments per kit.
QF-Pro Donor-Acceptor Pair Chromophore
▶ 15:18
Violet 3.0 FLIM Microscope
Benchtop instrument replacing room-sized FLIM systems; modulated diode lasers; FLIM camera; 4-channel LED; takes 4 FFPE slides or 1 multi-well plate.
Violet 3.0 FLIM
▶ 16:19
Ease of Use: Press-Button Hardware
No alignment or expert technician needed; power on, open lid, insert samples; all complexity handled in software.
Violet 3.0
▶ 17:30
Software Workflow & Data Acquisition
Experiment setup, navigation, ROI mapping, acquisition; donor-only lifetime on slice 1; donor+acceptor on consecutive slice 2.
FLIM Violet 3.0
▶ 18:34
QF-Pro Score = FRET Efficiency
Calculation: lifetime(donor+acceptor) / lifetime(donor alone); percentage score for correlation with clinical variables.
FRET Efficiency Clinical Cutpoint
▶ ACT 4
Research Applications — From Bench to Bedside
James Miles · 5 chapters
19:00 – 25:59
▶ 19:00
E-cadherin / β-catenin Interaction
Interaction predisposes cells to metastasis; strong expression in both patients but interaction only in Patient 2; spatial mapping of interaction state.
PPI Spatial Biology Tissue Heterogeneity
▶ 20:51
Colocalization vs FRET: False Positives
Merged IF images show overlap at 70–100 nm, but no FRET at 1–10 nm; colocalization lends itself to false positives.
Colocalization FRET
▶ 21:30
HER2/EGFR/HER3 Interactions in HNSCC
HER2-EGFR, HER3-EGFR, HER3-HER2 interaction mapping; ubiquitous expression but differential interaction states; therapeutic targeting implications.
HER2-HER3 EGFR HNSCC
▶ 23:00
Post-Translational Modifications (aFRET)
Protein kinase B (Akt) phosphorylation state; two-site FRET confirms activation; not susceptible to non-specific binding; EGF stimulation time-response curves.
aFRET PKB/Akt Protein Activation State
▶ 25:02
Drug Dose-Response Curves
Anonymized customer study; compound inhibition of phosphorylation state; dose-response and time-response curves; pharma drug validation application.
aFRET
▶ ACT 5
Clinical Validation — The JCO Evidence
James Miles · 6 chapters
26:00 – 33:23
▶ 26:00
The Immunotherapy Challenge
12% response rates in solid tumors; immune checkpoint mechanism explained; PD-1/PD-L1 interaction; cancer cell immune evasion; why expression-based biomarkers fail.
ICI Immune Checkpoint PD-1/PD-L1
▶ 28:21
JCO Study: PD-L1 Expression vs QF-Pro Interaction
188 NSCLC patients; IHC expression shows no correlation; QF-Pro interaction clearly separates responders from non-responders.
QF-Pro Applications IHC NSCLC
▶ 29:23
The 2.13% FRET Efficiency Cutoff
Standardized clinical cutoff; patients ≥2.13% respond significantly better; high expression + low interaction = overtreated; low expression + high interaction = undertreated.
Clinical Cutpoint FRET Efficiency Patient Stratification
▶ 30:30
Potential Impact: 280% Efficacy Increase
Rescuing misclassified patients could triple response rates, double OS in NSCLC; tip of the iceberg for QF-Pro applications.
Overall Survival Patient Stratification NSCLC
▶ 31:25
Expanding to Multiple Checkpoint Signatures
Sequential imaging of PD-1/PD-L1, TIGIT/CD155, CTLA-4/CD80, LAG-3/MHC-II; building functional immune signatures per patient.
PD-1/PD-L1 TIGIT/CD155 CTLA-4/CD80 LAG-3/MHC II
▶ 32:29
Broader Applications Beyond Oncology
Neurodegenerative diseases, diabetes, cardiovascular; QF-Pro as a service model; any mouse/rabbit antibody pair.
QF-Pro PPI
▶ Q&A
Audience Questions & Expert Answers
James Miles & Panel · 11 chapters
33:24 – 43:30
▶ 33:24
Antigen Retrieval Optimization
Standard antibody manufacturer recommendations work well; reproducible results with standardized methods.
▶ 34:07
Z-Focus Automation
Semi-automated focusing; user fine-tunes Z with focus control; machine calibration sets starting position.
Violet 3.0
▶ 34:44
Frozen Tissue Compatibility
Not yet tested but expected compatible with labeling optimization; FLIM publications support frozen tissue use; neuropathology applications discussed.
FFPE
▶ 35:55
Antibody QC & Clone Selection
IF screening of multiple clones; specificity testing; blocking experiments to validate FRET; different clones give different efficiencies but internally reproducible.
FRET
▶ 37:50
ROI Navigation & Tissue Mapping
DAPI nuclear stain; 2× raster scan; 20×/40× ROI selection marked on overview image.
TMA
▶ 38:43
FLIM Acquisition Speed
16 seconds per image; ~30 ROIs per sample; ~8 minutes per tissue sample.
FLIM
▶ 39:05
Tissue Thickness Range
3–5 µm standard (routine oncology); 50 µm for neuroscience not yet tested but of interest.
▶ 39:41
FRET Efficiency Range & Amplification
Donor-only ~3.2 ns; donor+acceptor ~2.7 ns; high FRET 30–45%; clinical cutoff at 2.1% (low single digits for significance).
FRET Efficiency aFRET
▶ 40:55
Cell Culture Dishes & Multi-Well Plates
Any standard multi-well plate with glass bottom (1.5 grade); 96-well automated mapping; Ibidi and Revvity confirmed compatible.
▶ 41:42
Pixel Thresholding & Background Removal
Intensity thresholding for cells; phasor plot analysis for tissue; separating real signal from autofluorescence; DAPI masking for nuclear proteins.
Phasor Plot Autofluorescence
▶ 42:57
Consecutive Tissue Slices for Donor vs Donor+Acceptor
Two subsequent slices from same block; biologically identical; validated with 6 consecutive slices showing identical donor-only lifetimes.
FFPE