Technique - (3) Fluorescence fluctuation spectroscopy (FCS / number and brightness / phasor FLIM)

Type: Experimental

Description: Photon-counting analysis of fluorescence fluctuations in live cells to extract diffusion, oligomeric state, molecular flow and lifetime-resolved contrast without localisation.

Department(s)/lab(s): EMBL Australia Node in Single Molecule Science, UNSW Medicine and Health | Gambin Single Molecule Biophysics Group @ UNSW
Summary:

Gambin was the first EMBL Australia group leader appointed to Single Molecule Science. His signature method combines cell-free protein expression with two-colour single-molecule coincidence and fluctuation spectroscopy, which sidesteps purification entirely: proteins are expressed, labelled and measured in lysate, an order of magnitude faster than conventional interaction assays. The biology is protein self-association and aggregation — alpha-synuclein in Parkinson's, cardiac and muscular disease proteins — where the size distribution of oligomers, not the mean, is the quantity of interest. Positioned against the established body of NV-ensemble quantum sensing work — DEER, nanoscale NMR and T1 relaxometry protocols operating at pT/sqrt(Hz) field sensitivity — the conceptual overlap with quantum biosensing is the insistence on distributions over averages, and his aggregation systems (paramagnetic-species-generating, redox-active amyloid) are a plausible target for T1-relaxometry-based NV detection at pT/sqrt(Hz) in the near term.

Department(s)/lab(s): School of Physics (joint with Biochemistry and Pharmacology) | Hinde Laboratory (Cell Nucleus Biophysics) @ UMelb
Summary:

Hinde is a fluorescence-fluctuation physicist embedded in cell biology: she uses pair-correlation function analysis, number-and-brightness, phasor-FLIM and FRET to read out chromatin compaction, protein-chromatin binding dynamics and nucleocytoplasmic transport in living nuclei, at spatial and temporal scales that conventional imaging averages away. The programme is a technique-pushing one — the emphasis is on extracting nanoscale structural information from photon statistics rather than on brute-force localisation — and it is now being coupled to quantum sensing through her QUBIC investigatorship, where the goal is to combine fluorescence readouts with NV-based magnetic and spin-noise contrast in the same cell. Positioned against the established body of NV-ensemble quantum sensing work — DEER, nanoscale NMR and T1 relaxometry protocols operating at pT/sqrt(Hz) field sensitivity — her role in QUBIC is to supply the cell-biological questions and the correlative optical readouts that make pT/sqrt(Hz)-class ensemble sensing biologically interpretable. Preferred attribute present: lifetime- and orientation-resolved methods pushing past the usual resolution limits.

Department(s)/lab(s): EMBL Australia Node in Single Molecule Science, UNSW Medicine and Health | Sierecki Protein Interaction Networks Group @ UNSW
Summary:

Sierecki co-developed the cell-free single-molecule interaction platform with Gambin and runs a group applying it to protein interaction networks: mapping which proteins bind which, with what affinity and in what stoichiometry, at throughput high enough to screen rather than characterise one pair at a time. Recent applications include viral protein-host interactions and transcription factor complexes. Positioned against the established body of NV-ensemble quantum sensing work — DEER, nanoscale NMR and T1 relaxometry protocols operating at pT/sqrt(Hz) field sensitivity — the relevance to a quantum-sensing candidate is as a source of well-characterised, quantitatively-defined biological targets: a pT/sqrt(Hz)-class sensor is only useful in biology if someone can tell you exactly what molecular species is present and at what concentration, which is what this platform delivers. Borderline inclusion — no quantum or physics-instrumentation component — kept because single-molecule technique development is the core of the group.