A 2026 bioRxiv preprint (Merle et al.) demonstrated absolute, sub-piconewton force spectroscopy deep inside living scattering tissue by optically trapping endogenous lipid droplets and combining back-focal-plane interferometry with square-wave active microrheology and fluctuation-dissipation-based calibration, validated in Drosophila pupa (about 600 µm thick), embryo, and leg tissue. The demonstration leaves five linked scientific gaps open: (1) the method is single-trap, and dual-trap or time-shared multi-trap operation in scattering media — with the required calibration and cross-talk control — is undeveloped; (2) the range of tissue optical properties (scattering coefficient, anisotropy, thickness) over which the optical memory effect preserves sufficient interferometric contrast for the calibration is uncharacterized; (3) the empirical observation that harmonics above the fifth order isolate intrinsic single-cell mechanics from collective neighbour-cell forces lacks a theoretical basis, frequency bounds, and tests in other tissues; (4) the global-fit viscoelastic model (Maxwell branch plus weak parallel elasticity plus fractional power law), validated on the Drosophila samples and on water and milk phantoms (milk being a casein emulsion with distributed relaxation spectra), is untested in tissue types beyond these samples; (5) tissues lacking endogenous lipid droplets require exogenous probes, and while bead-based square-wave FDT calibration has been demonstrated in scattering phantom media (milk), the calibration of exogenous probes inside tissue and the biological impact of introduced particles are unaddressed. No later work resolving any of these components is known, so the bundled generalization problem remains open.

Background

Optical tweezers use a tightly focused laser beam to trap and manipulate micron-scale objects; the forces they exert in vivo are small (piconewton scale) and measuring them absolutely inside living tissue is difficult because tissue scatters light. Merle et al. (bioRxiv, 2026, DOI 10.64898/2026.03.23.712846) addressed this by optically trapping endogenous lipid droplets (fat-storage organelles naturally present in many cells) inside Drosophila tissues and reading the trap-induced probe motion through back-focal-plane (BFP) interferometry — a standard technique in which the interference pattern of forward-scattered light in the laser beam's back focal plane reports the probe's displacement. Two ingredients made absolute calibration possible deep in scattering tissue. First, instead of relying on passive thermal fluctuations alone, they drove the probe with a square-wave displacement of the optical trap and used a fluctuation-dissipation-theorem-based framework: the fluctuation-dissipation theorem (FDT) is the statistical-mechanics relation linking a system's spontaneous thermal fluctuations to its response to an applied perturbation, so a combined active (driven) plus passive (thermal) measurement yields the absolute force scale without prior knowledge of the local trap stiffness or medium properties. Second, interferometric detection through millimetres of tissue remains possible because of the optical (angular) memory effect: within a finite angular range, tilting the illumination laser produces a translated — not decorrelated — speckle/interference pattern behind scattering media, so the BFP signal stays coherent over small trap displacements; the usable angular range shrinks as the thickness and scattering strength of the tissue grow (characterized for imaging by Schott et al., Optics Express 2015, and generalized by Osnabrugge et al., Optica 2017). With this instrument, the authors measured absolute forces and viscoelasticity inside Drosophila pupa (about 600 µm thick), embryo, and developing leg tissue. To extract mechanics they globally fitted a generic viscoelastic model — a Maxwell element (a spring and dashpot in series, describing stress relaxation) in parallel with a weak elastic element and a fractional power-law contribution (a response scaling as a non-integer power of frequency, capturing glassy-like broad relaxation) — and, in leg tissue, separated intrinsic single-cell properties from collective tissue-scale forces by filtering the response in the frequency domain: empirically, harmonics above the fifth order were largely insensitive to collective neighbouring-cell forces. Related prior art frames what is and is not solved. Time-shared optical tweezer microrheology (TiSOM; Català-Castro et al., Nature Nanotechnology 2025) already performs multi-probe, dual-trap viscoelastimetry in transparent or weakly scattering samples (condensates, cultured cells, C. elegans), but its force inference assumes a transmission geometry and has not been combined with absolute deep-tissue calibration in scattering media. Passive FDT-based microrheology in transparent living systems (Muenker et al., Nature Materials 2024) and bead-free in-vivo tweezer force measurement in transparent embryos (Chardès et al., JOVE 2018; Harlepp et al., Molecular Biology of the Cell 2017) likewise do not face the deep-scattering-tissue regime. The Merle et al. preprint itself states that its single-trap implementation, its Drosophila-only validation, its empirical harmonic-filtering observation, its fixed viscoelastic model, and its reliance on endogenous lipid droplets all leave open scientific questions, and its Discussion identifies time-sharing as a promising next step toward a scalable mechanical imaging modality. Bead-based square-wave FDT calibration with exogenous silica beads was likewise demonstrated by the source only in scattering phantom media (milk and water), not inside tissue.

Problem Statement

Advance the deep-tissue absolute force spectroscopy method of Merle et al. (bioRxiv 2026, DOI 10.64898/2026.03.23.712846) from its validated single-trap, Drosophila-only demonstration toward a generally valid quantitative method, by resolving its five open, linked components: (1) Multi-trap operation — develop the hardware, data-acquisition, and calibration procedures (including control of trap cross-talk in scattering media) for a dual-trap or time-shared multi-trap implementation of the square-wave interferometric approach, enabling high-throughput mechanical mapping and spatial correlation measurements inside tissue. (2) Optical validity domain — systematically characterize the tissue depth and the range of optical properties (scattering coefficient, anisotropy factor, thickness) over which the optical memory effect preserves sufficient interferometric contrast for the calibration to remain valid, and test the method in denser or thicker tissues than the Drosophila samples in which it was validated. (3) Harmonic localization theory — establish the theoretical basis and frequency bounds of the empirical observation that harmonics above the fifth order are largely insensitive to collective neighbouring-cell forces (so that they isolate intrinsic single-cell mechanical properties), and test its validity in tissue types with different mechanical coupling strengths and geometries. (4) Viscoelastic model generality — the generic model (Maxwell branch plus weak parallel elasticity plus fractional power law) was globally fitted to Drosophila pupa, leg, and embryo data and validated on water and on milk, a casein-emulsion phantom exhibiting distributed relaxation spectra; test whether this fixed model class remains adequate for other tissue types with more complex relaxation behavior (e.g., multiple distinct relaxation timescales). (5) Probe universality — devise strategies for absolute force measurement in cells or tissues lacking endogenous lipid droplets, and evaluate the tissue response to introduced exogenous particles. The common objective is a method whose absolute sub-piconewton force calibration is quantitatively trustworthy across tissue optical regimes, mechanical-coupling regimes, viscoelastic model classes, and probe classes, and which can measure multiple sites in parallel. All five components are jointly required, and each is an independently answerable sub-question: the task is resolved only when one method satisfies all five, i.e. when the calibration is quantitatively trustworthy across the regimes and probe classes spanned by components (2)–(5) while measuring multiple sites in parallel as in component (1), and when each component's claimed result is refutable, with definite failure conditions under which it would be refuted. Because the source fixes no universal numerical threshold, the solution must itself state and justify the operational definitions it adopts for the load-bearing terms: what makes the absolute force calibration 'quantitatively trustworthy', the minimum interferometric contrast (or signal-to-noise) at which the calibration remains valid in component (2), and the frequency bound and degree of insensitivity meant by 'largely insensitive' in component (3); each such threshold must be anchored to the sub-piconewton precision demonstrated in the source, and a component that fails at its own stated thresholds is refuted.

References

  1. Merle, Proag, Bouzignac, Dougados, Ould Moussa, Sentenac, Monier, Suzanne, Mangeat, 'Deep-tissue absolute force spectroscopy with sub-piconewton precision', bioRxiv preprint, 2026, DOI 10.64898/2026.03.23.712846, https://doi.org/10.64898/2026.03.23.712846
  2. Català-Castro, Ortiz-Vásquez, Martínez-Fernández, Pezzano, Garcia-Cabau, Fernández-Campo, Sanfeliu-Cerdán, Jiménez-Delgado, Salvatella, Ruprecht, Frigeri, Krieg, 'Measuring age-dependent viscoelasticity of organelles, cells and organisms with time-shared optical tweezer microrheology', Nature Nanotechnology 20:411-420, 2025, DOI 10.1038/s41565-024-01830-y, https://doi.org/10.1038/s41565-024-01830-y
  3. Schott, Bertolotti, Léger, Bourdieu, Gigan, 'Characterization of the angular memory effect of scattered light in biological tissues', Optics Express 23(10):13505-13516, 2015, DOI 10.1364/OE.23.013505, https://doi.org/10.1364/OE.23.013505
  4. Osnabrugge, Horstmeyer, Papadopoulos, Judkewitz, Vellekoop, 'Generalized optical memory effect', Optica 4(8):886-892, 2017, DOI 10.1364/OPTICA.4.000886, https://doi.org/10.1364/OPTICA.4.000886
  5. Muenker, Knotz, Krüger, Betz, 'Accessing activity and viscoelastic properties of artificial and living systems from passive measurement', Nature Materials 23:1283-1291, 2024, DOI 10.1038/s41563-024-01957-2, https://doi.org/10.1038/s41563-024-01957-2
  6. Chardès, Clement, Blanc, Lenne, 'Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo', Journal of Visualized Experiments (141):e57900, 2018, DOI 10.3791/57900, https://doi.org/10.3791/57900
  7. Harlepp, Thalmann, Follain, Goetz, 'Hemodynamic forces can be accurately measured in vivo with optical tweezers', Molecular Biology of the Cell 28(23):3252-3260, 2017, DOI 10.1091/mbc.E17-06-0382, https://doi.org/10.1091/mbc.E17-06-0382
  8. Creff et al., 'Goblet cells mechanically breach the epithelial barrier in gut homeostasis', Nature Communications 17:9078, 2026, DOI 10.1038/s41467-026-76034-0, https://doi.org/10.1038/s41467-026-76034-0