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Innovate the way we see life | Tomocube Inc. | May 2026

Collagen is everywhere in biology — from tumor invasion, fibrosis to tissue engineering and wound healing. Yet despite its importance, measuring it has always required a trade-off.

Fluorescence tells you where it is, but the labels can disturb the matrix. Second harmonic generation (SHG) gives you fibrillar signal without labels, but the intensity depends on fiber orientation and you lose cellular context. Confocal reflection shows polymerization dynamics, but the contrast is qualitative. None of these approaches directly answers a fundamental question: how much collagen mass is present in a given fiber, in real time and in 3D?

A study led by first author Sehyeon Lee in the labs of Prof. Deok-Ho Kim (Johns Hopkins), in collaboration with Prof. YongKeun Park's research team (KAIST), addresses exactly that problem using holotomography. In a single acquisition, the method simultaneously captures morphology and dry mass, fiber by fiber.

A physical measurement, not just an image

Because refractive index (RI) scales linearly with dry mass density, HT tomograms are not just images — they are physical measurements. The team reconstructed 3D RI volumes (~30 µm axial depth) of type I and type III collagen gels, then ran curvelet-based fiber segmentation (CT-FIRE) to extract per-fiber descriptors:

• Effective fiber width
• Fragment length and straightness
• Orientation angle
• Per-fiber dry mass

At matched concentrations (0.8 mg/mL), type I and type III networks were visually and quantitatively distinct. RI probability density analysis confirmed that fibrillar collagen occupies a distinct higher-RI population above background in both types. With increasing concentration, effective width and per-fiber dry mass shifted upward more strongly in type I than type III — consistent with its thicker, more bundled architecture.

Co-registration with SHG confirmed spatial alignment between HT-resolved fibrils and SHG-positive collagen. In regions with weaker SHG signal, HT resolved fibrillar features that fluorescence missed.
 

Live kinetics and drug-perturbed remodeling

The label-free, non-phototoxic nature of HT makes it well-suited to time-lapse work that fluorescence struggles with. The study demonstrates two dynamic applications:

• Polymerization kinetics. During type I collagen gelation, mean RI change (Δn) increased monotonically, providing a physically calibrated readout of fibrillar assembly as it happens and complementing earlier CRM- and SHG-based fibrillogenesis studies.
• Cell-driven remodeling under pharmacological perturbation. HT1080 fibrosarcoma cells were embedded in collagen and treated with either Y-27632 (a ROCK inhibitor that reduces cell contractility) or GM6001 (a broad-spectrum MMP inhibitor that blocks proteolytic matrix degradation). Time-lapse imaging at 5-minute intervals during the 0–2 h and 24–26 h windows captured spatially resolved, single-fiber-level differences between treated and vehicle-control conditions.

The authors position HT and SHG as complementary rather than competing: HT provides physically calibrated RI maps and dry mass; SHG provides molecular-level organizational information that RI contrast alone cannot supply. In reconstituted gels, where collagen dominates the high-RI signal, the readout is clean. The next steps — 3D fiber tracing, tissue-derived matrices, broader cell systems — point toward fibrosis drug screening, engineered-tissue QC, and quantitative mechanobiology.

The full methodology, fiber-level data, and time-lapse results are in the preprint. If collagen is part of your work — in any system — it's worth the read.