Journal of Biomedical Engineering and Biosciences (JBEB)

ISSN: 2564-4998

Collagen fiber bundles were visualized on a Zeiss microscope using Phase III contrast. Under identical laser power, the RoboLase laser-microscope system was used to transect thin bundles completely—as shown by the pre-ablation image in Figure 1a and the post-ablation image in Figure 1b. In other cases, partial damage without full transection was observed, with corresponding pre-ablation and post-ablation images shown in Figure 1c and Figure 1d, respectively.

Tissue thickness could not be quantified using conventional brightfield or phase-contrast microscopy. This limitation was addressed by coupling the microscope’s right-side port to a QuantEM camera and the metasurface-based quantitative phase imaging (QPI) system FOSSMM [13]. Inspired by avian “eagle-eye” optics, FOSSMM leverages polarization separation and solves the Transport of Intensity Equation (TIE) to generate label-free, high-resolution thickness maps of collagen tissue.

As illustrated in Figure 2, panels (a) and (b) showed quantitative phase images of collagen fibers imaged via QuantEM camera before and after ablation. Panels (c) and (d) were the quantitative phase images taken of the area indicated by the green dashed frames, and Panels (e) & (f) were the topological views of (c) & (d) respectively, clearly demonstrating the thickening of the tissue post-ablation. This remodelling may reflect laser-induced thermal denaturation, fiber swelling, or partial disruption of collagen crosslinks, triggering localized structural reorganization. This example revealed a significant post-ablation increase in collagen tissue thickness—from an average of 7.78 µm to 10.68 µm, marking a 30% rise.

The use of QPI allowed real-time monitoring without staining or destructive preparation, underscoring the platform’s potential for in situ diagnostics of fibrotic remodelling, wound healing, and laser-tissue interactions. While the system achieved excellent spatial accuracy, it was unable to resolve fine structural features under 1.12 µm—such as the narrow ablation line—highlighting the current resolution limit of the system.

Future iterations of this imaging platform may benefit from the integration of higher NA objectives and improved pixel-to-micron mapping to enhance its capacity for nanoscale tissue analysis. Overall, the findings demonstrated that even modest laser doses can induce robust and measurable changes in connective tissue morphology, and the robotic laser microscope system was well-positioned to monitor these changes non-invasively.

3.2 Drosophila Brain Tissue: Volume-Dependent Shockwave Resistance

In this study, Drosophila melanogaster brain tissue was imaged for the first time on a Zeiss fluorescence microscope and was subjected to multiple LIS exposures. Phase contrast images acquired pre-LIS (Figure 3(a)) and post-LIS (Figure 3(b)) demonstrated pronounced morphological alterations consistent with mechanical perturbation. Dead Red fluorescence images obtained pre-LIS (Figure 3(c)) and post-LIS (Figure 3 (d)) indicated an increased prevalence of membrane-compromised cells localized to the shocked region. Together, the phase and viability channels provided complementary evidence of LIS-induced structural and cellular injury in intact brain tissue.

To demonstrate precise, volume-based assessment of brain tissue responses, the robotic LIS microscope was used to acquire z-stepped phase and Dead Red fluorescence stacks at 5 µm intervals. Stack registration and voxel calibration were applied to compute tissue volumes and to track displacement after each shockwave exposure (Figure 4; panels a–h).

Two morphologically distinct Drosophila brains were analyzed: a wide–flat specimen (base area = 194,132.11 µm²) and a narrow–tall specimen (base area = 116,924.39 µm²) as shown in Figure 4. Despite the contrasting footprints, comparable in-plane displacements and similar EthD-1–positive signals were observed across exposures. Full displacement was reached after a cumulative depth of 15 µm (three LIS pulses) for the wide–flat brain, whereas 25 µm (five pulses) was required for the narrow–tall brain. Volumetric reconstructions yielded near-identical totals—2.91 × 10⁶ µm³ and 2.92 × 10⁶ µm³, respectively—indicating that total volume, rather than footprint or shape alone, governed resistance to mechanical shock.

Together, the phase and viability channels provided a synchronized readout of structural deformation and membrane compromise, while the z-stack workflow enabled precise, repeatable volume measurement. These results support the use of the LIS microscopy platform for quantitative, volume-resolved characterization of brain tissue mechanics and position it for applications in experimental TBI and tissue-level resilience studies.

3.3 Broader Implications and Technological Validation

Collectively, these results validated the robotic laser microscope systems as reliable tools for biomechanical studies at the tissue scale. The high reproducibility across replicates supported the robustness of the workflow. By integrating deep learning-assisted QPI with precision on the laser ablation system and LIS on the other system extended the scope from the cellular level to tissue-level mechanobiology, damage simulation, and diagnostics.

This versatility opened several research directions. First, the clear structure-function relationships uncovered in collagen and brain tissues suggested extensions patient-derived organoids, engineered scaffolds, and decellularized matrices. Second pairing laser ablation and LIS systems with outputs with machine-learning classifiers was expected to accelerate discovery of mechanical biomarkers. In addition, controlled perturbations could be used as programmable assays for preclinical drug screening and evaluation of targeted therapies in soft-tissue disease.

4. Conclusion

This study represented a pivotal step in extending the capabilities of the robotic laser microscope systems—originally designed for cellular-level investigations—into tissue-level applications with high spatial precision and functional relevance. By integrating femtosecond laser ablation and laser-induced shockwave (LIS) technologies with advanced imaging modalities such as quantitative phase imaging (QPI) and fluorescence microscopy, feasibility was demonstrated for tracking dynamic structural and biomechanical changes in extracellular matrices (collagen) and complex neuronal tissues (Drosophila brain).

Sensitivity to morphological adaptation was confirmed, including significant post-ablation thickening in collagen fibers, and a volume-dependent determinant of mechanical resilience was revealed in brains exposed to repeated shockwaves. Consistent displacement patterns across distinct brain geometries indicated that total tissue volume can serve as a predictive parameter for integrity under mechanical stress.

A recent Nanophotonics study (Aug 6, 2025) introduced a dual-mode varifocal Moiré metalens that combines TIE-based QPI with polarization-switchable edge-enhanced imaging, reporting ~1.3 µm minimum spatial resolution and QPI RMSE ≈0.015 rad [19]. This aligns with our current observation that features <~1.1 µm are difficult to resolve in our setup and motivates a higher-NA confirmatory set and explicit resolution benchmarking.

Present work uses ex vivo brain prep; ablation temperature not tracked; The future work will (i) repeat key runs with higher-NA optics and finer sampling with bead/target resolution controls, (ii) regress displacement vs. measured volume across additional tissue levels.

These findings carry implications for multiple fields, including neuroengineering, regenerative medicine, and tissue biomechanics. The ability to induce and monitor controlled microtrauma in both soft connective tissue and neuronal tissues provided a platform for studying injury response, repair processes and therapeutic mechanisms for testing biomaterials; for evaluating drug candidates; and for informing multiscale computational models of trauma.

Future development includes the integration of artificial intelligence and machine learning for automated damage classification and improved interpretation of phase and fluorescence signals, expansion in vivo for longitudinal studies of injury and healing, and coupling with optogenetic and electrophysiological readouts to enhance real-time mechanobiology. Future investigations will involve shockwaving similar brain areas for better comparisons.

In sum, tissue-level applicability of the robotic laser platforms was validated, and a pathway was established for broader use in next-generation biomedical research that links cellular phenomena to whole-tissue physiology. 

Acknowledgements

The authors would like to thank Dr. Lingyan Shi’s lab for providing the fruit flies (Drosophila), and Dr. Wei Xiong’s lab for providing the collagen samples. This study was based upon work supported by a gift from Beckman Laser Institute Inc. to LS & VGG. Special thanks to the private donors to our UCSD IEM BTC centre: Dr. Shu Chien from UCSD Bioengineering, Dr. Lizhu Chen from CorDx Inc., Dr. Xinhua Zheng, David & Leslie Lee for their generous donations.

References