Three-dimensional Multiphoton Microscopy Research Articles

223Ra Induces Transient Functional Bone Marrow Toxicity.

223Ra is a bone-seeking, α-particle-emitting radionuclide approved for the treatment of patients with metastatic prostate cancer and is currently being tested in a variety of clinical trials for primary and metastatic cancers to bone. Clinical evaluation of 223Ra hematologic safety showed a significantly increased rate of neutropenia and thrombocytopenia in patients, hinting at myelosuppression as a side effect. Methods: In this study, we investigated the consequences of 223Ra treatment on bone marrow biology by combining flow cytometry, single-cell RNA sequencing, three-dimensional multiphoton microscopy and bone marrow transplantation analyses. Results: 223Ra accumulated in bones and induced zonal radiation damage confined to the bone interface, followed by replacement of the impaired areas with adipocyte infiltration, as monitored by 3-dimensional multiphoton microscopy ex vivo. Flow cytometry and single-cell transcriptomic analyses on bone marrow hematopoietic populations revealed transient, nonspecific 223Ra-mediated cytotoxicity on resident populations, including stem, progenitor, and mature leukocytes. This toxicity was paralleled by a significant decrease in white blood cells and platelets in peripheral blood-an effect that was overcome within 40 d after treatment. 223Ra exposure did not impair full hematopoietic reconstitution, suggesting that bone marrow function is not permanently hampered. Conclusion: Our results provide a comprehensive explanation of 223Ra reversible effects on bone marrow cells and exclude long-term myelotoxicity, supporting safety for patients.

Reduced deep-tissue image degradation in three-dimensional multiphoton microscopy with concentric two-color two-photon fluorescence excitation

We theoretically demonstrate that enhanced penetration depth in three-dimensional multiphoton microscopy can be achieved using concentric two-color two-photon (C2C2P) fluorescence excitation in which the two excitation beams are separated in space before reaching their common focal spot. Monte Carlo simulation shows that, in comparison with the one-color two-photon excitation scheme, the C2C2P fluorescence microscopy provides a significantly greater penetration depth for imaging into a highly scattering medium.

Image heterogeneity correction in large-area, three-dimensional multiphoton microscopy

Large-area multiphoton laser scanning microscopy (LMLSM) can be applied in biology and medicine for high sensitivity and resolution tissue imaging. However, factors such as refractive index mismatch induced spherical aberration, emission/excitation absorption and scattering can result in axial intensity attenuation and lateral image heterogeneity, affecting both qualitative and quantitative image analysis. In this work, we describe an image correction algorithm to improve three-dimensional images in LMLSM. The method consists of multiplying the measured nonlinear signal by a three-dimensional correction factor, determined by the use of twophoton images of the appropriate specimens and specimen absorption and scattering properties at the excitation and emission wavelengths. The proposed methodology is demonstrated in correcting multiphoton images of objects imbedded in uniform fluorescent background, lung tissue, and Drosophila larva.