Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions

Abstract

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism

“…To confirm the impact of EGF on mitochondrial metabolism, we employed an approach based on label-free TPEF that has been recently characterized as a method to monitor subtle metabolic changes 22, 23 . In particular, the TPEF signal scales linearly with the concentration of mitochondrial cofactors, NAD(P)H and FAD, in the irradiated cell volume.…”

“…We employed a home-built multimodal optical microscope featuring epi-detected Two-Photon Excited Fluorescence (TPEF), along with linear transmission light. This setup has been described in detail elsewhere 23, 66 . Briefly, a compact Erbium-fiber multi-branch laser source (FemtoFiber Pro, Toptica Photonics, Germany) delivers 1560 nm pulses at a 40 MHz repetition rate with < 100 fs duration.…”

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism

“…To confirm the impact of EGF on mitochondrial metabolism, we employed an approach based on label-free TPEF that has been recently characterized as a method to monitor subtle metabolic changes 23 , 24 . In particular, the TPEF signal scales linearly with the concentration of mitochondrial cofactors, NAD(P)H and FAD, in the irradiated cell volume.…”

“…We employed a home-built multimodal optical microscope featuring epi-detected Two-Photon Excited Fluorescence (TPEF), along with linear transmission light. This setup has been described in detail elsewhere 24 , 74 . Briefly, a compact Erbium-fiber multi-branch laser source (FemtoFiber Pro, Toptica Photonics, Germany) delivers 1560 nm pulses at a 40 MHz repetition rate with <100 fs duration.…”

Deep ensemble learning and transfer learning methods for classification of senescent cells from nonlinear optical microscopy images

“…Compared to spontaneous Raman, SRS coherent signal generation greatly amplifies the Raman scattering processes, allowing for faster imaging speed, higher chemical sensitivity, and finer spatial resolution (Cheng et al, 2022). Moreover, SRS microscopy possesses intrinsic 3D optical sectioning capabilities and can be seamlessly coupled with other Nonlinear Optical (NLO) modalities, such as Two-Photon Excited Fluorescence (TPEF), also requiring ultrashort laser pulses in the red or near-infrared spectral range, for multimodal imaging (Lu et al, 2015;Talone et al, 2022). The study by Oh et al associates lipids and proteins upregulation to the TIS cells phenotype, but lacks any time resolution, since they investigated only one time point and no measurements were conducted during the first hours after treatment, at the onset of senescence.…”