Simultaneous label-free autofluorescence multi-harmonic microscopy driven by the supercontinuum generated from a bulk nonlinear crystal.

Nonlinear microscopy encompasses several imaging techniques that leverage laser technology to probe intrinsic molecules of biological specimens. These native molecules produce optical fingerprints that allow nonlinear microscopes to reveal the chemical composition and structure of cells and tissues in a label-free and non-destructive fashion, information that enables a plethora of applications, e.g., real-time digital histopathology or image-guided surgery. Because state-of-the-art lasers exhibit either a limited bandwidth or reduced wavelength tunability, nonlinear microscopes lack the spectral support to probe different biomolecules simultaneously, thus losing analytical potential. Therefore, a conventional nonlinear microscope requires multiple or tunable lasers to individually excite endogenous molecules, increasing both the cost and complexity of the system. A solution to this problem is supercontinuum generation, a nonlinear optical phenomenon that supplies broadband femtosecond radiation, granting a wide spectrum for concurrent molecular excitation. This study introduces a source for nonlinear multiphoton microscopy based on the supercontinuum generation from a yttrium aluminum garnet (YAG) crystal, an approach that allows simultaneous label-free autofluorescence multi-harmonic imaging of biological samples and offers a practical and compact alternative for the clinical translation of nonlinear microscopy. While this supercontinuum covered the visible spectrum (550-900 nm) and the near-infrared region (950-1200 nm), the pulses within 1030-1150 nm produced label-free volumetric chemical images of ex vivo chinchilla kidney, thus validating the supercontinuum from bulk crystals as a powerful source for multimodal nonlinear microscopy.

Acute irradiation induces a senescence-like chromatin structure in mammalian oocytes.

The mechanisms leading to changes in mesoscale chromatin organization during cellular aging are unknown. Here, we used transcriptional activator-like effectors, RNA-seq and superresolution analysis to determine the effects of genotoxic stress on oocyte chromatin structure. Major satellites are organized into tightly packed globular structures that coalesce into chromocenters and dynamically associate with the nucleolus. Acute irradiation significantly enhanced chromocenter mobility in transcriptionally inactive oocytes. In transcriptionally active oocytes, irradiation induced a striking unfolding of satellite chromatin fibers and enhanced the expression of transcripts required for protection from oxidative stress (Fermt1, Smg1), recovery from DNA damage (Tlk2, Rad54l) and regulation of heterochromatin assembly (Zfp296, Ski-oncogene). Non-irradiated, senescent oocytes exhibit not only high chromocenter mobility and satellite distension but also a high frequency of extra chromosomal satellite DNA. Notably, analysis of biological aging using an oocyte-specific RNA clock revealed cellular communication, posttranslational protein modifications, chromatin and histone dynamics as the top cellular processes that are dysregulated in both senescent and irradiated oocytes. Our results indicate that unfolding of heterochromatin fibers following acute genotoxic stress or cellular aging induced the formation of distended satellites and that abnormal chromatin structure together with increased chromocenter mobility leads to chromosome instability in senescent oocytes.

PGC-1α overexpression partially rescues impaired oxidative and contractile pathophysiology following volumetric muscle loss injury.

Volumetric muscle loss (VML) injury is characterized by a non-recoverable loss of muscle fibers due to ablative surgery or severe orthopaedic trauma, that results in chronic functional impairments of the soft tissue. Currently, the effects of VML on the oxidative capacity and adaptability of the remaining injured muscle are unclear. A better understanding of this pathophysiology could significantly shape how VML-injured patients and clinicians approach regenerative medicine and rehabilitation following injury. Herein, the data indicated that VML-injured muscle has diminished mitochondrial content and function (i.e., oxidative capacity), loss of mitochondrial network organization, and attenuated oxidative adaptations to exercise. However, forced PGC-1α over-expression rescued the deficits in oxidative capacity and muscle strength. This implicates physiological activation of PGC1-α as a limiting factor in VML-injured muscle's adaptive capacity to exercise and provides a mechanistic target for regenerative rehabilitation approaches to address the skeletal muscle dysfunction.

Novel Lipid Signaling Mediators for Mesenchymal Stem Cell Mobilization during Bone Repair.

INTRODUCTION: Mesenchymal stem and progenitor cells (MSCs), which normally reside in the bone marrow, are critical to bone health and can be recruited to sites of traumatic bone injury, contributing to new bone formation. The ability to control the trafficking of MSCs provides therapeutic potential for improving traumatic bone healing and therapy for genetic bone diseases such as hypophosphatasia. METHODS: In this study, we explored the sphingosine-1-phosphate (S1P) signaling axis as a means to control the mobilization of MSCs into blood and possibly to recruit MSCs enhancing bone growth. RESULTS: Loss of S1P receptor 3 (S1PR3) leads to an increase in circulating CD45-/CD29+/CD90+/Sca1 putative mesenchymal progenitor cells, suggesting that blocking S1PR3 may stimulate MSCs to leave the bone marrow. Antagonism of S1PR3 with the small molecule VPC01091 stimulated acute migration of CD45-/CD29+/CD90+/Sca1+ MSCs into the blood as early as 1.5 hours after treatment. VPC01091 administration also increased ectopic bone formation induced by BMP-2 and significantly increased new bone formation in critically sized rat cranial defects, suggesting that mobilized MSCs may home to injuries to contribute to healing. We also explored the possibility of combining S1P manipulation of endogenous host cell occupancy with exogenous MSC transplantation for potential use in combination therapies. Importantly, reducing niche occupancy of host MSCs with VPC01091 does not impede engraftment of exogenous MSCs. CONCLUSIONS: Our studies suggest that MSC mobilization through S1PR3 antagonism is a promising strategy for endogenous tissue engineering and improving MSC delivery to treat bone diseases.

Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy.

We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts.