Hung, H.-H. | Search bioRxiv

bioRxiv2023

Video-rate three-photon imaging in deep Drosophila brain based on a single Cr:forsterite oscillator

We have demonstrated 30-Hz three-photon imaging using a single 24-MHz mode-locked Cr:forsterite oscillator with a center wavelength at 1260 nm. By managing the dispersion distribution in the resonator using double-chirped mirrors, we have produced 32-fs pulses with 22-nJ pulse energy. Using the oscillator as a driving source, we have realized multi-color three-photon images using a GFP-labeled Drosophila brain and an AF647-labeled mouse brain. To demonstrate the capability of deep-tissue imaging, we have obtained a 10-times higher SBR from the three-photon images than the two-photon results at different depths in a GFP-labeled Drosophila brain dissection. Furthermore, we have shown the impact of excitation pulse width on three-photon deep-tissue imaging. Our results indicate the superiority of using shorter pulses for deeper-tissue imaging, especially in the Drosophila brain. In addition, we have recorded the three-photon calcium imaging in vivo from the Drosophila mushroom body in response to external electric shocks. We believe our demonstration provides a robust approach for high-speed three-photon microscopy applications, especially for intravital investigations in the Drosophila brain.

bioengineering↗

bioRxiv2020

Creb5 establishes the competence for Prg4 expression in articular cartilage

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-{beta} and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-{beta} and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-{beta}. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage.

molecular biology↗