Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known for its deep-brain structural and hemodynamic imaging capability. However, the largest imaging depth in MPM is limited by signal depletion in the deep brain. Here we demonstrate that quantum dots are an enabling material for significantly deeper structural and hemodynamic MPM in mouse brain in vivo. We characterized both three-photon excitation and emission parameters for quantum dots: the measured three-photon cross sections of quantum dots are 4–5 orders of magnitude larger than those of conventional fluorescent dyes excited at the 1700 nm window, while the three-photon emission spectrum measured in the circulating blood in vivo shows a slight red shift and broadening compared with ex vivo measurement. On the basis of these measured results, we further demonstrate both structural and hemodynamic three-photon microscopy in the mouse brain in vivo labeled by quantum dots, at record depths among all MPM modalities at all demonstrated excitation wavelengths.

Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3‐photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength‐dependent 3‐photon action cross section (ησ 3) measurement of SR101. 3‐photon fluorescence imaging of the SR101‐labeled vasculature enabled an imaging depth of 1340‐μm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal‐to‐background ratio limit encountered in 2‐photon fluorescence imaging. Visualization of astrocytes 910 μm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2‐photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101‐labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.

Background Stearoyl-CoA desaturase-1 (SCD1) is reported to play essential roles in cancer stemness among several cancers. Our previous research revealed significant overexpression of SCD1 in primary gastric cancer stem cells (GCSCs), with its functional role still unknown. Methods We stably established three primary GCSCs by sphere-forming assays and flow cytometry. Protein quantification and bioinformatics analysis were performed to reveal the differential protein pattern. Lentivirus-based small-interfering RNA (siRNA) knockdown and pharmacological inhibition approaches were used to characterise the function and molecular mechanism role of SCD1 in the regulation of GC stemness and tumour metastasis capacity both in vitro and in vivo. Results SCD1 was found to increase the population of GCSCs, whereas its suppression by an SCD1 inhibitor or knockdown by siRNA attenuated the stemness of GCSCs, including chemotherapy resistance and sphere-forming ability. Furthermore, SCD1 suppression reversed epithelial-to-mesenchymal transition and reduced the GC metastasis probability both in vitro and in vivo. Downregulation of SCD1 in GCSCs was associated with the expression of Yes-associated protein (YAP), a key protein in the Hippo pathway, and nuclear YAP translocation was also blocked by the SCD1 decrease. Conclusions SCD1 promotes GCSC stemness through the Hippo/YAP pathway. Targeting SCD1 might be a novel therapeutic strategy, especially to suppress GC metastasis and sensitise chemotherapy.

No abstract

Multiphoton action cross-sections are the prerequisite for excitation light selection. At the 1700-nm window suitable for deep-tissue imaging, wavelength-dependent 3-photon action cross-sections ησ for RFPs are unknown, preventing wavelength selection. Here we demonstrate: (1) ex vivo measurement of wavelength-dependent ησ for purified RFPs; (2) a multiphoton imaging guided measurement system for in vivo measurement; and (3) in vivo measurement of wavelength-dependent ησ in RFP labeled cells. These fundamental results will provide guidelines for excitation wavelength selection for 3-photon fluorescence imaging of RFPs at the 1700-nm window, and augment the existing database of multiphoton action cross-sections for fluorophores.