F-CUBIC: a rapid optical clearing method optimized by quantitative evaluation.

In recent decades, various powerful optical clearing methods have emerged to facilitate deep-tissue imaging. However, a rapid and safe protocol for millimeter-thick specimens is still desired. In this study, we propose a simple and economical chemical screening method that uses porcine small intestine tissue as the testing sample to quantify the clearing speed of different optical clearing reagents. By screening with this method, we developed a fast and versatile clearing protocol, termed F-CUBIC (adding formamide to CUBIC). F-CUBIC allows easy clearing of millimeter-thick tissues within 2-20 min by one-step immersion at room temperature. It introduces negligible tissue distortion and shows high compatibility with various fluorescent labeling techniques. Based on endoscopic human colon specimens, we successfully demonstrated the potential of F-CUBIC for nondestructive three-dimensional (3D) biopsy in combination with two-photon microscopy. This study would substantially benefit rapid 3D tissue mapping in biomedical research and clinics, such as instant histopathological examinations.

In vivo label-free two-photon excitation autofluorescence microscopy of microvasculature using a 520 nm femtosecond fiber laser.

Observing microvasculature in its native environment provides invaluable information to understand the initiation and development of microcirculatory related diseases. However, the lack of a high-resolution three-dimensional (3D) imaging technique hinders in vivo investigation of the microvasculature. Recently, we found that the red blood cells can emit autofluorescence signals with short-wavelength two-photon excitation. In this study, we exploited this property and developed a time-resolved two-photon excitation microscopy system using a homemade 520 nm femtosecond fiber laser as the excitation source. Using this system, we could achieve intravital high-resolution 3D imaging of a microvascular network noninvasively. In a mouse tumor model, tumorous blood vessels could be observed and distinguished clearly from the normal vessels.

In vivo identification of arteries and veins using two-photon excitation elastin autofluorescence.

Distinguishing arteries from veins in vivo has a great significance in clinical practices and preclinical studies. Optical imaging methods such as two-photon microscopy can provide high-resolution morphological information of tissue and are therefore extremely suitable for imaging small blood vessels. However, few optical imaging methods allow in vivo identification of arteries and veins merely utilizing the autofluorescence signal of blood vessels. In this report, we found the arterial wall generates a remarkably stronger two-photon excitation autofluorescence (TPEA) signal compared with the venous wall based on BALB/c mice. According to histological analysis and fluorescence characteristic measurement, the contrast signal is confirmed to be from elastin fibers. Employing this unique feature, we propose an objective and effective artery-vein separation strategy that considers the presence of the elastin-TPEA border as the indicator of arteries. Using this strategy, the arterial and venous networks of the dorsal skin and cerebral cortex of BALB/c mice are demonstrated to be excellently mapped and accurately separated in vivo without depending on any exogenous contrast agent, empirical knowledge, and algorithm. This study may provide a novel technique for mapping arterial and venous networks for anatomic research as well as an extra aid to basic researches on the mechanism, diagnosis, and treatment of blood vessel-related diseases.

UNC5C­knockdown enhances the growth and metastasis of breast cancer cells by potentiating the integrin α6/ß4 signaling pathway.

Unc­5 Netrin Receptor C (UNC5C) is a netrin­1 dependence receptor that mediates the induction of apoptosis in the absence of netrin­1. The present study found that UNC5C is heterogeneously expressed in breast cancer cell lines. By knocking down UNC5C in SK­BR­3 and ZR­75­30 cells and overexpressing UNC5c in MDA­MB­231 cells, it was demonstrated that UNC5C exerts an inhibitory effect on the growth and metastasis of breast cancer cells. The mechanism involved a UNC5C­knockdown­induced enhancement of matrix metalloproteinase (MMP)3, MMP7, MMP9 and MMP10 expression via activation of the PI3K/AKT, ERK and p38 MAPK signaling pathways. Notably, UNC5C directly interacted with integrin α6, which is involved in the growth and metastasis of breast cancer cells. Additionally, UNC5C­knockdown enhanced the phosphorylation of FAK and SRC, which are key kinases in the netrin­1/Unc5C and netrin­1/integrin α6/ß4 signaling pathways. This suggests that netrin­1 functions as an integrator for both the netrin­1/Unc5C and netrin­1/integrin α6/ß4 signaling pathways. UNC5C­knockdown potentiated netrin­1/integrin α6/ß4 signaling. Given that UNC5C­knockdown inhibited integrin­liked protein kinase phosphorylation at Thr­173, at least in SK­BR­3 cells, this may be an inhibitory phosphorylation site rather than activating phosphorylation site for relaying integrin signaling.

Multicolor re-scan super-resolution imaging of live cells.

BACKGROUND: Multicolor fluorescence microscopy has proved essential in biological studies. However, the application of conventional multicolor microscopy to imaging subcellular organelles is restricted by its diffraction-limited spatial resolution. Re-scan confocal microscopy (RCM), a novel super-resolution imaging technique, can effectively address this problem. However, previous multicolor RCM imaging methods usually led to spatial mismatch in images due to the sequential scanning of the sample with multiple excitation lasers. METHODS: We present a new RCM system to achieve multicolor super-resolution imaging. A spectrograph was used as the multicolor detection system, and a linear spectral unmixing algorithm was applied to separate different fluorophores in the spectral image. Moreover, since the image reconstruction process induced an artificial resolution improvement, a gamma correction was introduced to restore the multicolor super-resolution image. RESULTS: By imaging phalloidin-labeled F-actin in breast cancer cells, we found that the lateral resolution of our system is approximately 171 nm, which is a 1.8-fold improvement over that of wide-field imaging. The successful identification of three types of fluorescent beads indicated that our multicolor RCM can resolve different fluorophores whose spectra largely overlap with each other. Finally, we demonstrated that our method is suitable for imaging multicolor-labeled organelles of live cells. CONCLUSIONS: Our novel RCM system can acquire multicolor super-resolution images of live cells without spatial mismatch, obvious photobleaching or photodamage. This system may provide a new imaging tool for monitoring dynamic events involving interactions between multiple molecules and organelles in cells.

Spectrum- and time-resolved endogenous multiphoton signals reveal quantitative differentiation of premalignant and malignant gastric mucosa.

Early identification of premalignant and malignant gastric mucosa is crucial to decrease the incidence and mortality of stomach cancer. Spectrum- and time-resolved multiphoton microscopy are capable of providing not only structural but also biochemical information at the subcellular level. Based on this multidimensional imaging technique, we performed a systematic investigation on fresh human tissue specimens at the typical stages of gastric carcinogenesis, including normal, chronic gastritis with erosion, chronic gastritis with intestinal metaplasia, and intestinal-type adenocarcinoma. The results demonstrate that this technique is available to characterize the three-dimensional subcellular morphological and biochemical properties of gastric mucosa and further provide quantitative indicators of different gastric disorders, by using endogenous contrast. With advances in multiphoton endoscopy, it has the potential to allow noninvasive, label-free, real-time histological and functional diagnosis of premalignant and malignant lesions of stomach in the future.

Indocyanine Green-holo-Transferrin Nanoassemblies for Tumor-Targeted Dual-Modal Imaging and Photothermal Therapy of Glioma.

Active-targeted cancer imaging and therapy of glioma has attracted much attention in theranostic nanomedicine. As a promising tumor-targeting ligand, holo-transferrin (holo-Tf) has been applied for enhancing delivery of nanotheranostics. However, holo-Tf-based nanoassemblies for active targeting mediated multimodal imaging and therapeutics have not been previously reported. Here, we develop a one-step method for the preparation of holo-Tf-indocyanine green (holo-Tf-ICG) nanoassemblies for fluorescence (FL) and photoacoustic (PA) dual-modal imaging and photothermal therapy (PTT) of glioma. The nanoassemblies are formed by hydrophobic interaction and hydrogen bonds between holo-Tf and ICG, which exhibit excellent active tumor-targeting and high biocompability. The brain tumor with highly expressed Tf receptor can be clearly observed with holo-Tf-ICG nanoassemblies base on FL and PA dual-modal imaging in subcutaneous and orthotopic glioma models. Under the near-infrared laser irradiation, the holo-Tf-ICG nanoassemblies accumulated in tumor regions can efficiently convert laser energy into hyperthermia for tumor ablation. The novel theranostic nanoplatform holds great promise for precision diagnosis and treatment of glioma.

Effects of Slit3 silencing on the invasive ability of lung carcinoma A549 cells.

Slit proteins function as chemorepellents in axon guidance and neuronal migration by binding to cognate Robo receptors. The Slit/Robo signaling pathway is also involved in the regulation of tumor cell metastasis. However, whether the Slit/Robo signaling pathway exerts prometastatic or antimetastasis functions remains controversial. To date, most of the research on Slit/Robo has focused on Slit2, and the effects of Slit3 on metastasis remain largely unknown. Based on the Oncomine database, overall expression of Slit3 is low in tumor tissues compared to its level in normal tissues. The underlying mechanism for slit3 silencing in tumor tissues is likely related to hypermethylation of the slit3 promoter. However, lung carcinomas appear to be an exception. Several studies have reported that the frequency of Slit3 methylation in lung cancers is far lower than the frequency of Slit2. In the present study, high Slit3 expression at the mRNA level, yet not at the protein level, was detected in lung adenocarcinoma A549 cells. The function of Slit3 in tumor migration and invasion was examined by silencing of Slit3 expression in A549 cells. Silencing of Slit3 promoted proliferation, migration and invasion of A549 cells and induced epithelial-mesenchymal transition by downregulation of E-cadherin and upregulation of vimentin. The inhibitory effects of Slit3 on tumor migration and invasion are likely related to matrix metalloproteinases (MMPs). Silencing of Slit3 in the A549 cells enhanced MMP2 and MMP9 expression. These results indicate that Slit3 is a potential tumor suppressor in lung adenocarcinoma.