Dynamic Volumetric Imaging of Mouse Cerebral Blood Vessels In Vivo with an Ultralong Anti-Diffracting Beam.

Volumetric imaging of a mouse brain in vivo with one-photon and two-photon ultralong anti-diffracting (UAD) beam illumination was performed. The three-dimensional (3D) structure of blood vessels in the mouse brain were mapped to a two-dimensional (2D) image. The speed of volumetric imaging was significantly improved due to the long focal length of the UAD beam. Comparing one-photon and two-photon UAD beam volumetric imaging, we found that the imaging depth of two-photon volumetric imaging (80 µm) is better than that of one-photon volumetric imaging (60 µm), and the signal-to-background ratio (SBR) of two-photon volumetric imaging is two times that of one-photon volumetric imaging. Therefore, we used two-photon UAD volumetric imaging to perform dynamic volumetric imaging of mouse brain blood vessels in vivo, and obtained the blood flow velocity.

Ultra-long anti-diffracting beam volume imaging using a single-photon excitation microscope.

We studied a novel volumetric single-photon excitation microscope with an ultralong anti-diffracting (UAD) beam as illumination. Volumetric fluorescence image direct mapping showed that the axial imaging range of the UAD beam was approximately 14 times and 2 times that of conventional Gaussian and Airy beams, respectively, while maintaining a narrow lateral width. We compared the imaging capabilities of the Gaussian, Airy, and UAD modes through a strongly scattering environment mixed with fluorescent microspheres and agarose gel. Thick samples were scanned layer by layer in the Gaussian, Airy, and UAD modes, and then the three-dimensional structural information was projected onto a two-dimensional image. Benefiting from the longer focal length of the UAD beam, a deeper axial projection was provided, and the volume imaging speed was vastly increased. To demonstrate the performances of the UAD microscope, we performed dynamic volumetric imaging on the cardiovascular system of zebrafish labeled with green fluorescent proteins in the three modes and dynamically monitored substance transport in zebrafish blood vessels. In addition, the symmetrical curve trajectory of the UAD beam and the axial depth of the lateral position can be used for localization of micro-objects.

Aberration Correction to Optimize the Performance of Two-Photon Fluorescence Microscopy Using the Genetic Algorithm.

Due to less light scattering and a better signal-to-noise ratio in deep imaging, two-photon fluorescence microscopy (TPFM) has been widely used in biomedical photonics since its advent. However, optical aberrations degrade the performance of TPFM in terms of the signal intensity and the imaging depth and therefore restrict its application. Here, we introduce adaptive optics based on the genetic algorithm to detect the distorted wavefront of the excitation laser beam and then perform aberration correction to optimize the performance of TPFM. By using a spatial light modulator as the wavefront controller, the correction phase is obtained through a signal feedback loop and a process of natural selection. The experimental results show that the signal intensity and imaging depth of TPFM are improved after aberration correction. Finally, the method was applied to two-photon fluorescence lifetime imaging, which helps to improve the signal-to-noise ratio and the accuracy of lifetime analysis. Furthermore, the method can also be implemented in other experiments, such as three-photon microscopy, light-sheet microscopy, and super-resolution microscopy.

Low-power compact continuous-wave stimulated emission depletion microscopy.

Stimulated emission depletion (STED) microscopy can break the optical diffraction barrier and provide subdiffraction resolution. According to the STED superresolution imaging principle, the resolution of STED is positively related to the power of the depletion laser. However, high-laser power largely limits the study of living cells or living bodies. Moreover, the high complexity and high cost of conventional pulsed STED microscopy limit the application of this technique. Therefore, this paper describes a simple continuous-wave STED (CW-STED) system constructed on a 45 × 60 cm breadboard and combined with digitally enhanced (DE) technology; low-power superresolution imaging is realized, which has the advantages of reducing system complexity and cost. The low-system complexity, low cost, and low-power superresolution imaging features of CW-STED have great potential to advance the application of STED microscopy in biological research.

Erianin inhibits the growth and metastasis through autophagy-dependent ferroptosis in KRASG13D colorectal cancer.

Colorectal cancer (CRC) is the third most common cause of cancer mortality worldwide. Approximately 40% of CRC patients are KRAS sequence variation, including KRAS G13D mutation (KRASG13D) CRC patients, accounting for approximately 8% of all KRAS mutations in CRC patients and showing little benefit from anti-EGFR therapy. Therefore, there is an urgent need for new and effective anticancer agents in patients with KRASG13D CRC. Here, we identified a natural product, erianin, that directly interacted with purified recombinant human KRASG13D with a Kd of 1.1163 µM, which also significantly improve the thermal stability of KRASG13D. The cell viability assay showed that KRASG13D cells were more sensitive to erianin than KRASWT or KRASG12V cells. In vitro, results showed that erianin suppressed the migration, invasion and epithelial-mesenchymal transition (EMT) of KRASG13D CRC cells. Furthermore, erianin induced ferroptosis, as evidenced by the accumulation of Fe2+ and reactive oxygen species (ROS), lipid peroxidation, and changes in the mitochondrial morphology of KRASG13D CRC cells. Interestingly, we also found that erianin-induced ferroptosis was accompanied by autophagy. Moreover, the occurrence of erianin-induced ferroptosis is reversed by autophagy inhibitors (NH4Cl and Bafilomycin A1) and ATG5 knockdown, suggesting that erianin-induced ferroptosis is autophagy-dependent. In addition, we evaluated the inhibition of tumor growth and metastasis by erianin in vivo using a subcutaneous tumor model and a spleen-liver metastasis model, respectively. Collectively, these data provide novel insights into the anticancer activity of erianin, which is valuable for the further discussion and investigation of the use of erianin in clinical anticancer chemotherapy for KRASG13D CRC.

Ultralow Laser Power Three-Dimensional Superresolution Microscopy Based on Digitally Enhanced STED.

The resolution of optical microscopes is limited by the optical diffraction limit; in particular, the axial resolution is much lower than the lateral resolution, which hinders the clear distinction of the three-dimensional (3D) structure of cells. Although stimulated emission depletion (STED) superresolution microscopy can break through the optical diffraction limit to achieve 3D superresolution imaging, traditional 3D STED requires high depletion laser power to acquire high-resolution images, which can cause irreversible light damage to biological samples and probes. Therefore, we developed an ultralow laser power 3D STED superresolution imaging method. On the basis of this method, we obtained lateral and axial resolutions of 71 nm and 144 nm, respectively, in fixed cells with 0.65 mW depletion laser power. This method will have broad application prospects in 3D superresolution imaging of living cells.

Low-Power Two-Color Stimulated Emission Depletion Microscopy for Live Cell Imaging.

Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.

Nanodrug Transmembrane Transport Research Based on Fluorescence Correlation Spectroscopy.

Although conventional fluorescence intensity imaging can be used to qualitatively study the drug toxicity of nanodrug carrier systems at the single-cell level, it has limitations for studying nanodrug transport across membranes. Fluorescence correlation spectroscopy (FCS) can provide quantitative information on nanodrug concentration and diffusion in a small area of the cell membrane; thus, it is an ideal tool for studying drug transport across the membrane. In this paper, the FCS method was used to measure the diffusion coefficients and concentrations of carbon dots (CDs), doxorubicin (DOX) and CDs-DOX composites in living cells (COS7 and U2OS) for the first time. The drug concentration and diffusion coefficient in living cells determined by FCS measurements indicated that the CDs-DOX composite distinctively improved the transmembrane efficiency and rate of drug molecules, in accordance with the conclusions drawn from the fluorescence imaging results. Furthermore, the effects of pH values and ATP concentrations on drug transport across the membrane were also studied. Compared with free DOX under acidic conditions, the CDs-DOX complex has higher cellular uptake and better transmembrane efficacy in U2OS cells. Additionally, high concentrations of ATP will cause negative changes in cell membrane permeability, which will hinder the transmembrane transport of CDs and DOX and delay the rapid diffusion of CDs-DOX. The results of this study show that the FCS method can be utilized as a powerful tool for studying the expansion and transport of nanodrugs in living cells, and might provide a new drug exploitation strategy for cancer treatment in vivo.