Chemical & Biomedical Imaging最新文献

Nanoscale surface topography is an effective approach in modulating cell-material interactions, significantly impacting cellular and nuclear morphologies, as well as their functionality. However, the adaptive changes in cellular metabolism induced by the mechanical and geometrical microenvironment of the nanotopography remain poorly understood. In this study, we investigated the metabolic activities in cells cultured on engineered nanopillar substrates by using a label-free multimodal optical imaging platform. This multimodal imaging platform, integrating two photon fluorescence (TPF) and stimulated Raman scattering (SRS) microscopy, allowed us to directly visualize and quantify metabolic activities of cells in 3D at the subcellular scale. We discovered that the nanopillar structure significantly reduced the cell spreading area and circularity compared to flat surfaces. Nanopillar-induced mechanical cues significantly modulate cellular metabolic activities with variations in nanopillar geometry further influencing these metabolic processes. Cells cultured on nanopillars exhibited reduced oxidative stress, decreased protein and lipid synthesis, and lower lipid unsaturation in comparison to those on flat substrates. Hierarchical clustering also revealed that pitch differences in the nanopillar had a more significant impact on cell metabolic activity than diameter variations. These insights improve our understanding of how engineered nanotopographies can be used to control cellular metabolism, offering possibilities for designing advanced cell culture platforms which can modulate cell behaviors and mimic natural cellular environment and optimize cell-based applications. By leveraging the unique metabolic effects of nanopillar arrays, one can develop more effective strategies for directing the fate of cells, enhancing the performance of cell-based therapies, and creating regenerative medicine applications.

The visualization of the central nervous system (CNS) has proposed stringent criteria for fluorescent probes, as the inevitable production of reactive oxygen species (ROS) or heat generated from most photoluminescent probes upon excitation can disturb the normal status of relatively delicate CNS cells. In this work, a red-emitting fluorogen with aggregation-induced emission (AIE) characteristics, known as DTF, was chosen as the model fluorogen to investigate whether the side effects of ROS and heat could be suppressed through easy-to-operate processes. Specifically, DTF was encapsulated with different amphiphilic matrices to yield AIE nanoprobes, and their photoluminescent properties, ROS production, and photothermal conversion rates were examined. BSA@DTF NPs possessed 1.3-fold brightness compared to that of DSPE-PEG@DTF NPs and F127@DTF NPs but its ROS generation efficiency is markedly decreased to only 2.4% of that produced by F127@DTF NPs. Meanwhile, BSA@DTF NPs showed a negligible photothermal effect. These features make BSA@DTF NPs favorable for long-term live cell imaging, particularly for fluorescent imaging of CNS cells. BSA@DTF NPs were able to sustain the normal state of HT-22 neuronal cells with continuous illumination for at least 25 min, and they also preserved the cytoskeleton of microglia BV-2 cells as the untreated control group. This work represents a successful but easy-to-operate process to suppress the ROS generation of red-emissive AIEgen, and it highlights the importance of minimizing the ROS generation of the fluorescent probes, particularly in the application of long-term imaging of CNS cells.

The visualization of the central nervous system (CNS) has proposed stringent criteria for fluorescent probes, as the inevitable production of reactive oxygen species (ROS) or heat generated from most photoluminescent probes upon excitation can disturb the normal status of relatively delicate CNS cells. In this work, a red-emitting fluorogen with aggregation-induced emission (AIE) characteristics, known as DTF, was chosen as the model fluorogen to investigate whether the side effects of ROS and heat could be suppressed through easy-to-operate processes. Specifically, DTF was encapsulated with different amphiphilic matrices to yield AIE nanoprobes, and their photoluminescent properties, ROS production, and photothermal conversion rates were examined. BSA@DTF NPs possessed 1.3-fold brightness compared to that of DSPE-PEG@DTF NPs and F127@DTF NPs but its ROS generation efficiency is markedly decreased to only 2.4% of that produced by F127@DTF NPs. Meanwhile, BSA@DTF NPs showed a negligible photothermal effect. These features make BSA@DTF NPs favorable for long-term live cell imaging, particularly for fluorescent imaging of CNS cells. BSA@DTF NPs were able to sustain the normal state of HT-22 neuronal cells with continuous illumination for at least 25 min, and they also preserved the cytoskeleton of microglia BV-2 cells as the untreated control group. This work represents a successful but easy-to-operate process to suppress the ROS generation of red-emissive AIEgen, and it highlights the importance of minimizing the ROS generation of the fluorescent probes, particularly in the application of long-term imaging of CNS cells.

Super-resolution optical imaging overcomes the diffraction limit in light microscopy to enable the visualization of previously invisible molecular details within a sample. The realization of super-resolution imaging based on stimulated Raman scattering (SRS) microscopy represents a recent area of fruitful development that has been used to visualize cellular structures in three dimensions, with multiple spectroscopic colors at the nanometer scale. Several fundamental approaches to achieving super-resolution SRS imaging have been reported, including optical engineering strategies, expansion microscopy, deconvolution image analysis, and photoswitchable SRS reporters as methods to break the diffraction limit. These approaches have enabled the visualization of biological structures, cellular interactions, and dynamics with unprecedented detail. In this Perspective, an overview of the current strategies and capabilities for achieving super-resolution SRS imaging will be highlighted together with an outlook on potential directions of this rapidly evolving field.

Super-resolution optical imaging overcomes the diffraction limit in light microscopy to enable the visualization of previously invisible molecular details within a sample. The realization of super-resolution imaging based on stimulated Raman scattering (SRS) microscopy represents a recent area of fruitful development that has been used to visualize cellular structures in three dimensions, with multiple spectroscopic colors at the nanometer scale. Several fundamental approaches to achieving super-resolution SRS imaging have been reported, including optical engineering strategies, expansion microscopy, deconvolution image analysis, and photoswitchable SRS reporters as methods to break the diffraction limit. These approaches have enabled the visualization of biological structures, cellular interactions, and dynamics with unprecedented detail. In this Perspective, an overview of the current strategies and capabilities for achieving super-resolution SRS imaging will be highlighted together with an outlook on potential directions of this rapidly evolving field.

Macrophages exhibit a spectrum of behaviors upon activation and are generally classified as one of two types: inflammatory (M1) or anti-inflammatory (M2). Tracking these phenotypes in living cells can provide insight into immune function but remains a challenging pursuit. Existing methods are mostly limited to static readouts or are difficult to employ for multiplexed imaging in complex 3D environments while maintaining cellular resolution. We aimed to fill this void using bioluminescent technologies. Here we report genetically engineered luciferase reporters for the long-term monitoring of macrophage polarization via spectral phasor analysis. M1- and M2-specific promoters were used to drive the expression of bioluminescent enzymes in macrophage cell lines. The readouts were multiplexed and discernible in both 2D and 3D formats with single-cell resolution in living samples. Collectively, this work expands the toolbox of methods for monitoring macrophage polarization and provides a blueprint for monitoring other multifaceted networks in heterogeneous environments.

Macrophages exhibit a spectrum of behaviors upon activation and are generally classified as one of two types: inflammatory (M1) or anti-inflammatory (M2). Tracking these phenotypes in living cells can provide insight into immune function but remains a challenging pursuit. Existing methods are mostly limited to static readouts or are difficult to employ for multiplexed imaging in complex 3D environments while maintaining cellular resolution. We aimed to fill this void using bioluminescent technologies. Here we report genetically engineered luciferase reporters for the long-term monitoring of macrophage polarization via spectral phasor analysis. M1- and M2-specific promoters were used to drive the expression of bioluminescent enzymes in macrophage cell lines. The readouts were multiplexed and discernible in both 2D and 3D formats with single-cell resolution in living samples. Collectively, this work expands the toolbox of methods for monitoring macrophage polarization and provides a blueprint for monitoring other multifaceted networks in heterogeneous environments.

Over the last decades, various tissue-clearing techniques have broken the ground for the optical imaging of whole organs and whole-organisms, providing complete representative data sets of three-dimensional biological structures. Along with advancements in this field, the development of fluorescent markers for staining vessels and capillaries has offered insights into the complexity of vascular networks and their impact on disease progression. Here we describe the use of a modified water-soluble chitosan linked to cyanine dyes in combination with ethyl cinnamate-based optical tissue clearing for the 3D visualization of tissue vasculature in depth. The water-soluble fluorescent Chitosans have proven to be an optimal candidate for labeling both vessels and capillaries ex vivo thanks to their increased water solubility, high photostability, and optical properties in the near-infrared window. In addition, the nontoxicity of these markers broadens their applicability to in vitro and in vivo biological applications. Despite the availability of other fluorescent molecules for vascular staining, the present study, for the first time, demonstrates the potential of fluorescent chitosans to depict vessels at the capillary level and opens avenues for advancing the diagnostic field by reducing the complexity and costs of the currently available procedures.