Methods in microscopy最新文献

Coherent low-energy electrons have been demonstrated as a practical tool for imaging individual macromolecules and two-dimensional (2D) crystals. Low-energy electrons exhibit unique properties: low radiation damage to biological molecules and high sensitivity to the local potentials. In this study, we outline the conditions at which single isolated charge-free atoms can be imaged by low-energy electron holography. A single atom produces an interference pattern consisting of concentric fringes of finite diameter and of very weak intensity. The diffraction angle θ, determined as the first minimum of the concentric rings interference pattern, exhibits similar dependency on the source-to-sample distance zs as sinθ ∼ 0.3/zs ^1/2 for electrons of different energies (50, 100 and 200 eV) and scattered off different elements (Li, C, and Cs). The results are compared to the recently reported experimental holograms of alkali atoms intercalated into bilayer graphene and adsorbed on top of graphene.

This tutorial focuses on presenting experimental protocols for acquiring instrument response functions (IRF) and for calibrating the instruments using reference dyes with validated lifetime in time-domain fluorescence lifetime measurements. Step-by-step preparation of different samples used for the calibrations (scatter solutions, crystals generating second harmonic signal and reference dyes) and the corresponding instrument settings in one- and two-photon excitation are explained. The expected shape of the IRF curves and reference decays are shown using experimentally acquired examples, followed by troubleshooting of the instruments when the expected results are distorted. The discussions focus on the importance of using IRF and reference dyes for adjusting the acquisition parameters of the time-resolved instrument, for data analysis and for comparison and extrapolation of lifetime values between different biological systems.

In this review paper, we summarize the significant advancements in the field of fluorescence lifetime imaging microscopy (FLIM), particularly wide-field FLIM with single-molecule sensitivity, achieved using the time-correlated single-photon counting-based position-sensitive LINCam system. Fluorescence lifetime adds valuable information beyond conventional intensity-based imaging, enabling diverse applications across research fields. Here, we focus on three primary bioimaging applications: (I) single-molecule FLIM in the far-red spectral region, (II) fast and multiplexed super-resolution imaging of cells, and (III) three-dimensional super-resolution imaging with high axial localization precision. Recent advances in position-sensitive detector technologies offer exciting opportunities for high-throughput super-resolution imaging with enhanced localization precision.

Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields.

Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.

Live-cell imaging of fluorescent biosensors has demonstrated that space-time correlations in signalling of cell collectives play an important organisational role in morphogenesis, wound healing, regeneration, and maintaining epithelial homeostasis. Here, we demonstrate how to quantify one such phenomenon, namely apoptosis-induced ERK activity waves in the MCF10A epithelium. We present a protocol that starts from raw time-lapse fluorescence microscopy images and, through a sequence of image manipulations, ends with ARCOS, our computational method to detect and quantify collective signalling. We also describe the same workflow in the interactive napari image viewer to quantify collective phenomena for users without prior programming experience. Our approach can be applied to space-time correlations in cells, cell collectives, or communities of multicellular organisms, in 2D and 3D geometries.