The establishment of apicobasal or planar cell polarity involves many events that occur at or near the plasma membrane including focal adhesion dynamics, endocytosis, exocytosis, and cytoskeletal reorganization. It is desirable to visualize these events without interference from other regions deeper within the cell. Total internal reflection fluorescence (TIRF) microscopy utilizes an elegant optical sectioning approach to visualize fluorophores near the sample-coverslip interface. TIRF provides high-contrast fluorescence images with limited background and virtually no out-of-focus light, ideal for visualizing and tracking dynamics near the plasma membrane. In this chapter, we present a general experimental and analysis TIRF pipeline for studying cell surface receptor endocytosis. The approach presented can be easily applied to study other dynamic biological processes at or near the plasma membrane using TIRF microscopy.
Tetraspanin proteins are closely associated with high-curvature membrane structures and play key roles in organizing membrane domains and regulating membrane signaling in immune cells. However, their specific roles in regulating T cell membrane signaling, particularly within the microvilli often characteristic of these cells, remain poorly understood. Here, we used Jurkat T cells as a model system and investigated CD81 as a member of the tetraspanin family.
* The study introduces a method using fluorescent labeling and advanced imaging techniques to categorize sEVPs from the same cell type into seven distinct subpopulations, revealing differences in composition and features.
* Findings suggest that this new characterization approach can help differentiate sEVPs from cancerous and normal cells, potentially improving diagnostic and therapeutic strategies related to these vesicles.
Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25 nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously.
The TNF-TNFR1 signaling pathway plays a pivotal role in regulating the balance between cell survival and cell death. Upon binding to TNF, plasma membrane-localized TNFR1 initiates survival signaling, whereas TNFR1 internalization promotes caspase-mediated apoptosis. We previously reported that the α2-6 sialylation of TNFR1 by the tumor-associated sialyltransferase ST6GAL1 diverts signaling toward survival by inhibiting TNFR1 internalization.
We report a method for generating uniform, artifact-free total internal reflection fluorescence (TIRF) excitation via a photonic lantern. Our tapered waveguide, consisting of a multimode input and nine few-mode outputs, enables single-shot TIRF illumination from nine azimuthal directions simultaneously without the introduction of nonstationary devices. Utilizing the photonic lantern for multi-beam excitation provides a low-loss mechanism that supports a wide range of light sources, including high-coherence lasers and various wavelengths in the visible spectrum.
