How is endocytosis experimentally observed and measured?

Endocytosis can be studied and measured through multiple microscopy, biochemical, and biophysical methods. The choice of technique depends on the type of endocytic pathway being examined and whether the aim is to visualize the process live or quantify it across a population.

Microscopy-Based Observation and Measurement

Live-Cell Imaging Tools

Researchers use several fluorescence-based methods to track cargo and vesicles:

• Fluorescently labeled cargo:
  Specific molecules (like transferrin for clathrin-mediated endocytosis) are tagged with fluorescent dyes such as Alexa Fluor. Cells are then incubated with these labeled molecules, and internalization is visualized over time under a fluorescence microscope.
• pH-sensitive fluorescent probes:
  Dyes like pHrodo remain non-fluorescent at neutral pH but glow brightly in acidic environments such as endosomes and lysosomes. This helps in no-wash assays that measure only internalized cargo.
• Fluorescent membrane dyes:
  Lipophilic dyes like FM 1-43 or FM 5-95 insert into the cell membrane and fluoresce upon internalization. These are useful for tracking rapid vesicle cycling, especially in neurons.
• Photoactivatable fluorescent proteins:
  Proteins such as PA-mCherry are fused to a target receptor or protein. A laser activates fluorescence at specific membrane spots, allowing visualization of that protein’s movement during endocytosis.

Advanced Microscopy Techniques

To achieve higher spatial resolution:

• Total Internal Reflection Fluorescence (TIRF) Microscopy: Excites fluorophores only near the membrane surface, ideal for studying vesicle formation and budding events.
• Super-Resolution Microscopy (SIM, STORM): Enables visualization at below 50 nm resolution, revealing protein clustering, endocytic coat assembly, and cytoskeletal changes.
• Electron Microscopy (EM): Produces detailed static images of endocytic structures such as clathrin-coated pits in fixed cell sections.

Biochemical and Analytical Methods

To measure endocytosis at a population level:

• Flow Cytometry: Cells are incubated with fluorescent cargo, then cooled to halt uptake. Acid wash or quenching removes surface fluorescence, leaving only internalized signals to be measured by a flow cytometer.
• Whole-Cell ELISA: Used to quantify receptor internalization. After ligand stimulation and fixation, remaining surface receptors are detected using enzyme-linked antibodies and compared to unstimulated controls.
• Radiolabeled Ligands: Traditional method using radioisotope-tagged ligands (e.g., ({}^{125})I). After incubation, an acid wash removes external ligands, and internalized radioactivity is measured. Though effective, this is now mostly replaced by fluorescent or enzymatic assays.

Biophysical Techniques

• Membrane Capacitance Measurement: Using the patch-clamp technique, researchers measure changes in plasma membrane surface area. As vesicles form and the membrane area decreases, capacitance drops, providing real-time, high-resolution tracking—especially useful in neurons and other excitable cells.