TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Take a polymer-coated glass bottom dish containing IgG-opsonized red blood cells or RBCs adhered to its surface.

Place the dish on a total internal reflection fluorescence microscope stage, maintaining it at physiological temperature.

Add macrophages containing fluorescently labeled cytoplasmic peptides that bind to polymerized actin, facilitating cytoskeleton visualization.

During imaging, direct a laser beam at an angle superior to the critical angle, causing internal reflection and evanescent wave generation at the contact point.

The evanescent waves selectively illuminate the fluorophores at the glass-sample interface, allowing real-time visualization of phagocytosis.

Macrophage Fc receptors bind to the IgG-opsonized RBC, causing receptor clustering around the contact area.

Clustering triggers intracellular signaling pathways and GTPase activation, driving actin polymerization and dynamin recruitment, forming pseudopods.

The pseudopods extend around the RBC, forming a phagocytic cup.

Eventually, the pseudopod tips fuse. The accumulated dynamin mediates phagosome separation from the cell membrane, internalizing the opsonized RBC.

For non-covalent fixation of the SRBCs, pour 2 milliliters of the IgG-opsonized cells into each of the poly-lysine-coated dishes, and centrifuge the samples in a swinging rotor centrifuge. Discard the supernatants, and wash the particles one time with 2 milliliters of PBS plus 10% BSA.

Then, incubate the particles with 2 milliliters of fresh PBS plus 10% BSA for 30 minutes at room temperature, followed by three washes with 2 milliliters of PBS. After the last wash, replace the PBS with 2 milliliters of 37 degrees Celsius serum-free microscopy medium.

Place an SRBC-coded dish onto the microscope stage. Then, scrape the transfected cells of interest from the bottom of the culture dish and pipette the cells a few times to achieve a single-cell suspension. Add the transfected cells to the SRBC-coated dish.

Start the live acquisition software. Find a cell that expresses the fluorescently tagged proteins and adjust the position of the dish so that a cell of interest is in the middle of the field. Acquire 500 images at different angles from 0 to 5 degrees at 0.01-degree increments at one excitation wavelength.

To determine the critical angle for the incident light to be totally reflected at the glass-medium interface and generate an evanescent wave, open the image sequence in the appropriate imaging software. Select a region of interest in the cell with uniform fluorescence.

Then, under the Image tab, select Stacks and plot Z-axis profile. To plot the Z-axis profile mean fluorescence intensity measured in the region of interest with the function of the angles on the X-axis. Any angle value on the x-axis superior to the critical angle can then be used during the microscopy session to obtain a TIRF signal.