The overall goal of this protocol is to visualize phagosome formation, as well as the precise site of phagosome schism in 3D, in living macrophages, using Total Internal Reflection Florescence, or TIRF Microscopy. This method combines TIRF Microscopy with epifluorescence to monitor the step by step localization of two different fluorescent proteins during pseudopod extension and tip fusion. The technique provides a systematic method to determine the critical angle and obtain a TIRF signal which is crucial for drawing conclusions about the close to the plasma membrane.
The day before the TIRF microscope session, turn on the heating chamber to 37 degrees Celsius to allow a homogeneous heating of the microscope stage. The next morning, using 100 microliters of PBS, supplemented with 0.1%BSA to wash 7x10 to the 6th sheep red blood cells or SRBCs per 35-millimeter glass-bottom dish two times. After the second centrifugation, resuspend the cells in 500 microliters of rabbit IGG anti-SRBCs, at a sub-agglutinating concentration, in PBS supplemented with 0.1%BSA per 5 microliters of cells, and incubate the cells for 30 minutes with a slow rotation.
At the end of the incubation, wash the SRBCs two times in PBS plus BSA, and resuspend the cells in 2 milliliters of 37-degree Celsius, serum-free microscopy medium per dish. Then, treat 35-millimeter glass-bottom dishes with 2 milliliters of 0.1%poly-L lysine for 30 minutes at room temperature, followed by two washes with 2 milliliters of PBS. For non-covalent fixation of the SRBCs, pour 2 milliliters of the IGG opsonized cells into each of the polylysine-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 milliters 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-degree Celsius, serum-free microscopy medium.
Place an SRBC-coated 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 zero to 5%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 the 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. Next, in the live acquisition software, use the protocol editor to set the parameters of the acquisition.
Including the number of loop acquisition sequences, the fluorescent channels and their laser intensity, the TIRF angle, and the exposure time. Set the Z move of the objective to reach the epifluorescence mode plane. Then, in the epifluorescence mode, set the TIRF angle to 1, and the Z move of the objective back to the TIRF plane.
Then, obtain an image of the cell in the bright light LED mode. Now, find a cell of interest with a moderate level of fluorescently-tagged protein expression, engaged in phagocytosis with an SRBC, and move the cell into the middle of the field. Note that such a cell can be identified by an extended plasma membrane around the particle.
Enter the number of frames in the Loop Count tab to begin a streaming acquisition of 500 to 1, 000 frames. If necessary, change the laser intensity and exposure time to adapt to the different levels of fluorescence in the cell. To generate two separate image sequences corresponding to the two channels, open the sequences in the imagine software and click on the Image tab.
In the Hyperstacks menu, select Stack to Hyperstack. Then, in the pop-up window, enter xyctz for the Order, the number of fluorochromes used for the Channels, the number of Slices in the z axis, the number of images divided by the number of channels under Frames, and Grayscale for the Display Mode. Then, split the channels.
Here, a representative live-cell TIRF microscopy movie of a phagosome closure assay in raw 264.7 macrophages engulfing an IGG opsonized SRBC is shown. As the tips of the pseudopods opposed to the glass coverslip around the SRBC, an F-actin ring is detected in the TIRF area that progressively narrows until it closes. In parallel, the blurry F-actin signal detected by the epifluroescence, after shifting the stage three microns above the TIRF area corresponds to de-polymerization at the base of the phagocytic cup.
After three minutes, the SRBC is totally internalized, as confirmed by transmitted light imaging. Using this method, the of actin at the base of the phagocytic cup were the focal exocytosis of the intracellular compartments occurs, can be demonstrated. After its acquisition, a cell can be further processed for correlative electron microscopy, or the whole cell population can be fixed and frozen processed for classical immunofluorescence microscopy.
It is important to keep in mind that phagocytosis is very sensitive to temperature, and therefore, the temperature within the heating chamber and the culture dish medium should be maintained at a constant 37 degrees Celsius temperature throughout the procedure. After watching this video, you should have a good understanding of how to opsonize particles, coat cover slips with the particles, determine the critical angles for TIRF microscopy, and image the localization of proteins of interest during live cell phagocytosis.
