TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Overview

This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

Note: The plasmid used Lifeact-mCherry is a kind gift of Dr. Guillaume Montagnac, Institut Curie, Paris.

1. Cells and Transfection

Note: RAW264.7 macrophages are grown to subconfluency in complete medium (RPMI (Roswell Park Memorial Institute) 1640 medium, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 2 mM L-Glutamine and 10% FCS (Fetal Calf Serum)) in a 100 mm plate. They are transfected with plasmids encoding fluorescently tagged proteins by electroporation. Routinely approximately 5 - 6 x 10⁶ cells are transfected with 20 µg or 10 µg of plasmid for each transfection or co-transfection respectively. Note that other means of transfection based on electroporation or lipofection can be used as alternative approaches.

1. Scrape the cells with a cell lifter and resuspend them in 10 ml of culture medium by pipetting up and down several times.
2. Centrifuge the cell suspension 300 x g, 5 min at RT in swinging angle rotor.
3. Preheat 10 ml of complete medium supplemented with 10 µg/ml of gentamicin at 37 °C.
4. Following centrifugation, discard the supernatant and resuspend the pellet in 3 ml of "washing buffer A" from the electroporation kit.
5. Centrifuge the cell suspension 300 x g, 5 min at room temperature in swinging angle rotor.
6. In the meantime prepare the DNA mix at room temperature: 120 µl 2x Buffer B, 20 µg of plasmid DNA coding for the protein of interest, q.s.p. 240 µl H₂O.
7. Following centrifugation, discard the entire supernatant.
8. Resuspend the cell pellet in the DNA mix and transfer into 4 mm electroporation cuvettes.
9. Incubate at RT for 3 min.
10. Electroporate at 250 V, 900 µF.
11. Immediately resuspend the cells in the pre-warmed (37 °C) complete medium supplemented with gentamicin and plate them in a 100 mm dish.
12. Incubate the transfected cells overnight at 37 °C, 5% CO₂.
13. The next morning, replace the complete medium with 10 ml of serum-free microscopy medium (RPMI 1640 without phenol red, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 2 mM L-Glutamine).

2. Opsonization of Red Blood Cells

Note: As a model of particle target for macrophages, sheep red blood cells (SRBCs) are used. Usually, around 7 x 10⁶ SRBCs per 35 mm glass bottom dish is used.

1. Wash the SRBCs with 100 µl of 1x PBS (phosphate buffered saline)/0.1% BSA (bovine serum albumin) and centrifuge (600 x g, 4 min).
2. Following centrifugation, discard the supernatant and resuspend the SRBCs in 100 µl of 1x PBS/0.1% BSA and centrifuge (600 x g, 4 min).
3. Following centrifugation, discard the supernatant and resuspend the SRBCs in 1x PBS/0.1% BSA with rabbit IgG anti-SRBCs at sub-agglutinating concentration. Use 500 µl of solution containing antibody/5 µl of SRBCs.
   Note: The sub-agglutinating concentration of antibodies corresponds to the lowest concentration that did not induce agglutination detected as formation of a network. In general, the sub-agglutinating concentration is 8.2 µg/ml.
   1. Determine the concentration of IgG anti-SRBCs required to opsonize the SRBCs by a hemagglutination test. Serially dilute the IgG anti-SRBCs (stock at 13.1 mg/ml) between 1/50 - 1/25,600 in a microplate. Add 2 x 10⁶ SRBCs in each well. Incubate the plate in the dark at RT for several hr.
4. Incubate at RT for 30 mins with slow rotation.
5. After two washes in 1x PBS/0.1% BSA as described above, resuspend the IgG-opsonized SRBCs (IgG-SRBCs) in pre-warmed serum-free microscopy medium (2 ml/dish).

3. Poly-lysine Coating of Coverslips

1. In the meantime, treat 35 mm glass bottom dishes with 2 ml of 0.01% poly-L-lysine for 30 min at room temperature.
2. Wash the dishes two times with 2 ml of 1x PBS.

4. Non-covalent Fixation of SRBCs on Glass Bottom Dishes

1. Pour 2 ml of SRBCs suspension per 35 mm glass bottom dish.
2. Centrifuge with a swinging rotor at 500 x g during 2 min onto 35 mm glass bottom dishes.
3. Remove the supernatant and wash once with 2 ml of 1x PBS/10% BSA.
4. Incubate the particles for 30 min with 2 ml of 1x PBS/10% BSA per dish.
5. Wash the dishes three times with 2 ml of 1x PBS.
6. Replace 1x PBS with 2 ml of pre-warmed (37 °C) serum-free microscopy medium.

5. Phagocytosis Visualized by TIRFM

1. Microscope
   Note: TIRFM was performed using a microscope equipped with an oil-immersion objective (N 100X, NA1.49), a heating chamber with CO₂, and two single photon detection cameras EMCCD (Electron Multiplying Charge Coupled Device) coupled with a 1.5X lens.
   1. The day before the TIRF microscope session, turn on the heating chamber at 37 ºC to allow a homogeneous heating of the microscope stage.
2. Critical Angle Determination
   Note: ImageJ Color Profiler software is used to process TIRF image streams.
   1. Place a 35 mm glass bottom dish containing opsonized SRBC with serum-free microscopy medium under the microscope.
   2. Scrape the cells and resuspend them in the medium before adding them (100 - 500 µl) in the dish.
   3. Use the "Live Acquisition" software to control the microscope and perform excitation with a 491 nm and/or a 561 nm laser to identify cells expressing fluorescently tagged proteins.
   4. Identify a cell that expresses the fluorescently tagged proteins.
   5. Place it in the middle of the field and acquire 500 images at one excitation wavelength, with different angles starting from 0º up to 5º, with an increment of 0.01º (Figure 1A). The angles are automatically changed by the microscope system.
   6. Determine the critical angle allowing the incident light to be totally reflected at the glass/ medium interface and generate the evanescent wave. Using ImageJ Color Profiler software, open the image sequence by clicking on "File", "Open" and select the file.
   7. Select a region of interest (ROI), with the rectangular tool, in the cell with a uniform fluorescence (Figure 1B yellow 1).
   8. Plot the "Z axis profile" mean fluorescence intensity measured in the ROI with function of the angles on the x axis by clicking on "Image" tab, "Stacks" in the drop down menu and "Plot Z-axis Profile" (Figure 1B red 2).
      Note: The critical angle is the angle leading to the maximum fluorescence before a sharp decrease in fluorescence.
   9. Use any value of angle on the x-axis superior to the critical angle during the microscopy session to obtain a TIRF signal as example 2.00 (Figure 1C).
3. Acquisitions
   1. Using Live Acquisition software, set up the parameters of acquisition with the module "Protocol Editor" (Figure 2A).
      1. Create a protocol with a "loop" comprising "Multi Channels" acquisition to acquire fluorescent signal from proteins of interest in the TIRF region. Enter the TIRF angle (for example 2.00), the exposure time (for example 50 msec) and the laser intensity (for example 50%) (Figure 2B 1).
      2. Introduce a "Z move" of the objective 3 µm above the TIRF region to obtain signal acquisition in epifluorescence with the "Multi Channels "tool. Enter an angle below the critical angle (as example 1.00), the time of exposition (50 msec) and the laser intensity (50%) (Figure 2B, 2 and 3).
      3. Next add a "z move" of the objective 3 µm below, to return in the TIRF region (Figure 3B 4). Add a "snapshot" of the cell in bright light LED (Light-Emitting Diode) (Figure 2B 5).
   2. Find a cell of interest with a moderate level of fluorescently tagged protein expression that initiates phagocytosis of SRBC by extending plasma membrane around the particle.
   3. Place it in the middle of the field.
   4. Start streaming acquisition of 500 - 1,000 frames. In the "loop count" tab, enter "750 frames" (Figure 2B 1).
      Note: ImageJ Color Profiler software is used to process TIRF image streams.
   5. Open the sequence images in Image J software by clicking "File", "Open" and choosing the file.
   6. Separate the channels by clicking on the tab "Image ", "Hyperstacks" drop down menu and on "Stack to hyperstack" function. Complete the appeared window: Order: xyctz; Channel (c): number of channels (ex: "2" if you have two fluorochromes); Slices (z): number of slices in z axis (ex: "1" if there is no movement in the z- axis); Frames (t): number of images divided per number of channels; Display mode: Grayscale.
      Note: Two separate image sequences are generated, corresponding to the two different channels.

Representative Results

Figure 1. Schematic Representation of the "Phagosome Closure Assay" Analyzed by TIRFM. The phagosome closure assay is performed using macrophages transiently expressing one or two fluorescently tagged protein. Macrophages are deposited on IgG-opsonized SRBCs non-covalently fixed on poly-lysine coated coverslips. Images are recorded in TIRF mode to detect the site of phagosome closure and in epifluorescence mode to detect the base of the phagocytic cup.

Figure 2: Determination of the Critical Angle for TIRFM. (A) Using "Live acquisition", a cell expressing fluorescently tagged proteins is placed in the middle of the field (region 1 in red). With the TIRF Stack option, images were acquired at one excitation wavelength, with different angles starting from 0° up to 5°, with an increment of 0.01° (region 2 in red). (B) The sequence of images is opened using ImageJ Color Profiler software and the mean fluorescence intensity of a region of interest (ROI) is plotted with function of the angles on the x axis using "Stacks" and "PlotZ axis profile" (region 1 in red). (C) The x position of the peak of fluorescence on the plot corresponds to the critical angle: 1.98° (black dotted line). Any value after this angle can be used. As an example, 2.00° can be chosen (red line).

Figure 3. Workflow Process using Live Acquisition Module: "Protocol Editor". (A) In the "Protocol Editor" window, a workflow canvas is created. (B) This protocol comprises a "Loop" of actions that the microscope will repeat the number of times decided by the user. As an example: 750 (1). One loop included: "Multi Channels" acquisition with laser excitation of fluorescent proteins of interest in TIRF mode. As example: Laser 491 nm intensity 50% -TIRF angle 2.00 (2); "Z move" of the objective 3 µm (3); "Multi Channels" acquisition in epifluorescence mode (4); "Z move" of the objective back to the TIRF region (5) and a "Snapshot" in transmitted light (6).

Materials

Name	Company	Catalog Number	Comments
Anti-sheep red blood cells IgG	MP Biomedicals	55806
Bovine Serum Albumin heat shock fraction, pH 7, ≥98%	Sigma	A7906
Cell lifter	Corning	3008
Cuvettes 4mm	Cell project	EP104
DPBS, no calcium, no magnesium	Thermo Fischer Scientific	14190-094	Room temperature
Electrobuffer kit	Cell project	EB110
100mm TC-Treated Cell Culture Dish	Corning	353003
Gene X-cell pulser	Biorad	165-2661
Gentamicin solution	Sigma	G1397
Glass Bottom Dishes 35 mm uncoated 1.5	MatTek corporation	P35G-1.5-14-C Case
iMIC	TILL Photonics		Oil-immersion objective (N 100x, NA1.49.), heating chamber with CO2, a camera single photon detection EMCCD ( Electron Multiplying Charge Coupled Device) and a 1.5X lens
Poly-L-Lysine Solution 0.1%	Sigma	P8920-100ml	Dilution at 0.01% in water
RPMI 1640 medium GLUTAMAX Supplement	Life technologies	61870-010
RPMI 1640 medium, no phenol red (10x500 ml)	Life technologies	11835-105	Warm in 37°C water bath before use
Sheep red blood cells (SRBCs)	Eurobio	DSGMTN00-0Q	Conserved in Alsever buffer at 4°C before use