Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

1. Membrane Sheet Preparation and Fixation

1. Prepare membrane sheets labeled with iRFP-PAmCherry1-PH_PLCδ1
   1. Prepare the DNA plasmid encoding iRFP-PAmCherry1-PH_PLCδ1²³ using standard molecular biology techniques.
   2. Culture INS-1 cells on #1.5 18 mm round coverslips pre-coated with 30 µg/ml fibronectin to 50-70% confluency following standard INS-1 cell culture protocols^38,39. Transfect the cells a day after 50 to 70% confluency is reached.
   3. Transfect cells with iRFP-PAmCherry1-PH_PLCδ1 using liposome transfection reagent following manufacturer's protocols. After transfection, allow the cells to grow for 48 hr.
   4. On the day of the experiment, coat coverslips (on one-side) with ~0.5 – 1 ml of 500 µg/ml Poly-D-Lysine (PDL; diluted in dH₂O) for 1-2 hr. Then drain PDL by placing a tissue paper on the edge of the coverslip. Place the coverslips on a pre-chilled (4 °C) metal plate for later use.
   5. Wash pre-transfected INS-1 cells growing on coverslips with ~0.5 – 1 ml ice-cold phosphate-buffered saline (PBS) containing 1 mM EGTA. Repeat the wash three times and drain PBS.
   6. Place the coverslips with cultured cells (cell-side facing down) on the PDL-coated coverslips on a pre-chilled metal plate with a pair of tweezers. Leave the plate in a refrigerator (4 °C) for 7 – 10 min to allow cells to attach to the PDL-coated coverslips (Figure 2).
      NOTE: The incubation time should be in the range of 7 – 10 min for better attachment of the cells to the PDL-coated surface. The refrigerator environment is dry and incubation should not be too long.
   7. Remove the metal plate from the refrigerator. Gently peel off the coverslip containing the pre-transfected cells using tweezers. This produces a thin layer of cell membrane sheet on the PDL-coated coverslip. Gently wash the membrane sheets with ~0.5 – 1 ml ice-cold PBS and then fix with ~0.5 – 1 ml ice-cold 4% paraformaldehyde (PFA) + 0.2% glutaraldehyde (GA) in PBS for 15 min at 4 °C.
      Caution: Paraformaldehyde and glutaraldehyde are toxic. Handle them in a fume hood with skin and eye protection.
   8. After fixation, image the membrane sheets immediately (see section 3) or store in ~0.5 – 1 ml PBS at 4 °C.
      NOTE: After fixing the membrane sheet, there will be no equilibrium binding of PH probes from cytosol to PI(4,5)P₂. The bound PH probes will gradually dissociate from the membrane sheet and diffuse into the solution (although it might take days to weeks). Therefore, it is recommended to image the fixed samples right after sample preparation.
2. Prepare membrane sheets for PI(4,5)P₂ specific antibody labeling
   1. Culture INS-1 cells on #1.5, 18 mm round coverslips pre-coated with 30 µg/ml fibronectin to 50%-70% confluence. Transfect cells with DNA plasmid the next day as described in step 1.1.3.
   2. Repeat steps from 1.1.4 to 1.1.7 as described above.
      NOTE: Perform the following procedures at 4 °C.
   3. Wash the coverslips containing fixed PM sheets three times with ~0.5 – 1 ml ice-cold PBS containing 50 mM NH₄Cl. Quench the sheets with ~0.5 – 1 ml 0.1% sodium borohydride in PBS for 7 min, and wash with ~0.5 – 1 ml PBS (without 50 mM NH₄Cl).
   4. Block the samples with ~0.5 – 1 ml blocking solution (PBS solution containing 5% (v/v) normal goat serum, 5% (v/v) bovine serum albumin and 50 mM NH₄Cl) for 45 min. Then, incubate with ~0.5 – 1 ml primary PI(4,5)P₂ antibody (1:300 dilution) in blocking solution for 1 hr. Wash three times (10 min each time) with ~0.5 – 1 ml PBS containing 50 mM NH₄Cl.
   5. Incubate samples with ~0.5 – 1 ml secondary F(ab')2-goat-anti-mouse antibody conjugated with fluorophores (1:300) in blocking buffer for 1 hr. Wash three times with ~0.5 – 1 ml PBS.
   6. Post-fix samples with 4% PFA + 0.2% GA for 15 min, then wash samples three times with PBS (7 min each time). Proceed to Section 3 for imaging or store in ~0.5 ~ 1 ml PBS at 4 °C for later use.
      NOTE: Prepare samples at 4 °C. Use 4% PFA + 0.2% GA fixative to preserve the physiological distribution of phosphoinositides in the PM. RT preparation or fixation with 4% PFA alone may cause significant artifacts (Figure 4).

2. Cell Culture Preparation for Live-cell Imaging with mEOS3.1-PH_PLCδ1

1. Prepare the DNA plasmid encoding the mEOS3.1-PH_PLCδ1 as described previously ²³_.
2. Culture INS-1 cells on #1.5 18 mm round coverslips pre-coated with 30 µg/ml fibronectin until they reach 50%-70% confluency following standard INS-1 cell culture protocols^38,39.
3. The following day, transfect INS-1 cells with the mEOS3.1-PH_PLCδ1 DNA plasmid using liposome transfection reagent following manufacture's protocol. Incubate for 48 hr before imaging.

3. PALM Image Acquisition of Membrane Sheets and Live Cells

1. PALM image acquisition of membrane sheets
   NOTE: Here, a total internal reflection fluorescence (TIRF) microscope based SMLM system (100X oil, APO, NA = 1.49, WD 0.12 mm) ²³ is used for all image acquisition.
   1. Before imaging, dilute 1 µl of fluorescent bead solution in 10 ml PBS + 50 mM MgCl₂. Add 200 µl diluted solution into the imaging chamber containing cell samples (from steps 1.1.8/1.2.6) for 10 min. Then, wash with PBS three times.
   2. Start the PALM imaging system. In the associated imaging software, select the "iRFP channel" button (642 nm laser excitation, 700/75 nm emission). Find the cell membrane expressing PH probes in the iRFP channel.
      NOTE: The membrane sheet should have a fluorescent bead nearby. This is required for drift correction later.
   3. Use the "Capture" button to collect a conventional TIRF image of the cell membrane in iRFP channel as a reference for future image reconstruction. Set a normal TIRF angle (i.e., after reaching the critical angle, turn another 1.5 degrees) by typing in 2140 in the TIRF angle setting for imaging. Use this angle setting for all steps unless otherwise noted.
      NOTE: In the TIRF system described here, 3,500 is the vertical up angle, 2,200 is the TIRF critical angle, and 2140 is the normal TIRF angle, which is 1.5 degree more after TIRF critical angle. The narrow TIRF angle mentioned below in 3.2.3 is 2120, which is another 2 degree after reaching TIRF critical angle.
   4. Switch to PAmCherry1 channel by clicking the RFP channel button (561 nm excitation, 600/50 nm emission filter). Adjust the scroll bar in the "AOTF" pad all the way to the right to have full power illumination of 561 nm laser for 10-20 sec to bleach the background membrane fluorescence.
   5. Set the optimal camera setting (2×2 binning) in the "Format" tab and the fast acquisition protocol in "ND sequence acquisition" tab (typically 10,000-40,000 images at 20 Hz).
   6. Start acquisition of PAmCherry1 images (561 nm laser at full power, 50 msec/frame) with simultaneous 405 nm laser activation at a low level (0.1% – 1%) by clicking the "Run now" button. Adjust the intensity of 405 nm laser so that spatially isolated individual points in each frame are easily identifiable.
      NOTE: The number of activatable PAmCherry1 molecules decreases gradually during acquisition. 405 nm laser intensity should be gradually increased to keep the optimal density of individual molecule signals in each frame.
   7. Continue acquiring images until no PAmCherry1 single molecule signal is activated.
2. PALM imaging in live cells
   1. Before imaging, dilute fluorescent beads into the imaging chamber for 10 min as described in step 3.1.1. Then start PALM imaging by opening the associated imaging software.
      NOTE: Use a magnetic quick-release imaging chamber for live-cell imaging. Maintain the center of the imaging field at 35 °C with a temperature controller under constant perfusion with extracellular buffer (Extracellular Solution: 135 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 3 mM glucose, and 20 mM HEPES; pH =7.3).
   2. Choose the GFP channel button (525/50 nm emission). Identify the cell membrane expressing fluorescent probes based on green fluorescent from mEOS3 under 488 nm laser excitation. Make sure that fluorescent beads nearby are also included during imaging as this is required later for offline drift correction.
   3. Click the "Capture" button to collect a TIRF image of the cell membrane in mEOS3 (green) channel as a reference image. Use a shallow evanescent wave excitation illumination (after reaching the critical angle, turn another 2 degrees) by typing in 2120 in the TIRF angle setting for live-cell imaging.
      NOTE: This minimizes the fluorescence from the unbound mEos3-PH in the cytosol adjacent to the plasma membrane.
   4. Switch to the "RFP channel" button (561 nm excitation, 600/50 nm emission). Use the full power 561 nm laser to bleach background fluorescence of the membrane sheets for 10 ~ 20 sec with the scrollbar in the "AOTF pad" (see 3.1.4).
   5. Start image acquisition by clicking the "Run Now" button in the red channel (561 nm full power, set at 10 msec/frame in the "camera" setting tab) with simultaneous 405 nm laser activation. Adjust the 405 nm laser intensity to activate spatially separated points in each frame.
      NOTE: As acquisition proceeds, un-converted mEOS3.1-PH_PLCδ1 molecule number slowly decreases. Gradually increase the 405 nm laser power to optimize the single molecule signal. The imaging acquisition length depends on the experimental purpose. Acquire images continuously for 5 min under normal conditions. If a longer acquisition is required, collect multiple image stacks instead of a single, large image stack. The file size of each image stack should not exceed 4 GB or it will affect software analysis later.

4. SMLM Image Processing and Reconstruction

1. Transfer the image files (.tif image stacks) to an image analysis station. Launch the image reconstruction program (custom-written) in Matlab as described previously³⁷ and load the image stack by clicking the "File" tab in the main menu, then "open", then "new file".
2. Identify and localize individual molecular events from each frame. Set filtering intensity threshold with a threshold number (1-10) in the "wavelet". Verify the optimal parameter settings for point detection visually before the reconstruction. Then go to the "PALM" tab and click "one step process" to start image reconstruction.
   NOTE: Intensity threshold for point detection is arbitrary and depends on personal experience. Several trials may be required to generate an optimal detection threshold that neither picks up too many unqualified (dim) points nor filters out too many qualified (bright) points. Adjust other parameters to optimize data analysis. Here, neighborhood distance (fluorescence points in the neighboring frames within a distance are combined) is set as 65 nm (1/2 pixel width) and fitted as a single molecule. Gap frames (individual molecule events that occurred within these frames and neighborhood distance were combined and fitted as a single molecule event) is set as 26 frames (1.3 sec) for PAmCherry1 imaging⁴⁰. Adjust these parameter according to the properties of single molecule probes and acquisition rate to avoid over-counting⁴⁰ in SMLM. Apply drift correction with fluorescent beads during the reconstructions.
   1. For imaging the fixed membrane sheets labeled by iRFP-PAmCherry1-PH_PLCδ1, reconstruct the whole image stacks from the same membrane sheet into a single super-resolution image³⁷.
   2. For live cell samples labeled by mEOS3.1-PH_PLCδ1, separate the whole image stack into several smaller stacks of 1,000 frames so that 1,000-consecutive-frame stack is reconstructed as a single super-resolution image, which has a temporal resolution of 10 sec. In the end, combine all the time series images into the final time series stack²³.
      NOTE: In live-cell PALM imaging, a small number of activated probes may move or dissociate from PI(4,5)P₂ in the PM before bleaching. To account for the oversampling of the probes, combine individual molecule signals in the neighboring frames within 130 nm (instead of 65 nm) into a single emission event during image analysis.
3. After obtaining the reconstructed images, carry out further image analysis with the custom-written program in Matlab to quantify molecule density, micro-domain density/size, and cluster analysis with pair-correlation⁴¹.