The overall goal of this work is to emit the nanoscale distribution of Phosphatidylinositol 4, 5-bisphosphate in the cell plasma membrane using single molecule fluorescence localization microscopy. This method helps answer key questions about phosphoinositide signaling. Such as what's the nanoscale organization of phosphoinositides under the signaling complex in cell membrane?

The main advantages of this technique are the high spatial resolution and the bypassing of detergent treatment which is a major concern in phospholipid morphological studies. This approach includes three major steps. Step one:The plasma membrane sheet preparation from live cells.

Step two:Phosphoinositide labeling and single molecule florescence imaging. Step three:Single molecule localization and imaging reconstruction. On the day of the experiment, coat cover slips with one half to one milliliter of PDL.

One or two hours later absorb the excess PDL with tissue paper from the edge of the cover slip. Then store the cover slips on a metal plate at four degrees Celsius. Next wash pre-transpected INS-1 cells that have been grown on cover slips with one half to one milliliter of ice cold PBS containing one millimolar EGTA.

Repeat this wash three times and absorb the excess PBS. Now using tweezers place the cover slips with the cultured cells onto the PDL coated cover slips, cell side down. Do this on the chilled metal plate.

Then transfer the plate to four degrees Celsius for seven to ten minutes to give the cells time to attach. Next use tweezers to gently peal off the cell loaded coverslip which will leave behind a thin layer of cell membrane on the PDL coated coverslip. Then gently wash the membrane sheets with one half to one milliliter of ice-cold PBS.

Now fix each membrane sheet in approximately 150 microliters of ice-cold four percent PFA with 0.2 percent glutaraldehyde in PBS. Allow the membranes to fix for 15 minutes at four degrees Celsius. After the fixation either immediately proceed with imaging or cover the preparations with ice-cold PBS and store them at four degrees Celsius.

If pH probes are in use, immediate imaging is strongly recommended. For this procedure a TIRF microscope based SMLM system is put to use. Before imaging the membranes, dilute one microliter of fluorescent bead solution in 10 milliliters of PBS with 50 millimolar of magnesium chloride.

Then load a sample into the imaging chamber and add 200 microliters of the diluted solution then wait 10 minutes. Next wash the sample with PBS three times. Now start the PALM imaging system.

In the associated imaging software select the iRFP channel button. Then find the cell membrane expressing the pH probes in the iRFP channel, use the fluorescent beads for drift correction. Set a normal TIRF angle as 2140 which is 1.5 deeper than the TIRF critical angle and should be default angle.

Then use the capture button to collect a conventional TIRF image in the iRFP channel as a reference. Next switch to the PAm-Cherry1 channel by clicking the RFP channel button. Then adjust the scroll bar in the AOTF pad all the way to the right for full illumination and apply the laser for about 10 to 20 seconds to bleach out the background membrane florescence.

Next set the optimal camera setting under the format tab to two by two binning. Then set the fast acquisition protocol under the ND Sequence Acquisition tab to 30 000 images at 20 hertz. Then apply 0.1 to one percent power to the 405 nanometer laser during the image acquisition so that spatially isolated points in each frame are easily identifiable.

Now acquire PAm-Cherry1 images by clicking the Run Now button. The number of molecules that can be activated will decrease gradually during the acquisitions. In response, gradually increase the intensity of the 405 nanometer laser to maintain an optimal density of signals.

Acquire images until all of the PAm-Cherry1 signal is activated. Later when undergoing image processing and reconstruction, identify and localize the individual molecular events from each frame. Set the filtering intensity threshold with the threshold number in the wavelet.

Further details are provided in the text protocol. PIP2 spatial distribution is sensitive to different fixation with proper fixation described in the text, PIP2 probes showed a consistent distribution when labeled by PIP2 antibody or specific pH domains. By comparison live cells labeled with EGFP tagged pH domains do not image much differently.

All samples showed an even distribution of probes. In contrast non-optimal fixation resulted in sharp-dense PIP2 clusters and a decrease in signal intensity. Under optimal fixation conditions the super resolution images of PIP2 in fixed cells revealed a homogeneous distribution of probes in a significant portion of the plasma membrane with only limited concentration gradients.

Some membrane patches enriched with PIP2 probes where sparsely distributed and had various sizes. Live cell PALM images displayed a similar spatial distribution as fixed cells. Detailed analysis of the PIP2 signals revealed that their dynamics were fast in local areas but this didn't result in significant changes in their overall abundance.

This technique introduce a new way for researchers in the field of phospholipids to explore the distribution and activity of other phosphoinositide subtypes under physiological condition.