A step-by-step protocol for performing LIVE-PAINT super-resolution imaging of proteins in live cells using reversible peptide-protein interactions

Super-resolution imaging of proteins inside live cells is a powerful tool for investigating protein behavior. We have developed a super-resolution method we call LIVE-PAINT, which uses reversible peptide-protein interactions to achieve super-resolution inside live cells. This method is particularly useful for studying proteins which do not tolerate large genetic fusions, such as direct fusion to a uorescent protein. Here, we provide a detailed protocol for the use of LIVE-PAINT in S. cerevisiae. of GAL2 gene, which is important for making the GAL1 promoter response linear with respect to galactose concentration. In our work, we tagged and imaged Cdc12p, Cof1p, and though this be done with the desired protein of interest. Below we show an example strain for tagging and imaging Cdc12p and an example strain for imaging a user's desired protein of interest.


Introduction
Here we present a detailed protocol for the use of LIVE-PAINT in S. cerevisiae including all necessary steps: cell growth, microscope and sample preparation, data acquisition, and data processing.
The below yeast strains are examples of those that can be used with the LIVE-PAINT method. In our strains we express the uorescent protein construct under control of the GAL1 promoter and delete the GAL2 gene, which is important for making the GAL1 promoter response linear with respect to galactose concentration. In our work, we tagged and imaged Cdc12p, Cof1p, and actin, though this can be done with the user's desired protein of interest. Below we show an example strain for tagging and imaging Cdc12p and an example strain for imaging a user's desired protein of interest. Procedure 1) Yeast cell growth 1.1) Prepare a stock of synthetic complete media: 6.7 g/L yeast nitrogen base with amino acids, 10 g/L ra nose, plus the desired concentration of galactose (usually 0.05 g/L). Typically we would prepare 10 mL stock to use for a set of experiments.
1.2) Pipette 600 μL of synthetic complete media into a culture tube. Pick one yeast colony from an agar plate into the media, and pipette to mix the cells evenly.
1.3) Prepare a second 400 μL aliquot of synthetic complete media, in a culture tube. Add 100 μL of cells from the rst suspension of yeast cells to the fresh 400 μL aliquot, to make a 1:5 dilution of the culture.
This second culture is likely to produce the desired log phase cells approximately 16 hours after starting the overnight culture.
1.4) Grow the 500 μL cultures in 15 mL tubes overnight in a 30°C shaking incubator. One could set up overnights with different dilutions, to best match the desired time to mid-log phase.
2) Prepare microscope 2.1) Use a TIRF microscope equipped with a 488 nm laser and tted with a 'perfect focus' system. (The 'perfect focus' autocorrects any z-stage drift during imaging).
2.2) Rotate the mirrors in the beamline path to align the laser and direct it parallel to the optical axis. We have used a 1.49 NA TIRF objective (CFI Apochromat TIRF 60XC Oil, Nikon, Japan), mounted on an inverted Nikon TI2 microscope (Nikon, Japan).

2.3)
Use an appropriate camera to collect uorescence output from the microscope. For example, we have used an EMCCD camera (Delta Evolve 512, Photometrics, Tucson, AZ, USA) operating in frame transfer mode (EMGain = 11.5 e-/ADU and 250 ADU/photon).
3.2) Secure frame-seal slide chambers (9 × 9 mm 2 , Biorad, Hercules, CA) to a coverslip. There are two plastic sides. One has a square cutout for the well, while the other side has no cutout. Carefully remove the plastic side with no well cutout using forceps. This will reveal a sticky square frame-seal chamber.
3.3) Lower the exposed sticky side of the frame-seal slide chamber onto a cleaned coverslip. Press gently around the edge of the well with forceps to make a secure seal to reduce the chance of buffer leakage in later steps. 5.2) Set the desired exposure time and laser power for the experiments. For example, we chose an exposure time of 50 ms with a laser power density of 3.1 W/cm 2 , using a 488 nm laser to excite green uorescent proteins.

5.
3) Bring the sample into focus and then adjust the laser angle to achieve TIRF. When in TIRF you will likely start to see single localization blinking events inside the cell.
5.4) Image the cells in TIRF for the desired amount of time. For example, we typically use 1,000 to 6,000 frames with an exposure time of 50 ms.
5.5) Ensure at this stage that you can see individual blinking events. If you cannot see these single blinking events, try varying the TIRF angle in order to decrease the illumination volume, increasing the laser power, or waiting for some of the uorescent proteins to photobleach, if you have an abundance of uorescence signal.
5.6) Save the output as a tiff stack.
6) Image analysis 6.1) Load a tiff stack acquired during imaging into Fiji.
6.2) Use the Peak Fit function of the Fiji GDSC SMLM plugin to analyze single localization events. Typical parameters we used were a signal strength threshold of 30, a minimum photon threshold of 100, and a precision threshold of 20 nm.

Time Taken
Anticipated Results