The overall goal of this fluorescent imaging technique is to visualize and quantify cellular proteins in fixed cells at subdiffraction-limited resolution using a form of super-resolution microscopy called ground state depletion microscopy and individual molecule return. This method helps answer key questions in the field of cell biology and membrane biophysics, as it circumvents diffraction-limited resolution, which helps investigators more precisely define, visualize and quantify the distribution of cellular proteins. The main advantage of this protocol is that it enables the acquisition of images with an approximate ten-fold improvement in resolution over conventional fluorescence microscopy imaging approaches, such as confocal microscopy.
Demonstrating the procedure will be Oscar Vivas, a post-doc in my laboratory, and Karen Hannigan, a post-doc in Rose Dixon's laboratory. To begin, prepare the cover slips and cells as described in the accompanying text protocol. Then fix the samples and perform immunocytochemistry in order to label the plasma membrane and/or intracellular membrane proteins of interest.
Next, prepare the oxygen scavenging Glock solution by first adding 12.5 microliters of a catalase stock solution, 3.5 milligrams of glucose oxidase, and 0.5 microliters of one molar Tris to 49.5 microliters of PBS in a 1.5 milliliter microcentrifuge tube. Close the tube and briefly vortex it to dissolve the components. Then centrifuge the solution for three minutes.
Now, prepare the photo switching inducing thial solution by dissolving beta-mercaptoethylamine solution into PBS to a concentration of 100 millimolar and modify the pH to 8.0. Aliquot and store the thial solution in the freezer for up to one week. Immediately before imaging, mix the thial and oxygen scavenging components together with a saline buffer and store the solution on ice.
Using 100 microliters of the prepared photo switching buffer, mount cover slips containing stained cells onto the depression slides. Next, seal the edges of the cover slip with a silicone glue to prevent rapid oxidation of the imaging buffer. Wait several minutes until the silicone glue has fully cured, prior to imaging.
Otherwise, the cover slip will drift. To begin, select an appropriate laser to illuminate the sample, based on the chosen fluorophore. For an Alexa 647 fluorophore, use a 642 nanometer laser.
Next, chose the appropriate dichroic imaging cube and objective lens. Open the imaging software. Select the 2D acquisition mode then set the exposure time to 11 milliseconds.
Also, set the EM Gain to 300 and select an appropriate detection threshold. Next, turn on Auto Event Control and set the number of Events Per Images to eight. Enter the Pixel Size as 20 nanometers.
In the GSD Tools tab, go to the High Resolution Image Calculation options and ensure that Histogram Mode is selected. To image proteins at the plasma membrane, select TIRF mode and modify the incidence angle to determine the penetration depth. Then, set the laser intensity to 100%in order to send electrons to the dark state.
Next, select Single Molecule Detection while pumping and set to acquire automatically when the frame correlation is 0.20. For acquisition, set laser intensity to 50%and the number of acquisition frames to 60, 000. Then begin taking images.
To quantify protein cluster size, open the image file of a 10 nanometer histogram single molecule localization map in ImageJ. In the Image menu, go to Adjust and use the Auto option to optimize the brightness and contrast of the image. Then, under the Type menu, choose 8-bit.
Next, open the Analyze menu, click Set Scale and enter the values shown here. Then, in the Analyze menu, select Set Measurements and check the box beside the Area option. Back in the Image menu, select Adjust, Threshold, and then select Over/Under.
Move the maximum value to 255, the minimum value to one and click Apply. Finally, open the Analyze menu and select Analyze Particles. Place a check mark to select Pixel units, Display results and Summarize.
Then set the size to four to infinity, select the Bare Outlines option and click OK.The cell imaged here was transfected with the endoplasmic reticulum protein, mCherry-Sec61 beta, and imaged using both defraction-limited TIRF microscopy and super-resolution GSD microscopy in TIRF mode. These curves represent the diameter of the ER tubules. The image on the right shows an improvement in the lateral resolution and represents a more accurate representation of the structure of ER tubules.
The improvement in resolution offered by super-resolution GSD imaging is further demonstrated in these images, showing a labeled cardiac myocyte with an anti-CAV 1.2 antibody. Using GSD mode, the improvement in the lateral resolution is prominent and separate clusters of channels are easier to identify. Once mastered, this protocol can be completed in less than three days.
The super-resolution imaging of a single cell takes 10 to 30 minutes, depending on the number of frames required. Following this procedure, other methods like electromicroscopy can be performed in order to answer addition questions like how the fluorescent protein of interest interacts with the complex cellular environment. After watching this video, you should have a good understanding of how to prepare, acquire and analyze samples to visualize one or more cellular proteins for super-resolution microscopy using ground state depletion followed by individual molecule return.
