Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

1. Primary cultures

1. Culture mouse hippocampal primary neurons in chambered coverslips (such as Ibidi µ-Slide 8 Well or Nunc Lab-Tek Chambered Coverglass) that match the objective requirement for #1.5 (0.17 mm) coverslip thickness.
2. Coat chambered coverslips with 100 µL of poly-L-lysine (100 µg/mL).
3. The next day, wash the chambered coverslips twice with sterile phosphate-buffered saline (PBS).
4. To obtain mouse primary neurons, isolate hippocampi from P1-P4 pups²³.
5. Place dissected hippocampi in 10 mL of Dissection Media (Table 1) and let them deposit at the bottom of the tube.
6. Using a sterile pipette, carefully remove the Dissection Media, leaving the hippocampi undisturbed at the bottom of the tube.
7. Add 10 mL of Media 1 (Table 1) to the hippocampi and incubate for 30 minutes at 37 °C.
8. Using a sterile pipette, carefully remove Media 1, leaving the hippocampi undisturbed at the bottom of the tube.
9. Add 10 mL of Media 2 (Table 1) and leave the (capped) centrifuge tube under the hood horizontally for 45 minutes.
10. Let the centrifuge tube stand vertically to allow the tissue to settle at the bottom of the tube.
11. Using a sterile pipette, carefully remove Media 2, leaving the hippocampi undisturbed at the bottom of the centrifuge tube.
12. Add 2 mL of Media 3 (Table 1).
13. Using a p1000 pipette with a filtered tip, mechanically dissociate cells from the tissue.
14. Transfer the supernatant, in which are located the isolated neurons, to a 15 mL centrifuge tube.
15. Centrifuge the cell suspension for 2 min at 300 x g at room temperature (RT).
16. After centrifugation, cells are located at the bottom of the centrifuge tube. Using a sterile pipette discard the supernatant.
17. Resuspend cells in 1 mL of Media 4.
18. Use a 70 µm filter to eliminate undissociated cells.
19. Count viable cells in a Bürker chamber by adding 1 µL of 0.4 % Trypan blue solution to 19 µL of the cell suspension.
20. Plate cells at 70,000 cells/well in a volume of 200 µL per well.
21. Allow the cells to attach for 2 h in a humidified incubator at 37 °C and 5% CO₂.
22. Take out the chambered coverslips from the incubator and carefully replace the medium with 200 µL of Culture Media.
23. Leave the chambered coverslips in a humidified incubator at 37 °C and 5% CO₂.
24. Replace one third of the medium with fresh culture media every 5-7 days.
25. Wait until hippocampal primary neurons are fully matured (12-14 days after plating) to perform co-localization studies.

2. Immunofluorescence staining

1. Take the chambered coverslips from the incubator.
2. Remove the medium.
3. Quickly wash the wells with 200 µL of PBS.
4. Add 4% paraformaldehyde (PFA) in PBS (200 µL/well) to neurons to fix them quickly.
5. Incubate the cells for 15 min at RT.
6. Remove the PFA solution.
7. Permeabilize the cells by adding PBS with 0.2% Triton X-100 (200 µL/well).
8. Incubate for 1 min at RT.
9. Remove the solution and incubate the samples with 1% bovine serum albumin (BSA) in PBS (200 µL/well) for 1 h at RT to passively cover all free binding surfaces of the plate with an irrelevant protein for the analysis. A BSA-based blocking buffer without Triton X-100 reduces the antibody background more efficiently than the same buffer with 0.2% Triton X-100.
10. Remove the solution.
11. Add the primary antibody of choice diluted in a PBS solution containing 1% BSA and 0.2% Triton X-100 (120-200 µL/well, depending on the antibody dilution and availability). Incubate the samples for 2 h.
    1. As a negative control, do not add any primary antibody to one of the wells. Multiple antibodies of different species against different targets may be used at the same time. Use an antibody against MAP2 (a neuronal marker) raised in chicken, an antibody against either PSD95 or synaptophysin raised in mouse, and an antibody against a target protein raised in rabbit. This allows three-color SIM analyses.
12. Quickly wash the wells three times with PBS (200 µL/well).
13. Add secondary antibodies (dyLight and Alexa secondary antibodies can both be used) diluted in a PBS solution containing 1% BSA and 0.2% Triton X-100 (200 µL/well). Incubate the samples for 1 h at RT.
14. Quickly wash the wells three times with PBS (200 µL/well).
15. Add Hoechst dye at a concentration of 1 μg/mL diluted in PBS (200 µL/well) to stain nuclei. Incubate the samples for 10 minutes at RT.
16. Quickly wash the wells twice with PBS.
17. Mount cells using a SIM-compatible mountant. Use 10 µL/well of ProLong Glass Antifade Mountant.
18. Cover and protect the cells with a coverglass (e.g., a round coverglass with a diameter of 8 mm). Square ones can also be used.
19. Store the chambered coverslips at RT and wait at least 48 h before acquiring images. Diamond Glass requires at least two days of curing before super-resolution acquisitions.

3. Antibody specificity control

NOTE: Use two strategies to assure antibody specificity. The first strategy is to use at least two different antibodies targeting the same substrate. The second strategy is antibody neutralization by incubation with the purified protein target or the epitope used to raise the antibody.

1. Incubate the antibody of choice with five times excess of the recombinant target or epitope for 1 h at RT in 1% BSA in PBS.
2. After the incubation, use the neutralized antibody at the usual concentration for staining as described above from 2.11.

4. Microscope calibration

NOTE: We routinely use an N-SIM Super-Resolution Microscope System manufactured by Nikon for the super-resolution studies. However, several other companies also offer super-resolution microscopes in their catalogues. Although specific indications for Nikon’s N-SIM system are described, the instructions that follow can be generalized to other systems. Before the acquisition of SIM images, the system requires a proper calibration with specific sub-resolution size fluorescent beads. An example is the TetraSpeck microspheres. These beads are stained with different fluorescent dyes to allow the calibration of different lasers with one sample.

1. In a water bath sonicate around 1.8 x 10⁸ fluorescent microspheres for 10 minutes. Nikon’s N-SIM system requires a sparsely populated multicolor beads sample for the calibration. This could differ for other systems that require a dense single layer of sub-resolution size fluorescent beads. Adjust the number of fluorescent particles accordingly.
2. Dilute the fluorescent microspheres 1:500 in double distilled water.
3. Sonicate a second time for an additional 10 minutes.
4. Pipet 15 µL of the diluted beads into a well of a chambered coverslip.
5. Let the solution dry for 5 minutes at RT.
6. Add 10 µL of the mounting solution and place an 8 mm coverslip on top.
7. Wait at least 48 hours to allow proper curing.
8. Turn on the microscope and lasers.
9. Let the system warm up to reach the thermal equilibrium of all microscope components. N-SIM Super-Resolution Microscope System requires at least 3 hours.
10. Select the 100x objective.
11. Start the calibration by aligning the lasers to the center of the diffraction grating block. In the N-SIM system, a micrometer knob and a dedicated camera allow centering of the light beams to the target.
12. Insert the chambered coverslip in the microscope for viewing. Set the system to the chambered coverslip thickness by adjusting the objective correction collar. NIS software, the proprietary software provided with N-SIM Super-Resolution Microscope systems, has an automatic function to regulate correction collars.
13. Adjust grating block focus for each channel to ensure focused structured pattern illumination on the sample. NIS software provides an automatic function for this task.
14. Next, acquire raw 3D-SIM images of the multicolor microspheres. Reconstruct the raw images to obtain a super-resolved image using the microscope software or the open-source software platform for the analysis of biological images ImageJ²⁴ and the plugin fairSIM²⁵.
15. Calculate, for each separated wavelength, the Fourier transform of the super-resolved image obtained in 4.14. If the transformed image fails to obtain a correct flower-like pattern, restart calibration from 4.11 since super-resolution has not been achieved.
16. In the super-resolved image, select a single microsphere and calculate its intensity profile for each channel to measure the resolution achieved. It should now be close to 100 nm laterally.
17. Next, perform channel registration by overlaying a multichannel acquisition of the microspheres. The goal is to collimate all channel signals laterally and axially. This will eliminate chromatic aberrations due to the misalignment of the different channels and help the co-localization analysis.
18. Confirm the quality of calibration by using the functions “Illumination Phase Steps” and “Illumination Pattern Focus” of SIMcheck²⁶, a suite of plugins for the open-source application ImageJ. To this end, prepare a chambered coverslip to obtain a dense single layer of TetraSpeck microspheres and acquire a 3D-SIM image of the sample. Analyze the image in ImageJ and, if aberrations are detected, restart microscope calibration from step 4.11.

5. Acquisition

1. Start analyzing the sample using a 40x objective in confocal or widefield mode. This allows navigation to the sample, maintaining good details and a large field of view.
2. Use MAP2 antibody signal to identify an area representing neuronal processes.
3. Acquire images of the sample in confocal mode to determine the quality of the staining. Poor confocal quality will reflect in poor SIM quality, therefore requiring the samples to be discarded.
4. If the area and the quality of the images are satisfactory, switch the objective to 100x.
5. Apply oil to the 100x objective.
6. Acquire a widefield or confocal image that will be used later to assess the quality of the super-resolved image (Figure 1A,B).
7. Switch to 3D-SIM mode.
8. Using dialog windows to set parameters for acquisition, select the highest bit-depth setting available to maximize color information. Typically, 16-bit is the standard choice. Moreover, to improve signal-to-noise ratio, select a low frequency value for acquisition, such as 1 MHz.
9. Using histogram windows, set lasers power to obtain a linear response of signal. To avoid loss of information, limit saturated pixels in the images. The N-SIM system uses an Andor iXon3 camera. When working at 16-bit, choose a target intensity of 16,000 to ensure the linear response of the camera. Alternatively, choose a range between 30,000-45,000 to maximize the dynamic range of the acquisition.
10. Set laser power between 0.1% and 50% when imaging the samples and exposure times between 50 ms and 2 s. Laser powers above 50% may cause rapid photobleaching of the fluorophores in use.
11. Start acquiring the images in 3D-SIM mode.
12. Use SIMcheck, a suite of free plugins for ImageJ, to assess the quality of acquisition of the raw images.
13. If SIMCheck does not detect any artifacts or quality issues, acquire a minimum of 10 images from 4 technical replicates to allow statistical analysis.

6. Post-production: image reconstruction

NOTE: 3D-SIM acquired images are raw images that need to be processed to obtain reconstructed super-resolved images. Incorrect reconstruction of raw images can lead to artifacts that would affect the analysis of the samples. Great attention should therefore be paid to properly choosing reconstruction parameters.

1. Process the raw images using the microscope reconstruction analysis software to obtain a super-resolved image (Figure 1C). Alternatively, use the freely available ImageJ plugin fairSIM to reconstruct raw images.
2. Calculate the Fourier transform of the super-resolved images using the microscope reconstruction software or ImageJ plugin SIMCheck. A good reconstructed image should return, for each channel, a flower-like image. If the reconstructed images fail to recreate a flower-like shape, restart from the raw images and reconstruct them by modifying the reconstruction parameters such as Wiener filtering, apodization and zero-order suppression²⁷. In NIS software, using the preview to monitor how changing the parameters affects the final resolved image, modify the parameters i) Illumination Modulation Contrast, ii) High Resolution Noise Suppression and iii) Out of Focus Suppression.
3. Next, analyze the reconstructed image to unbiasedly detect artefacts by using NanoJ-SQUIRREL²⁸, an ImageJ-based plugin to assess the quality of super-resolved images.
4. If NanoJ-SQUIRREL detects artifacts, restart from the raw images and reconstruct them by modifying the reconstruction parameters such as Wiener filtering, apodization and zero-order suppression. In NIS software, using the preview to monitor how changing the parameters affects the final resolved image, modify the parameters Illumination Modulation Contrast, High Resolution Noise Suppression and Out of Focus Suppression.
5. Use the super-resolved images to calculate the co-localization profile and/or Pearson’s and Mander’s coefficients.

7. Co-localization with profile analysis

NOTE: As a first step to study co-localization between synaptic markers and a protein of interest, take a super-resolved image and analyze a single locus to determine signal overlap.

1. Identify a single locus on the super-resolved image.
2. Obtain the intensity profiles of the fluorescent signals of the locus of interest.
3. Export the data.
4. Use GraphPad Prism, or a similar analysis software, to normalize all signal peaks and obtain comparable signal intensities for each channel with the final goal of determining locus specific co-localization.

8. Quantification of Pearson’s and Mander’s coefficients

NOTE: If profile analysis has suggested single locus co-localization, a more general analysis of the whole image can be carried out by calculating Pearson’s and Mander’s coefficients^29,30.

1. Use JACoP³¹, an ImageJ plug-in, to determine the two parameters of co-localization: Pearson’s and Mander’s.

9. Statistical analysis

1. Use GraphPad Prism, or a similar analysis and graphing software, to process data collected with JACoP.
2. Use at least 40 SIM images for each condition analyzed to obtain graphs and for statistical relevance.