Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

All experimental procedures involving animals were optimized to reduce animal suffering and were approved by the Commission for Animal Experimentation, University of Amsterdam, DEC protocol # DED204 and DED250.

1. Coverslip Preparation

1. Cut down the coverslips to a size of 15 mm x 15 mm using a carbide or diamond scribe, so that they fit into the wells of a 12-well plate.
2. While working in a fume hood, start coverslip coating by fully submerging coverslips in concentrated nitric acid solution (70% wt/wt) in a glass container. To ensure even distribution shake, then incubate for a minimum of 4 hr. It is possible to reuse the concentrated nitric acid solution for approximately 2 times, but it can loose color by exposure to light.
3. Remove the concentrated nitric acid solution and wash the coverslips four times in distilled water for 30 min, carefully shaking after each wash.
4. With caution remove the water.
   Note: the next three steps are performed in a sterile laminar flow cabinet.
5. Soak the coverslips in 96% EtOH. Remove the coverslips from the EtOH solution and let them dry.
6. Flame the coverslips and put them in a glass container. Use fine forceps to handle the coverslips (Dumont style no.5 forceps for example).
7. Cover the container with aluminum foil and bake in a dry-heat oven for 12-16 hr at 180 °C. Baked coverslips can be stored at room temperature for up to 1 month if covered tightly.

2. Coverslip Coating

The coverslip coating procedure favors neuron attachment to the glass surface and dendritic arborization ¹⁴.

1. In a sterile laminar hood, use sterile small forceps to place the individual coverslips in single wells of a 12-well plate.
2. Add 500 μl of Poly-L-lysine solution (or sufficient amounts to submerge the coverslips).
3. Wrap the plate in aluminum foil to prevent evaporation and leave it overnight at room temperature.
4. Before starting the culture, aspirate the Poly-L-lysine solution carefully in a sterile laminar hood.
5. Wash each well with 1 ml of sterile water twice, whilst preventing them to dry out.
6. Aspirate water completely, add 1 ml of plating medium and leave the coverslips in the tissue culture incubator until ready to plate the cells. It is recommended to plate the cells within 24 hr.

3. Removal of Brains from E16-E19 Rat Embryos

1. Sterilize the surgical instruments by heating them in a dry sterilizer overnight or washing them with 70% EtOH. Dry thoroughly if EtOH is used.
2. Prepare several 30 mm dishes with 1x HBSS buffer and keep them on ice.
3. Euthanize the rat dam with an intraperitoneal injection of Euthasol (160 mg/kg Euthasol in a volume of ± 0.4 ml).
4. Check for the absence of reflexes.
5. Spray the dam’s abdomen with 70% EtOH.
6. Make an incision along the abdomen and remove the uterus.
7. Remove the embryos from the uterus and place them in a 100 mm diameter petri dish.
8. Remove the heads of the embryos with large scissors and place the heads in a new petri dish containing cold 1x HBSS buffer.
   Note: from here to step 7.1 the procedure is performed under sterile conditions in a laminar flow cabinet.
9. Hold down the heads along the sides with big forceps.
10. With small scissors, make a sagittal cut in the skin on top of the head, then laterally peel the skin down with a large forceps.
11. Use the same approach as in step 3.10 to remove the skull. Make a sagittal cut starting at the caudal end; gently opening the skull without damaging the brain tissue. Fold the two halves of the skull away laterally exposing the brain.
12. With the blunt spatula, scoop out the brain and place it in fresh cold 1x HBSS.

4. Dissection of the Hippocampi

It is very important that the dissection is done as quickly as possible in sterile conditions to ensure cell viability. Keep the samples cold on ice.

1. Remove and discard the cerebellum with the fine scissors.
2. Separate the two hemispheres of the brain by making a sagittal cut along the midline.
3. Take each hemisphere and place both in a new 30 mm dish containing fresh cold 1x HBSS.
4. Place each hemisphere such that the temporal lobe faces the bottom of the dish.
   NOTE: From here on, it is recommended to use a dissecting microscope.
5. Gently hold the midbrain using a small forceps and remove the midbrain with another pair of forceps. Leave the remainder of the hemisphere intact containing the cortex and the hippocampus.
6. Turn over the tissue, so that the hippocampus is now facing the bottom of the dish.
7. Gently hold the hemisphere in place with a fine forceps. By using another fine forceps, carefully and gently remove the meninges. It is easier to start at the olfactory bulb. Use caution so as not to damage the hippocampus.
8. Orient the tissue so that the hippocampus is now facing up. The hippocampus can now be seen by its characteristic C-shaped structure.
9. By using fine forceps, dissect out the hippocampus. Collect it in a new 30 mm dish containing fresh cold 1x HBSS.

5. Cell Dissociation and Plating

1. Count the total number of hippocampi, then cut them into small pieces.
2. Collect the pieces in a 15 ml centrifuge tube containing 3 ml 1x HBSS.
3. Centrifuge at 300 x g for 5 min and carefully remove the supernatant.
4. Add 6 μl Trypsin per hippocampus.
5. Incubate for max 20 min at 37 °C. Swirl after 3 min.
6. Wash two times with 5 ml of fresh cold 1x HBSS and discard the supernatant.
7. After the second wash, add 1.5 ml of plating medium, pre-warmed to 37 °C. The serum in the plating medium will inactivate Trypsin activity.
8. Slowly triturate 30x with a fire-polished Pasteur pipette until all pieces of tissue are homogenously dispersed into single cells. Avoid any bubbling.
9. Add 5 ml of plating medium pre-warmed to 37 °C.
10. Count cells using a Trypan blue vital staining.
11. Seed 50,000 cells per well of a 12-well plate in 1 ml plating medium, prepared in Section 2 (total volume 2 ml).
12. Gently rock the plate to evenly distribute the cells.
13. Incubate at 37 °C, 5% CO₂. After 2-3 days, replace half of the plating medium (0.5 ml) with culture medium containing 10 µM FUDR.
    Note: Dendritic spine imaging is performed 16-17 days after plating (16-17 days in vitro, DIV).

6. Rat Hippocampal Primary Neuron Transfection using Lipofectamine

On DIV 14-15 neurons are transfected using the following protocol:

1. Prepare plasmid DNA expressing GFP and incubation medium (10 ml of Neurobasal medium with 100 μl of glutamax).
2. Pre-warm the incubation medium to 37 °C.
3. Prepare DNA mix (Tube A). For each coverslip add 1 μg of DNA in 100 μl of plain Neurobasal medium. Gently mix.
4. Prepare Lipofectamine mix (Tube B). For each coverslip add 2 μl Lipofectamine in 100 μl of plain Neurobasal medium. Gently mix.
5. Add the Lipofectamine mix to the DNA mix dropwise.
6. Incubate in the laminar flow hood for 30 min at room temperature.
7. Add 1 ml of pre-warmed incubation medium to plate 1.
8. Store plate 2 for 5 min at 37 °C, 5% CO₂.
9. Gently add dropwise 200 μl of the DNA/Lipofectamine mix to each well and incubate for 45 min at 37 °C, 5% CO₂.
10. With the use of small forceps lift the coverslips containing the neurons and rinse them by dipping them in a 3 cm dish containing fresh warm Neurobasal medium and move them to plate 2.
11. Incubate the transfected neurons at 37 °C, 5% CO₂ for 48 hr.
12. Check for transfection efficiency 24 hr after transfection.

7. Immunostaining and Mounting of Rat Hippocampal Primary Neurons

To improve fluorescence intensity in transfected cells, perform an immunostaining protocol to enhance GFP detection 48 hr after transfection.

1. Prepare 4% PFA and 0.05 M TBS.
2. Warm the 4% PFA solution to 37 °C.
3. Gently aspirate the medium from the wells containing the coverslips.
4. Add 500 μl of warm 4% PFA carefully to prevent damaging the dendrites.
5. Incubate at room temperature for 15 min.
6. Wash with 1x HBSS 3 times for 5 min.
   NOTE: at this point samples could be stored up to 3 weeks at 4 °C or immunostaining can be started immediately.
7. If samples were stored, wash with 0.05 M TBS 3 times for 5 min.
8. Block with TBS-BSA (1%) solution at room temperature for 30 min.
9. Wash with 0.05 M TBS 3 times for 5 min.
10. Add the primary antibody diluted in incubation mix.
11. Incubate the plates for 1 hr at room temperature.
12. Further, Incubate overnight at 4 °C with gentle shaking.
13. Remove the primary antibody.
14. Wash with 0.05 M TBS 3x for 5 min.
    NOTE: from this point on, keep the coverslips protected from light.
15. Add the secondary fluorescent-conjugated antibody diluted in incubation mix.
16. Incubate at room temperature for 2 hr.
17. Remove the secondary antibody.
18. Wash with 0.05 M TBS 3 times for 5 min.
19. Wash with 1X TB 2 times for 5 min.
20. Use fine tweezers to remove to coverslips from the wells.
21. Dry any excess of TB with a tissue and mount the coverslips using mounting medium.
22. Seal with nail polish to prevent evaporation of mounting medium.

8. Dendritic Spine Imaging using Structure Illumination Microscopy

Dendritic spine imaging using the SIM system described in the materials has a lateral resolution (XY) value of approximately 85-110 nm and an axial (Z) resolution value between 200 – 250 nm, providing a factor of 2 times improvement in resolution compared to wide-field microscopy.

NOTE: Dendritic spine imaging using SIM is done typically 2 days after step 7.22, but could be done up to 3 weeks later if samples are kept in the dark and under a controlled temperature of 22 – 23 °C.

1. Turn on the 488 nm laser, the mercury lamp, the stage controller, the piezo controller, the halogen lamp for transmitted light and the PC and start up the SIM software in the “ANDOR for N-SIM” mode.
2. Clean the 100x TIRF objective with 95% ethanol three times, and if necessary with petroleum ether.
3. Use the following filter settings: 520 LP with a 488 dichroic.
4. Put a drop of immersion oil on the objective. Check that there are no air-bubbles in the oil-drop. Move the objective upwards until the oil touches the sample.
   Note: Place a cover over the stage to protect the sample from ambient light and dim the lights in the room as much as possible.
5. Set the correction collar of the objective to 37 °C, 200 µm, to obtain the best symmetry of PSF. In order to set the correct collar position, first position the objective ring at the optimal nominal position and then check a 100 nm bead sample. According to the best PSF’s symmetry, slightly change the collar position around the nominal one.
6. For illumination, use 3D-SIM grating (3D 1layer 100X/1.49 all wavelengths). To begin the grating alignment place the selected grating block into the SIM illuminator, with the 100X 1.49 objective in place. Use a 100 nm bead sample mounted in media, with a concentration that can allow isolating 10-15 beads for a field of view (FOV). After setting the objective correction collar to the desired position, select the 3D-SIM illumination and start the software-guided alignment procedure. It will run (5 phases) x (1 direction) x (100 z-planes) images, from which it will reconstruct the FOV with beads. After selecting a single bead via an appropriate ROI, covering the entire bead including the out-of-focus blurred light, the software will start an automatic PSF fitting and it will adjust the grating position according to the result.
7. To check the performance of the microscope, repeat the grating alignment every 2 weeks, because of the possible misalignment caused by table movements and/or temperature drifting. Furthermore, also check the laser intensity and stability according to the manufacturer’s suggestions.
8. Clean the sample surface with 95% ethanol three times.
   Note: For the next steps see figure 3 for an overview of the control panels and the correct settings within the SIM software.
9. In the SIM software, select the Optical Configuration Eye FITC and select an empty filter block in Turret 1 for visual inspection with white light.
10. Set the focusing speed and the travel speed of the XY table to “Fine”.
11. Open the shutter and quickly focus on the sample.
12. Close the shutter again and set the Z-coordinate to zero.
13. Select the green filter in Turret 1 for visual inspection using the green channel.
14. Set the intensity of the mercury lamp to the lowest setting.
15. Move to the border of the sample carefully, making sure that the objective does not touch the sealant.
16. Open the shutter and quickly scan through the sample with the lowest possible intensity.
17. Upon encountering a sufficiently bright dendrite of interest and centering a segment of interest in the field of view, close the shutter.
18. Set the software to Optical Configuration 3D-SIM 488 and the camera settings to read-out mode EM, gain 1 MHz 16-bit, exposure time 100 msec, laser power 5% and EM gain 200.
    NOTE: Check that the green filter in Turret 2 is selected and that Turret 1 is empty.
19. Check that the grating is set to ‘Moving’ and click Live to view the sample with laser light and through the camera.
20. Activate the Look Up Table.
21. Center the object of interest if necessary and focus with the focusing speed set to Extra Fine.
    NOTE: in the read-out mode EM gain 1 MHz 16-bit, the target intensity for a good SIM image is between 30,000-45,000.
22. Quickly adjust the camera settings to get an intensity value between 30,000-45,000 in the read-out mode EM gain 1 MHz 16-bit. Initially use:
    1. Laser power: 0% – 20% (with samples prepared as described above, 5% or 2.6 mW is sufficient)
    2. Exposure time: 50 msec-2 sec
    3. Read-out mode: EM gain 1 MHz 16-bit
    4. EM gain: 0-300
    5. Conversion gain: 1x – 5.1x
    6. Format for Live: No binning
    7. Format for Capture: No binning
       NOTE: with these settings 6.3% ± 1.3% bleaching is routinely achieved, within a 10% limit of acceptable maximum bleaching, which could significantly affect the image quality ¹⁵.
23. Click Stop to turn off Live view.
24. Configure the settings of the 3D Z stack in the ND Sequence panel to:
    1. Range: 2 µm
    2. Set size: 120 nm
    3. Z plane
    4. Click Home position
    5. Select the Optical Configuration 3D-SIM 488 in the Lambda section
25. Select 3D-SIM as the acquisition mode in the N-SIM pad.
26. Run the ND Sequence acquisition and save the raw data.
27. Select the Optical Configuration Eye FITC again and repeat steps 8.15-8.27 until the entire sample has been imaged
28. 3D Image reconstruction: The data acquired in step 8.23 can either be reconstructed right away or later on. Start by reconstructing the Z stack with the default reconstruction settings in Reconstruct Slice or Reconstruct Stack mode and adjust if necessary.
    NOTE: for best results, Z stacks should be made with the indicated step size and reconstructed in Reconstruct Stack mode. Always check the validity of the reconstructed image by comparing it to the raw data or, preferably, a wide-field image. The parameters that can be adjusted for the reconstruction process are the contrast and high frequency noise suppression. Both of them influence how different raw image properties are taken into account during the reconstruction process. In case of low modulation depth raw data, the contrast parameter plays an important role. If the user has datasets with a low signal-to-noise ratio, then the high frequency noise suppression parameter can influence the reconstruction quality severely.
29. When imaging in finished, center the XY stage and move the objective all the way down to its resting position.
30. Unmount the sample and clean both the sample and objective with 95% ethanol.
31. Shut down the software and the PC and switch off all other devices.
32. 3d reconstruction and spine classification of the acquired images can be carried out after converting the files to TIFF as described before using NeuronStudio software (see Figure 4) ¹⁶.
33. Use the following parameters for reconstruction in the NeuronStudios software:
    1. Volume: Voxel dimensions: X: 0.03 μm; Y: 0.03 μm; Z: 0.120 μm.
    2. Dendrite detection: Attach ratio: 1.3; Minimum length: 5 μm; Discretization Ratio: 1; Realign junctions: Yes.
    3. Spine detection: Minimum height: 0.2 μm; Maximum height: 5.001 μm; Maximum width: 3 μm; Minimum stubby size: 10 voxels; Minimum non-stubby size: 5 voxels.
    4. Spine Classifier: Neck ratio (head-neck ratio): 1.1; Thin ratio: 2.5; Mushroom size: 0.35 μm;
34. NeuronStudio parameters used for rendering: Neurite vertex shape: solid eclipse; Neurite vertex color: by type; Neurite edge shape: line; Neurite edge color: single color; Spine shape: solid eclipse; Spine color: by type.
    Note: After 3D reconstruction is it also possible to set the volume render. The default threshold was set to 20 with the ‘Regenerate volume rendering’ option active. Opacity was set by ‘Automatic Intensity’ checked. ‘Surface only’ and ‘Use Point Pre-Rendering’ options were also active