The overall goal of this procedure is to image dendritic spines in super resolution. This is accomplished by first preparing a rat primary hippocampal called the second step is to visualize the dendritic spines by means of GFP transfection and staining. Next, the acquisition of GFP labeled dendritic spines is carried out in super resolution using a SIM microscope.
The final step is to reconstruct the raw image data into 3D ready for subsequent image analysis. Ultimately, GFP expressing primary hippocampal neurons imaged in super resolution is used to show subtle changes in dendritic spine morphology. The main advantage of this technique over existing methods like conventional confocal microscopy, is that it allows you to study dendritic spine morphology in more detail.
This method can help examine key ongoing questions in neuroscience such as molecular and pharmacological dependent alterations in the shape of dendritic spines. Structured elimination microscopy is based on the projection of line patterns onto the sample, and this gives extra information that can be extracted by a computer program and gives extra sharp images and that allows you to look at MI mitochondria and other small structures in the cell. A visual demonstration of this method is important in order to achieve a valid reconstruction of a SIM image is crucial to go through the steps that give you a good quality of the raw data.
An important preparatory step involves coating the cover slips, working in a sterile laminar hood. Use small sterile forceps to place the individual cover slips into single wells of a 12 well plate. Then add 500 microliters of polylysine solution to each well to submerge the cover slips.
After washing the wells, add one milliliter of plating medium and leave the cover slips in the tissue culture incubator for up to 24 hours prior to plating the cells. The procedure will be demonstrated by HU is SH a PhD student in our group. After isolating the of a rat embryo, use fine forceps to remove and discard the cerebellum from the brain.
Separate the two hemispheres by making a sagittal cut along the midline under a dissecting microscope, use one pair of small forceps to gently hold the midbrain and remove the midbrain. With another pair of forceps, leave intact the remainder of the hemisphere containing the cortex and the hippocampus. Next, carefully remove the meninges, reorient the tissue so that the hippocampus is facing up.
The hippocampus can be identified by its characteristic CS shaped structure. Using fine forceps, dissect out the hippocampus and place it in a new 30 millimeter dish containing fresh cold one X-H-B-S-S. After removing the hippo campi from all of the embryos, count the total number of hippo campi and then cut them into small pieces.
Collect the pieces in a 15 milliliter centrifuge tube containing three milliliters of one X-H-B-S-S. After centrifuging and removing the supernatant, add six microliters of trypsin per hippocampus. After incubating and washing the hippocampal fragments as described in the text protocol, slowly tate the fragments 30 times with a fire polished pastor pipette until all pieces of tissue are homogenously dispersed into single cells.
Take care to avoid any bubbling. Next, add five milliliters of plating medium, pre-warned to 37 degrees Celsius. After counting the cells using a trian blue vital staining seed, 50, 000 cells per well of a 12, well plate in one milliliter of plating medium for a total volume of two milliliters.
After rocking the plate to distribute the cells evenly, incubate the cells at 37 degrees Celsius, 5%carbon dioxide. After two to three days, replace 0.5 milliliters of the plating medium with culture medium containing 10 micromolar FUDR. To transfect, the neurons add a lipectomy.
Mix dropwise to A DNA mix and incubate at room temperature for 30 minutes. In a laminar flow hood, 10 minutes before the end of the 30 minute DNA lipectomy mixture, incubation, pipette the conditioned medium from the original or first culture plate to a second 12. Well plate and store this plate at 37 degrees Celsius, 5%carbon dioxide in the first culture plate.
Swiftly replaced the conditioned medium with one milliliter of prewarm incubation medium to prevent the cells from drying out. Place both plates back into the incubator at 37 degrees Celsius, 5%carbon dioxide. At the end of the 30 minute DNA lipectomy incubation, the DNA lipectomy mix is ready to be added to the cells.
Gently add dropwise 200 microliters of the DNA lipectomy mix to each well and incubate for 45 minutes at 37 degrees Celsius, 5%carbon dioxide. After the 45 minute incubation, use small forceps to lift the cover slips holding the neurons. Rinse them by dipping them in a three centimeter dish containing fresh, warm neuro basal medium and place them in a well.
In the second 12, well plate containing the conditioned medium. Then incubate the transfected neurons at 37 degrees Celsius, 5%carbon dioxide for 48 hours. To prepare for immuno staining, gently aspirate the medium from the wells containing the cover slips.
Then add 500 microliters of warm 4%PFA carefully to prevent damaging the dendrites. After incubating and washing the cells block with 1%T-B-S-B-S-A solution at room temperature for 30 minutes after washing again, add the primary antibody diluted in incubation, mix and incubate at room temperature for one hour. Then incubate overnight at four degrees Celsius with gentle shaking.
The next morning, remove the primary antibody and wash again from here on, keep the cover slips protected from light. Next, add the secondary fluorescent conjugated antibody diluted in incubation. Mix and incubate at room temperature for two hours.
Then remove the secondary antibody and wash the cells using fine tweezers. Remove the cover slips from the wells and dry any excess TBS with a tissue. Finally mount the cover slips using mounting medium and seal with nail polish.
To begin the SIM imaging, turn on the 488 nanometer laser, the mercury lamp, the stage controller, the pizo controller, and the halogen lamp for transmitted light. Start the SIM software in the and or for nsim mode. After putting a drop of immersion oil on the objective, check that there are no air bubbles in the oil drop.
Place the cover slip with the hippocampal neurons on the microscope stage. Move the objective upwards until the oil touches the sample. Remember to place a cover over the stage during the acquisition procedure to protect the sample from ambient light and dim the light in the room as much as possible.
In the SIM software, select the optical configuration IFI and select an empty filter block inter one for visual inspection with white light setting sample focus. Subsequently switch off the white light and switch the mercury lamp on. Open the shutter and select the green filter to search for a sufficiently bright dendrite.
Upon encountering a sufficiently bright dendrite of interest and centering a segment of interest in the field of view, close the shutter. Set the software to optical configuration 3D SIM 4 88, and the camera settings as follows. Set the readout mode to em.
Gain one megahertz 16 bit. Set the exposure time to 100 milliseconds. Set the laser power to 5%Set the EM gain to 200.
With the focusing speed set to extra fine, focus on the object of interest. Quickly adjust the camera settings as follows. To get an intensity value between 30, 040 5, 000, set the laser power to 0%to 20%Set the exposure time to 50 milliseconds to two seconds.
Set the readout mode to em. Gain one megahertz 16 bit. Set the EM gain to zero to 300.
Set the conversion gain to one x to 5.1 x. Set the format for live to no Benning. Set the format for capture to no Benning.
Next, configure the settings of the three DZ stack in the ND sequence panel as follows, set the range to two microns. Set the step size to 120 nanometers and click the home position. Then select the optical configuration, 3D SIM 4 88 in the lambda section.
After selecting 3D SIM as the acquisition mode, run the ND sequence acquisition and save the raw data dendrites and dendritic spines imaged with confocal and sim microscopy are shown here. Acquisitions were reconstructed from confocal and SIM images. Dendrites were traced and spines were classified automatically with the software after adjusting main parameters such as neck length, neck diameter, and head diameter.
The boxed areas show one individual dendritic spine imaged with confocal and sim microscopy and reconstructed from their corresponding Z stacks, depicting differences in resolution and accuracy of the resulting 3D reconstructions. According to SIM's, higher resolution, quantitative analysis of both head and neck diameter revealed that sim microscopy measures significantly smaller dimensions than confocal microscopy for the same dendritic spines indicating that SIM is capable of detecting smaller changes in dendritic spine morphology. While attending this procedure, it is important to remember to acquire raw images of high quality for a more valid subsequent reconstruction of the same image.
Structured illumination microscopy can also be effective for live cell imaging, allowing investigation of dynamics of cells like hela cells, neurons, or e coli bacteria. After watching this video, one should have a good understanding of how to prepare, visualize, and image standard explains from primary hippocampal neurons in a super resolution.
