The overall goal of this procedure is to allow full field measurement of in vitro neuronal volume with submicrometric axial resolution required for the analysis of neurites in dynamic structures implied in neuronal growth. This method can help answer key questions in the field of cellular neuroscience regarding the dynamics of volume changes at the subsididal scale during the nongrowth or axon degeneration. The main advantage of this technique is that it provides submicrometric axial resolution combined with full fit observation and time scale to hours or days.

To begin the procedure, demold the PDMS block under a chemical hood by pouring isopropanol on it. Next, place it on the stainless steel bench of the hood. Cut around the silicone wafer and demold the PDMS replica.

Then, cut around the chip and leave a two millimeter margin using a scalpel or a razor blade. Punch the inlet by pressing the 1.5 millimeter diameter puncher firmly and actuate it in order to cut and carve the hole of the inlet. Do the same at the four dedicated zones of the chip where liquid will be injected.

Afterward, clean the chip by sticking and peeling the adhesive tape on the microstructured side. Sprinkle isopropanol on both sides. Then, dry the chip with pressurized nitrogen using an air blow gun.

In this procedure, place each coverslip on a spin-coater located in a clean room environment. Apply a drop of the positive photoresist to cover about 75%of the coverslip and spin-coat at 4000 rpm for 30 seconds to reach a final thickness of 0.45 micrometer. Place the coverslips on a hotplate with a surface temperature of 115 degrees Celsius for one minute.

Using a mask aligner, expose each coverslip at a wavelength of 435 nanometer through the dedicated mask according to the fabricant parameters. Next, prepare two glass crystallizing dishes, one containing the developer without dilution, the other containing deionized water. Dip the coverslips one by one in the developer for one minute while continuously and gently stirring the crystallizing dish.

Then, submerge the wafer in deionized water for about five seconds. Transfer the wafer to an absorbent paper and dry it with pressurized nitrogen using an air blow gun. Place both the PDMS chip and the glass dish on which it will be bonded into the plasma chamber for surface activation.

Next, deposit a drop of water on a rectangular thick microscope glass slide and stick the coverslip on the glass slide by capillarity. Under a microscope, mark the location of the photoresist stripes at the rear of the glass slide. Place the patterned glass coverslip on the mask holder of the mask aligner.

Rely on the mark made to center the photoresist reference objects. To increase the optic contrast, place a silicon wafer beneath the PDMS chip. The silicon wafer should remain firmly attached on the chuck during the alignment process.

Then, place the PDMS chip on the mobile substrate holder of the mask aligner. Subsequently, lift the chuck enough to visualize both the chip and the array of photoresist stripes located on the coverslip while staying behind the mechanical contact. Perform alignment and achieve mechanical contact between the chip and the coverslip by lifting the chuck until the surface of PDMS pillars touches the glass coverslip.

After that, lower the chuck. Remove the coverslip bonded to the chip from the mask holder. Then, place the device into a 35 millimeter Petri dish and transfer everything into the oven for five to ten minutes to increase the bonding strength.

Thirty minutes after bonding, place a ten microliter pipette tip filled with PLO solution at the inlet. Then, load the liquid until forming a drop on top. Repeat the same operation at the outlet and reservoirs.

Subsequently, add PBS in the Petri dish all around the chip. Seal the Petri dish with a paraffin film and incubate at room temperature with a minimum of five hours. Then, press a ten microliter tip cone lightly into each injection hole and suck up the excess liquid.

Next, replace PLO as well as the PBS surrounding the chip by the culture medium. Afterward, place the chip in the incubator regulated at 37 degrees Celsius and 5%carbon dioxide until neurons seed with a minimum of five hours incubation time. In this procedure, draw up the culture medium from the chip.

Collect two to three microliters of the freshly resuspended cell solution and inject it at the inlet. Immediately repeat the same operation at the outlet. Inject about the same volume of culture medium in each reservoir.

Quickly observe the chip under the microscope to check the density of the cells. The order of magnitude of the optimal cell density corresponds to about five to ten cells within the square surface delimited by four pillars. Repeat the procedure of injection of 0.5 to 1 microliter of cell suspension to reach the targeted cell density.

For imaging, replace the culture medium with the imaging medium. Then, place the chip under an epifluorescence microscope. Acquire images of cells from single to multiple successive images in case of time-lapse experiments.

Shown here are the representative observations of a calibration photoresist stripe and neurons. Higher magnification images and cross-sections of these objects are displayed on the right. The representative neurites and a three micrometer high central chamber are shown here.

The volume of neurite slice can be computed as a function of neurite width. And here are the representative growth cones and a three micrometer high central chamber. Their substructures emerge clearly from an optical background noise of about 20 nanometers.

Time-lapse observations of life act neurons seeded into a 12 micrometer high chamber to follow the cycle of shrinking and reactivation of the growth cone within a time scale of a few tens of minutes. While attempting this procedure, it's important to have an access to a deep ion etching equipment to fabricate the silicon mud. After watching this video, you should have a good understanding of how to align a structured glass coverslip with a PDMS chip.

Once mastered, this technique can be reproducibly applied to the quantification of neurology in 3d. This technique paved the way for researchers in the field of pharmacology to explore the effect of various drugs with actual caliber all in closed cone shape. Don't forget that working with primary neurons require a lot of care to avoid contamination and shatters.

Be always gentle and quick when changing media in order to limit the time they spend outside incubator.

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Volume Imaging

Fluorescence Exclusion