TITLE: Scanning electron microscopy (SEM) protocol for imaging living materials
AUTHORS: Hans Priks, Tobias Butelmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Scanning electron microscopy (SEM) can be used to image cells and colonies immobilized inside hydrogels after supercritical carbon dioxide (CO</span><span style = "vertical-align:sub;">2</span><span>) extraction. Supercritical CO</span><span style = "vertical-align:sub;">2</span><span> extraction can also be used on suspension cells after filtering the sample onto a 0.2 μM filter attached into the extractors carriers. This protocol gives an overview on how different techniques can be used to characterize triblock copolymer hydrogels.</span></div></div>

[STEPS]
?. [Sample fixation]
Prepare fixation solution ( formaldehyde in  phosphate buffer)
?. [Sample fixation]
Submerge sample into the fixation solution and incubate at  for
37 Room temperature
?. [Sample fixation]
Replace fixation solution and incubate at for
37 Room temperature
?. [Sample Dehydration]
Prepare ethanol (EtOH) dilutions in milli-Q water as indicated below.
?. [Sample Dehydration]
Samples are dehydrated at  in an ascending EtOH series (40 – 90 %, 10 % steps; 96 %, 99.5 %).Submerge sample into EtOH solution, let it incubate (minimum  per step), change the EtOH solution.EtOH steps: EtOH EtOH EtOH EtOH EtOH EtOH EtOH EtOH () (for storage)
37 Room temperature
?. [Supercritical CO2 extraction]
Cool the critical point dryer (E3100, Quorum Technologies) to  with a thermostat (Proline RP 1845, LAUDA) using thermostat external temperature probe.
15 °C
?. [Supercritical CO2 extraction]
Open the critical point dryer and mount the samples. Close the critical point dryer according to producers instructions.
?. [Supercritical CO2 extraction]
Slightly open the outlet and purge the chamber for(bubbling inside the external EtOH bottle should not be too intensive).After purging close the outlet first then the inlet (to avoid pressure drop inside the chamber).
?. [Supercritical CO2 extraction]
Connect the critical point dryer outlet to a bottle containing EtOH (half full) under fume hood (it is used to capture residues during extraction and to estimate the gas realese speed).
?. [Supercritical CO2 extraction]
The chamber should be purged with fresh CO2  6 – 8 times in 30 – 60 min intervals (let the EtOH diffuse out of the structure and purge it out of the chamber, use shorter intervals at the beginning of this process).open the CO2 inlet, then slightly open the outlet  purge for  close the outlet, then close the CO2 inletrepeat 6-8 times in 30 – 60 min intervalspurge for close the outlet, then close the CO2 inletrepeat 6-8 times in 30 – 60 min intervals
?. [Supercritical CO2 extraction]
Increase the thermostat temperature to  (inlet and outlet of the chamber should be closed at this point).  Do not leave the critical point dryer unattended while the temperature is rising as the pressure can exceed the safety limit of the chamber. Control the internal pressure so it does not exceed 110 bar by opening the chamber outlet (should be done slowly as too fast gas release can cool the reactor and turn supercritical state back to liquid state). Adjust the outlet so that the pressure gauge stays stable around 105 bar as the temperature is rising Leave the outlet open as it is, when  is achieved (do not open the outlet more, as the faster gas release can cool the reactor).
37 °C
37 °C
?. [Supercritical CO2 extraction]
Let the chamber pressure drop until it reaches 60 bar (at this point CO2 has turned from supercritical state into gas state and opening the outlet will not result in temperature drop) .
?. [Supercritical CO2 extraction]
Open CO2 inlet and fill the chamber with liquid CO2.
?. [Supercritical CO2 extraction]
Leave the outlet open until the chamber is ready to be opened ().  As the pressure drops so does the bubbling.Adjust the outlet so that there is always slight bubbling (do not over do it as it can result in pore formation figure 1B)
?. [Supercritical CO2 extraction]
Before opening the chamber remove outlet tube from EtOH bottle that is situated under the hood (to avoid sucking EtOH into the chamber while opening it).Remove samples from the chamber and store in a sealable container (ependorf, glass vial, falcon tube)
?. [Supercritical CO2 extraction]
Remove the outlet from the external EtOH bottle as it can splatter under the hood at this step. Open outlet so the remaining pressure is released within 3-5 min (the generated pores depends on the speed of gas release)
?. [Supercritical CO2 extraction]
Before opening the chamber remove outlet tube from EtOH bottle that is situated under the hood (to avoid sucking EtOH into the chamber while opening it).Remove samples from the chamber and store in a sealable container (ependorf, glass vial)
?. [Sample cutting and mounting]
Attache conductive double sided carbon tabs/tape on aluminum SEM pin stubs and then lable them with a sharpie marker.
?. [Sample cutting and mounting]
?. [Sample cutting and mounting]
Fast incision (Figure 3: A, B) -  for acquiring artifact-free cross-sectionsImmerse the sample with forceps and scalpel into liquid N2 for  and instantly cut with fast incision (N2 cooled scalpels can break during sample cutting. Wear protective eyewear!).
?. [Sample cutting and mounting]
Slow incision (Figure 3: C, D) - for acquiring information of colony-material interactions, colony size and shapeImmerse the sample with forceps and scalpel in liquid N2 for  and cut after  at room temperature with slow incision.
?. [Sample cutting and mounting]
Using forceps, pick up the cut sample and gently press it onto the two-sided carbon tape.
?. [Sputter Coating]
Coat the sample with a  gold layer using a high vacuum sputter coater (EM ACE600, Leica Microsystems).
?. [SEM imaging]
Gold-coated samples were imaged with a tabletop scanning electron microscope (TM3000, Hitachi). The imaging was done under a high vacuum and 15 kV accelerating voltage.
?. [SEM imaging]
Gold-coated samples were imaged with a tabletop scanning electron microscope (TM3000, Hitachi). The imaging was done under a high vacuum and 15 kV accelerating voltage.
?. [Sputter Coating]
Coat the sample with a  gold layer using a high vacuum sputter coater (EM ACE600, Leica Microsystems).
?. [Sample cutting and mounting]
Using forceps, pick up the cut sample and gently press it onto the two-sided carbon tape.
?. [Sample cutting and mounting]
Slow incision (Figure 3: C, D) - for acquiring information of colony-material interactions, colony size and shapeImmerse the sample with forceps and scalpel in liquid N2 for  and cut after  at room temperature with slow incision.
?. [Sample cutting and mounting]
Fast incision (Figure 3: A, B) -  for acquiring artifact-free cross-sectionsImmerse the sample with forceps and scalpel into liquid N2 for  and instantly cut with fast incision (N2 cooled scalpels can break during sample cutting. Wear protective eyewear!).
?. [Sample cutting and mounting]
?. [Sample cutting and mounting]
Attache conductive double sided carbon tabs/tape on aluminum SEM pin stubs and then lable them with a sharpie marker.