TITLE: Flow Imaging Microscopy of Elastin-like Polymers
AUTHORS: Eva Rose Balog, Laura Marvin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the methods used by the Balog lab at the University of New England to evaluate elastin-like polymer microparticles using the FlowCam. </div></div>

[STEPS]
?. [Sample Preparation]
Prepare a bucket of ice for keeping samples, solvents, and focus beads cold.
?. [Instrument Setup]
Turn on the computer, then the FlowCam. Open the Visual Spreadsheet software.
?. [Flow Cell Cleaning]
Sonicate the flow cell in DI water for 180 s.
?. [Focusing]
Click the Setup|Focus icon to open the Setup and Focus Mode viewer window. This shows you what the camera sees. Inspect the area for contamination, bubbles, or anything else that might be problematic. Return to the cleaning procedure in step 5 if needed.
Imperfections in the glass may or may not be an issue. Wait to see if they get mistakenly captured as particles.
?. [Running Your Sample]
Add the solvent your sample is resuspended in (not the sample!) to the pipette tip. Go to Setup > Pump > Prime. Aspirate 0.5 mL.
?. [Running Your Sample]
Click the Context icon to choose your context settings. Set the minimum ESD diameter to 2 µm. An example context file is attached. It can be opened and viewed with a text editor.
?. [Sample Preparation]
Weigh out the desired mass of freeze-dried ELP required, typically 0.8-1.5 mg per experiment.
?. [Sample Preparation]
Resuspend ELP in the previously calculated volume of solvent. Help the ELP go into solution by alternately chilling on ice and pipetting up and down until solid material is no longer visible. Sample may remain on ice during cleaning and focusing procedures but should warm to room temperature at least 10 min before it is run.
Example calculations: Desired concentration: ~1.4 mg/mL Desired volume: at least 1 mL Added 1.12 mL of sterile-filtered, ice cold H2O to 1.6 mg of freeze-dried ELP.
?. [Instrument Setup]
Place a 1 mL pipette tip on the top of the flow cell tubing. Make a mark about halfway up the pipette tip using a Sharpie.
?. [Instrument Setup]
In Visual Spreadsheet, go to Setup > Pump > Prime. Aspirate 0.5 mL of water.
?. [Instrument Setup]
Repeat step 6 two more times.
It is important to aspirate the flow cell at the beginning of each experiment to determine whether there are flow issues. Visually confirm that the liquid level is going down during aspiration. Check for tiny bubbles in the tubing downstream of the flow cell or air pockets in the syringe. If water is not flowing, the flow cell may be broken or there could be something blocking the flow. Return to step 5 for additional cleaning procedures.
?. [Instrument Setup]
Add DI water to the pipette tip until the liquid level is above the mark.
Unless the flow cell is being removed for cleaning or storage, add liquid as necessary to keep the liquid level above this mark during operation!
?. [Instrument Setup]
Within the Prime window, click Evacuate Syringe.
?. [Instrument Setup]
Install the 20X objective, 20X collimator, and the 0.5 mL syringe according to instructions in the FlowCam manual.
?. [Instrument Setup]
Install the flow cell according to instructions in the FlowCam manual.
?. [Flow Cell Cleaning - Additional Procedures (optional)]
If the flow cell is not clean, fill the flow cell with isopropyl alcohol and sonicate again. Repeat this step as many times as necessary until the flow cell is clean.
In desperate times, the flow cell can be cleaned with a detergent solution. However, the presence of detergents may affect protein behavior, so care should be taken to rinse flow cells thoroughly after washing with detergents.
?. [Focusing]
When the flow cell is clean, add water and aspirate again to confirm that the flow is acceptable.
?. [Focusing]
Return to the Setup and Focus Mode window. Add 100 µL of focus beads. Go to the Pump Control window and set the flow rate to 0.030 mL/min. Visually confirm that beads are flowing through the flow cell.
?. [Focusing]
Manually focus on the beads by very gently adjusting the position of the flow cell.
?. [Focusing]
In the Setup and Focus Mode window, go to Tools > Autofocus to open the Auto Focus Assistant. Follow the steps provided. A pop-up will appear telling you whether the Autofocus is successful.
If the Autofocus is unsuccessful, add more beads and try again.
?. [Running Your Sample]
Click the AutoImage icon and follow the instructions provided. Select "Stop when user terminates experiment or fluid is aspirated." Assign the run a name using the following format or similar: date - solvent - run number
?. [Running Your Sample]
Click "Save" to automatically start the run.
The first run should be considered a "practice run" to identify potential problems. In addition to flow issues, this is when you can determine whether imperfections in the glass are being detected as particles. Also, look at the Capture Y vs Capture X scatter plot to confirm that particles are being captured across the entire area. Inspect the particle edge trace and binary image overlays to determine whether particles are being captured properly, and adjust particle capture and segmentation threshold context settings accordingly.
?. [Running Your Sample]
After the run is complete, aspirate again, adding solvent if necessary.
?. [Running Your Sample]
Add 100 µL of your sample to the pipette tip.
?. [Running Your Sample]
Add another 100 µL of sample and repeat steps 22-24 as many times as desired.
?. [Running Your Sample]
After you are finished running samples, add DI water and aspirate again.
?. [Running Your Sample]
Remove the flow cell from the flow holder and repeat step 4 (sonication bath).
Make sure to return the flow cell to the box labeled "proteins only."
?. [Flow Cell Cleaning]
Visually inspect the 50 µm flow cell for damage. If there are no apparent cracks, proceed to step 5.
?. [Initial Post-Processing - Bead Removal]
In VisualSpreadsheet, open the .lst file for the experiment. Click the "Open View" button to open the View window.
?. [Initial Post-Processing - Bead Removal]
Select three images of beads.
?. [Initial Post-Processing - Bead Removal]
Go to Filter > Like selected particles (Statistical). This will re-sort all images by their filter score based on similarity to the selected images.
?. [Initial Post-Processing - Bead Removal]
Manually select all images of beads. Go to Edit > Delete Images Selected.
?. [Initial Post-Processing - Bead Removal]
Go to File > Save As List... to save as a new .lst file.