Week 2: Trial, Error, and Standardization

If Week 1 was the “mise en place,” Week 2 was the test cook. This week, I moved from prepping materials to actually running the protocol. In science, you can’t just run an experiment once and hope for the best. You have to prove that your method is repeatable before you even touch your test variables. On top of that, my results also have to be repeatable.

Locking in the SOP:
The biggest milestone this week was finalizing the Standard Operating Procedure (SOP) for the thin-film hydration method. While the concept is simple, the actual execution is quite delicate. It involves drying the lipids, adding water, and shaking it up to perfection.

I spent the early part of the week conducting trial runs using just DI water and dPBS (Dulbecco’s Phosphate-Buffered Saline) to work out the kinks and resolve any issues in my procedure. One immediate challenge was the vacuum desiccation step. I noticed that if the vacuum pressure was applied too quickly, the solvent bubbled aggressively rather than evaporating smoothly, creating an uneven lipid film and a non-uniform spread lipid layer. By adjusting the pressure ramp-up time, I managed to create a uniformly smooth film on the tracing paper to essentially ensure that there is consistent hydration later on.

Microscopy:

I also finished optimizing the fluorescence microscope setup that I had started working on last week. It is tempting to adjust the brightness for every picture to make it look “good,” but that ruins the data and doesn’t allow for consistency amongst the samples. To ensure scientific validity, I locked in the exposure time and gain settings. I tested this on my trial dPBS batch and confirmed that I can clearly distinguish the Rhodamine-labeled vesicles from the background noise without over-saturating the image.

I have successfully created MLVs in my control groups (Water and dPBS), which is a huge relief since it means the chemistry is functional. I executed a final “dry run” of my image processing workflow. I took the images from the dPBS trial, stitched them into a tile scan, and ran them through my ImageJ macro to ensure the counting algorithm picks up the vesicles correctly. Since this is now verified, I’ll start working on my experimental variables. Next week, I will start the actual data collection with Sodium Chloride and Calcium Chloride.

Thanks for following along!

– Samahith