ALine Modular Device: in situ Small Molecule Permeability Optimization (Confocal Microscope)

All new fluorescent small molecules need to optimized for concentration, exposure time, and laser power. Concentration can usually start around the concentrations used for traditional permeability experiments. However, sometimes they will need to be increased for improved signal on a confocal microscope.

Optimize using non-porous chips. We try to keep the laser power and exposure times low to start. Exposure time is usually 500 ms, laser power depends on molecule. Start at 5% and go from there. Ultimately, you want to end up with three things:

1. A good dynamic range (at least 5000 is what we usually work with, but it can be bigger)
2. You want to make sure that you are in the linear range of concentration for the molecule on your scope
3. There is no photobleaching of the molecule with your settings over the course of the assay.

For concentration, start around 500 µg/ml and go down in serial dilutions to 75 ug/ml to start (or find what is used in the literature and generate a range based on that). For imaging these, fill the well of the non-porous device first with the lowest concentration and snap a “background” picture. Then fill the bottom channel and take a “max concentration” picture. Repeat with the next highest concentration and so on. Do a couple devices for each concentration. Check the fluorescence intensity and make sure it increases linearly. Subtract the “background” from “max” to determine your dynamic range. If you immediately find you have too low a signal, increase the laser power. If you still struggle, try upping exposure time.

Sometimes it takes a couple sessions to figure out optimal conditions. It is a little challenging because all parameters play together, and it is hard to decide what to adjust first. There are probably several combinations that work so find one you like.

Once you have a good combination of exposure time, concentration, and laser power, you need to double check photobleaching. Fill the channel of a non-porous chip with 10-20% of max concentration of the dye (simulating what will go across a cell layer) and run the full permeability assay with your optimized settings. Make sure you do not lose signal over the course of the assay. I put blank media in the well for this, you can probably just leave it empty.

Other confocal microscopes might need other settings to be optimized. If possible, work with a microscopy expert to ensure the set up is valid.