Imaging chemical gradient

1. Shade correction (correction for vignetting)
In microscopy, vignetting is a gradual darkening at the outside edges of the image. It can be caused by poorly aligned optical system or out of placed condenser.
In brightfield imaging, Koehler illumination correction can produce uniform illumination. But in epifluorescence imaging, the excitation light and emission light passes through the same objective. The optical components in the microscope and the relay lens also contribute to the possible causes of vignetting. 
One can try to analyse all the possible causes and still not be able to resolve the vignetting completely.
Through image processing, we can try to correct for this by setting a correction image or through post processing.

It is also very important to correct for vignetting in application of stitching after tile scanning.
Many confocal microscopy applications, such as Zeiss, resolve this problem by setting at higher overlap % and increase magnification to use the central portion of each image where the intensity is stable.

Obtaining a uniform illumination image is important to analyse chemical gradient distribution based on fluorescence intensity.

2. tiling scan on Nikon Ti-E microscope

3. Measuring the diffusion coefficient of a protein

4. Measuring the electrophoretic mobility of a protein

5. Measuring the charge of a protein
Prof. Tom Laue U new hampshire.
protein charge is important to colloidal stability.
only charge contributes to activation energy to aggregation

Debye Hueckel charge zDHH to Zeffective 

6. Plotting surface plot of chemical gradient