Nucleation

Evolution of Sub-Critical nuclei to Crystals of Protein

The knowledge of evolution of sub-critical nucleus is important in the process of understanding the classical nucleation and two – step nucleation processes. By examining the events where protein molecules come close to each other to form the critical nucleus, the size (or the number of molecules) of the critical nucleus can be determined using a supersaturated protein solution. Most of the proteins being sub-resolution particles the above objective is to be achieved by studying the fluctuations of the intensity of fluorescently labeled proteins using Widefield Fluorescence Microscopy.

Experiments

A model system of fluorescently labeled beads of different diameters inside emulsion drops (approximately of 5pl volume) is to be used before experimenting with proteins. The initial experiments will be carried out using the 300um diameter fluorescently labeled beads which are larger than the width of the point spread function (PSF) which is 200um for a high numerical aperture lens to image through time for couple of different bead concentrations. This would allow the particle tracking in real space and hence determining the diffusion coefficient of the beads. Experiments are to be continued for several different smaller diameters to resemble the real protein sizes (20-30um) and eventually carry out for fluorescently labeled proteins.

The challenge in the project is to fabricate a sample holder where the emulsion drops can be confined which would necessarily provide a flat area with the thickness approximately the height of the PSF in the microscope optical axis (0.5um) for the imaging purposes. In the process of fabricating such, there are few approaches to be tried out.

1. Made a wedge using a line of spacer beads in one corner (0.5um diameter) of a microscope slide, in between the two surfaces, one being the microscope slide and the other being the cover slide where the drops are confined in....