Advanced Microscopic Methods to Measure Dynamic Patterns of Intracellular and Intranuclear Proteins in Living Mammalian Cells
The appropriate functioning of living cells depends on a variety of dynamic processes that necessitate delicate motion, transportation, association, and disassociation in time and space. For proteins, it is important to know:
- Is there a mobile fraction and a fixed (bound) fraction?
- What is the ratio between these two sub-populations?
- What is the concentration of each one
- Other relevant factors such as the number of binding sites.
In addition, both for proteins and other structures (such as DNA loci), it is important to know different dynamic patterns such as the type of their dynamics – is it a directed motion, is it normal diffusion or restricted diffusion. It is important to know the length scale of the dynamics, the diffusion coefficients and more. These parameters serves as a tool for exploring biochemical processes. Although there are methods such as fluorescent recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS), these methods can only give partial information from the list given above.
We developed a method that combines intensity measurement with time-resolved measurement. It uses a confocal microscope setup and require labelling a tested protein (or structure) with a fluorescent dye (such as GFP) in live cells. Then, the sample (live cells) is placed under the microscope and using the confocal setup, the intensity is measured at a given point in the sample, normally for about 20-60 seconds. During the measurement, the life time of the emitted photons are also measured. Then, the data is processed by combining few different methods. It gives many answers related to “the problem” described above. This includes
- The ratio of bound and free sub-populations of the protein.
- The diffusion coefficient of the mobile fraction.
- The concentration of the mobile fraction and the bound fraction.
- The parameters are confirmed by two different methods, both from the fluorescence intensity as well as from the fluorescence lifetime.