Characterizing proteins in their cellular environment: Examples of recent advances in quantitative fluorescence...

Newton's third law states that every action has an equal and opposite reaction. Consider a swimmer pushing off the side of a pool. They push against the wall of the pool with their feet and accelerate in the direction opposite to that of their push. This occurs because the wall exerts an equal and opposite force on the swimmer. Here, the forces do not cancel out each other as they are acting on different systems. In this case, there are two systems: the swimmer and the wall. If we select...

Solving problems that involve forces is easy using free-body diagrams. A free-body diagram is a sketch showing all the external forces that are acting on an object or system. The object or system is represented by a single isolated point (or free body). Only those forces acting on it that originate outside of the object or system—the external forces—are shown. The forces are represented by vectors extending outward from the free body. Imagine a person sitting on a chair. Here, the...

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...

Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.

Metal ions can be separated from one another by complexation with organic ligands–the chelating agent– to form uncharged chelates. Here, the chelating agent must contain hydrophobic groups and behave as a weak acid, losing a proton to bind with the metal. Since most organic ligands used in this process are insoluble or undergo oxidation in the aqueous phase, the chelating agent is initially added to the organic phase and extracted into the aqueous phase. The metal-ligand complex is...