Multiphoton excited fluorescence spectroscopy of biomolecular systems

Molecules possess discrete energy levels called quantum states. Unlike atoms, which have simpler energy levels, molecules possess additional rotational and vibrational energy levels. Each energy level is separated by an energy gap, with the gaps between adjacent electronic, vibrational, and rotational levels varying significantly. The three types of energy levels in a diatomic molecule are shown in Figure 1.

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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 developed.

Photoluminescence is a process where a molecule absorbs light energy and re-emits it in the form of light. This phenomenon occurs when a substance absorbs photons, promoting its electrons to higher energy level excited states, followed by a relaxation process in which the electrons return to their original ground state energy levels and emit light. Photoluminescence is widely observed in various materials, including semiconductors, and organic and inorganic compounds.
A pair of electrons in a...

Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with...