Time Correlated Single Photon Counting
ChiSquare fluorescence lifetime systems are built on the core technology of time correlated single photon counting (TCSPC). It is a technology that gives rise to dynamic information that is in the picoseconds to nanoseconds time scale. With TCSPC, it is possible to derive information about excited state decay dynamics, fluorescence anisotropy decay, and photon correlation. The key characteristic of the fluorescence signal that a ChiSquare instrument focuses on is the excited state lifetime. When it is combined with Förster resonance energy transfer (FRET), it provides quantitative information on the molecular spatial scale of 2 – 8 nanometers.
Basic Principle
The general principle is to derive excited state lifetime from the histogram of emitted photons, and the histogram is built up from numerous excitation-emission cycles over an experimental time period. To achieve TCSPC, a femtosecond- or picosecond-pulse laser and a ultra-fast detector are necessary components of the hardware setup. Each photon that is detected has a time-tag associated with it. This time-tag is measured as illustrated in the diagram below. This histogram represents the photon emission probability of a particular molecule in a certain molecular state in a certain microenvironment. The fluorescence lifetime derived from this histogram reflects the molecular state. Changes in the molecular state, such as a change to the molecular structure due to binding to or dissociation from a ligand or due to changes to pH/redox state, can lead to a detectable change in the fluorescence lifetime.
