Modern single molecule fluorescence microscopy offers new, highly quantitative ways for studying the systems biology of cells while keeping the cells healthy and alive in their natural environment. In this context, a quantum optical technique, photon antibunching, has found a small niche in the continuously growing applications of single molecule techniques to characterize small molecular complexes. Here, we review some of the most recent applications of photon antibunching in biophotonics research, and we provide a guide for how to conduct photon antibunching experiments at the single molecule level by applying techniques borrowed from time-correlated single photon counting (TCSPC). We provide a number of new examples for applications of photon antibunching to the study of multichromophoric, molecules and small molecular complexes.

We demonstrate time-gated confocal imaging as a means to separate coherent anti-Stokes Raman scattering (CARS) microscopy data from multi-photon excited endogenous fluorescence in tissue. CARS is a quasi-instantaneous process and its signal decay time is only limited by the system's instrument response function (IRF). Signals due to two-photon-excited (TPE) tissue autofluorescence with excited state lifetimes on the nanosecond scale can be identified and separated from the CARS signal by employing time-gating techniques. We demonstrate this improved contrast on the example of CARS microscopy of intact roots of plant seedlings as well as on rat arterial tissue. (c) 2007 Optical Society of America.

We present a novel scheme to simultaneously detect coherent anti-Stokes Raman scattering ( CARS) microscopy signals in the forward ( F) and backward (epi-E) direction with a single avalanche photodiode (APD) detector using time-correlated single photon counting (TCSPC). By installing a mirror at a well-defined distance above the sample the forward-scattered F-CARS signal is reflected back into the microscope objective leading to spatial overlap of the F and E-CARS signals. Due to traveling an additional distance the F-CARS signal is time delayed relative to the E-CARS signal. TCSPC then allows for the two signals to be resolved in the time domain. This results in an efficient, simple, and compact method of CARS signal detection. We demonstrate this technique by analyzing forward and backward CARS signals obtained by imaging living adipocyte cells derived from human mesenchymal stem cells. (c) 2008 Optical Society of America.