Figure 2.

Single-molecule multiparameter photon stamping spectroscopy. (A) Experimental home-built four-channel single-molecule setup for measuring multidimensional conformational dynamics. Basically, it consists of an inverted confocal epi-illumination configured microscopy, a femtosecond pulse laser, four Si avalanche photodiode detectors, a time-correlated single photon counting module, and several optics. 532 nm green linearly polarized pulse laser is used to excite Cy3-Cy5 labeled T4 lysozyme. The emissions (yellow and red) from Cy3 and Cy5 are discriminated by dichroic mirrors. The polarization of the light emitted is further distinguished into parallel (∥) and vertical (⊥) components (relative to the polarization of the laser excitation) by two polarizing beam splitter cubes. (B) Single-molecule photon counting images of individual Cy3-Cy5 labeled T4 lysozymes. Dual-color (Cy3 and Cy5) and dual-polarization (∥ and ⊥) images are captured. DM: dichroic mirror; APD: avalanche photodiode; PBS: polarizing beam splitter cubes; LPF: long pass filter; WP: wave plate; L1/L2: lens; PC: personal computer.