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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Microsc Res Tech. 2013 Jan 7;76(3):282–289. doi: 10.1002/jemt.22165

Figure 1.

Figure 1

Schematic of the widefield FD-FLIM system that permits multi-harmonic FLIM measurements. A) A 10ps, 20MHz supercontinuum laser (SC390, Fianium Inc, USA) was coupled into a commercial Olympus TRIF microscope (Olympus, USA) via a fiber. Bandpass filters were used to select proper excitation wavelengths. A heat blocker (KG-5, Edmund Optics, USA) was added into the excitation path to reduce the heat. B) The excitation beam was reflected to the samples by a Z488/594RPC TIRF dichroic mirror (Chroma, USA). C) Experiments were done with a 60x (NA=1.45) oil-immersion TIRF objective (Olympus, USA). The fluorescence signal was collected by the same objective. D) The bottom prism reflected this signal to a LI2CAM MD Intensified CCD (Lambert Instruments, Netherlands). The ICCD was controlled by a computer for modulation of intensifier and data acquisition. E) The DDS (409B, Novatech) was synchronized with the white laser and controlled by the same computer to generate the sinusoidal modulation signal. A FPGA based pulse generator was developed for the pulse modulation approach.