Construction and application of a single-molecule fluorescence microscope

In this paper, we describe the construction, optimization, and testing of an epifluorescence microscope that has single-molecule (SM) resolution and sensitivity. The microscope makes use of a novel new type of wide-area charge-coupled device (CCD) photodetector with on-chip multiplier gain. Sensitivity and spatial resolution of the instrument are demonstrated by imaging individual Rhodamine 6G (R6G) molecules and characterizing their basic photophysical behaviour under a variety of imaging conditions. A simple, general method for calibrating the photodetector (correlating CCD counts with incident photons) using a highly attenuated laser beam is presented, and the performance of the photodetector is compared with that of other detectors commonly used in SM fluorescence imaging applications. We also demonstrate the versatility of the microscope system by characterizing the SM photophysical behaviour of several other fluorescent molecules, including bodipy-FL and the enhanced green fluorescent protein (EGFP).