In this paper a versatile, high sensitivity spectrograph is presented for use in fluorescence microscopy. The high sensitivity is achieved by using a prism for the dispersion in combination with a state-of-the-art back illuminated charge-coupled device (CCD) camera. The spectrograph, including the CCD camera, has a detection efficiency of 0.77 ± 0.05 at 633 nm. Full emission spectra with a 1-5 nm spectral resolution can be recorded at a maximum rate of 800 spectra per second. Two applications are shown, in which the spectrograph is fiber-coupled to a commercial confocal laser scanning microscope. In the first example, Förster resonance energy transfer imaging experiments were carried out on double-labeled actin filaments in the <i>in vitro</i> motility assay. A 160 × 160 point image was recorded in 1.5 min at 3 ms dwell time per image point. In the second application, a time-resolved study of single quantum dots is presented at 5.2 ms time resolution.
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