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 in vitro 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.
P. L. T. M. Frederix, M. A. H. Asselbergs, W. G. J. H. M. van Sark, D. J. van den Heuvel, W. Hamelink, E. L. de Beer, and H. C. Gerritsen, "High Sensitivity Spectrograph for Use in Fluorescence Microscopy," Appl. Spectrosc. 55, 1005-1012 (2001)