High sensitivity spectrograph for use in fluorescence microscopy

In this paper a versatile, high sensitivity spectrograph is presented for u se in fluorescence microscopy. The high sensitivity is achieved by using a prism for the dispersion in combination with a state-of-the-art back illumi nated charge-coupled device (CCD) camera. The spectrograph, including the C CD camera, has a detection efficiency of 0.77 +/- 0.05 at 633 nm. Full emis sion 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 s pectrograph is fiber-coupled to a commercial confocal laser scanning micros cope. In the first example, Forster resonance energy transfer imaging exper iments were carried out on double-labeled actin filaments in the in vitro m otility assay. A 160 X 160 point image was recorded in 1.5 min at 3 ms dwel l time per image point. In the second application, a time-resolved study of single quantum dots is presented at 5.2 ms time resolution.