Video-rate spectral imaging system for fluorescence microscopy

We describe fluorescence spectral-imaging results with the computed-tomography imaging spectrometer (CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, the CTIS can be used to image dynamic phenomena involving multiple, spectrally overlapping fluorescence probes. This system is also optimal for simultaneously monitoring changes in spectral characteristics of multiple probes from different locations within the same sample. This advantage will provide additional information about the physiological changes in function from populations of cells which respond in a heterogeneous manner. The results presented in this paper consist of proof-of-concept imaging results from the CTIS in combination with two different systems of fore-optics. In the first configuration, raw image data were collected using the CTIS coupled to an inverted fluorescence microscope. The second configuration combined the CTIS with a confocal microscope equipped with a fiber-optic imaging bundle, previously for in vivo imaging. Image data were collected at frame rates of 15 frames per second and emission spectra were sampled at 10-nm intervals with a minimum of 29 spectral bands. The smallest spatial sampling interval presented in this paper is 0.7 μm.