Computed tomography-based spectral imaging for fluorescence microscopy

The computed tomography imaging spectrometer (CTIS) is a non-scanning instr ument capable of simultaneously acquiring full spectral information (450 -7 50 nm) from every position element within its field of view (75 mum x 75 mu m) The current spatial and spectral sampling intervals of the spectrometer are 1.0 mum and 10 nm, respectively. This level of resolution is adequate t o resolve signal responses from multiple fluorescence probes located within individual cells or different locations within the same cell. Spectral ima ging results are presented from the CTIS combined with a commercial inverte d fluorescence microscope. Results demonstrate the capability of the CTIS t o monitor the spatiotemporal evolution of pH in rat insulinoma cells loaded with SNARF-1. The ability to analyze full spectral information for two-dim ensional (x, y) images allows precise evaluation of heterogeneous physiolog ical responses within cell populations. Due to low signal levels, integrati on times up to 2 s were required. However, reasonable modifications to the instrument design will provide higher system transmission efficiency with i ncreased temporal and spatial resolution. Specifically, a custom optical de sign including the use of a larger format detector array is under developme nt for a second-generation system.