Computed tomography-based spectral imaging for fluorescence microscopy.

The computed tomography imaging spectrometer (CTIS) is a non-scanning instrument capable of simultaneously acquiring full spectral information (450-750 nm) from every position element within its field of view (75 microm x 75 microm). The current spatial and spectral sampling intervals of the spectrometer are 1.0 microm and 10 nm, respectively. This level of resolution is adequate to resolve signal responses from multiple fluorescence probes located within individual cells or different locations within the same cell. Spectral imaging results are presented from the CTIS combined with a commercial inverted fluorescence microscope. Results demonstrate the capability of the CTIS to monitor the spatiotemporal evolution of pH in rat insulinoma cells loaded with SNARF-1. The ability to analyze full spectral information for two-dimensional (x, y) images allows precise evaluation of heterogeneous physiological responses within cell populations. Due to low signal levels, integration times up to 2 s were required. However, reasonable modifications to the instrument design will provide higher system transmission efficiency with increased temporal and spatial resolution. Specifically, a custom optical design including the use of a larger format detector array is under development for a second-generation system.

Midwave-infrared snapshot imaging spectrometer.

We report results from a demonstration of a midwave-infrared, nonscanning, high-speed imaging spectrometer capable of simultaneously recording spatial and spectral data from a rapidly varying target scene. We demonstrated high-speed spectral imaging by collecting spectral and spatial snapshots of blackbody targets and combustion products. The instrument is based on computed tomography concepts and operates in a midwave-infrared band of 3.0-5.0 mum. We record raw images at a frame rate of 60 frames/s, using a 512 x 512 InSb focal-plane array. Reconstructed object cube estimates were sampled at 46 x 46 x 21 (x, y, lambda) elements, or 0.1-mum spectral sampling. Reconstructions of several objects are presented.

High-speed spectral imager for imaging transient fluorescence phenomena.

We describe fluorescence spectral imaging results with the microscope computed-tomography imaging spectrometer (muCTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, muCTIS can be used to image dynamic phenomena. The results presented consist of proof-of-concept imaging results with static targets composed of 6-mum fluorescing microspheres. Image data were collected with integration times of 16 ms, comparable with video-frame-rate integration times. Conversion of raw data acquired by the muCTIS to spatial and spectral data requires postprocessing. The emission spectra were sampled at 10-nm intervals between 420 and 710 nm. The smallest spatial sampling interval presented is 1.7 mum.

Demonstration of a high-speed nonscanning imaging spectrometer.

We report results from a field demonstration of a nonscanning high-speed imaging spectrometer [computed-tomography imaging spectrometer (CTIS)] capable of simultaneously recording spatial and spectral information about a rapidly changing scene. High-speed spectral imaging was demonstrated by collection of spectral and spatial snapshots of a missile in flight. This instrument is based on computed-tomography concepts and operates in the visible spectrum (430-710nm). Raw image data were recorded at video frame rate (30frames / s) and an integration time of 2ms. An iterative reconstruction of the spatial and spectral scene information from each raw image took 10s. We present representative missile spectral signatures from the missile firing. The accuracy of the high-speed spectrometer is demonstrated by comparison of extended-source static-scene spectra acquired by a nonimaging reference spectrometer with spectra acquired by use of CTIS imaging of the same static scenes.

Demonstration of a computed-tomography imaging spectrometer using a computer-generated hologram disperser.

We have constructed a computed-tomography imaging spectrometer that uses a phase-only computer-generated hologram (CGH) array illuminator as the disperser. This imaging spectrometer collects multiplexed spatial and spectral data simultaneously and can be used for flash spectral imaging. The CGH disperser has been designed to maintain nearly equal spectral diffraction efficiency among a 5 x 5 array of diffraction orders and to minimize diffraction efficiency into higher orders. Reconstruction of the (x, y, lambda) image cube from the raw, two-dimensional data is achieved by computed-tomography techniques. The reconstructed image and spectral-signature data compare favorably with measurements by other spectrometric methods.