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Free keywords:
Confocal microscopy; Confocal spectroscopy; Hadamard; Imaging spectroscopy; Fluorescence microscopy; Spatial light modulator
Abstract:
We report the use of a programmable array microscope (PAM) for the acquisition of spectrally resolved and high throughput optical sections. The microscope is based on the use of a spatial light modulator for defining patterns of illumination and/or detection. To obtain optically sectioned spectral images, the entrance slit of an imaging spectrograph and a line illumination pattern defined with a spatial light modulator are placed in conjugate optical positions. Compared to wide-field illumination, optical sectioning led to greater than 3x improvement in the rejection of out-of-focus fluorescence emission and nearly 6x greater peak-to-background ratios in biological specimens, yielding better contrast and spectral characterization. These effects resulted from a reduction in the artifacts arising from spectral contributions of structures outside the region of interest. We used the programmable illumination capability of the spectroscopic system to explore a variety of excitation/detection patterns for increasing the throughput of optical sectioning microscopes. A Sylvester type Hadamard construction was particularly efficient, performing optical sectioning while maintaining a 50% optical throughput. These results demonstrate the feasibility of full-field highly multiplexed confocal spectral imaging.
