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 excitation and/or detection of fluorescence. For obtaining 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 3 X improvement in the rejection of outof-focus fluorescence emission and nearly 6 X 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.

PDF Article

Cited By

You do not have subscription access to this journal. Cited by links are available to subscribers only. You may subscribe either as an OSA member, or as an authorized user of your institution.

Contact your librarian or system administrator
Login to access OSA Member Subscription