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Controlling the depth-of-field in 3D spatial-frequency-projection fluorescence microscopy with holographic refocusing (Conference Presentation)
Author(s): Jeffrey J. Field; Randy A. Bartels
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Paper Abstract

While fluorescence microscopy has revolutionized a broad range of biological studies, one of several challenges that remain is the need to increase image acquisition rates to image in dynamic specimens. Spatial-frequency-projection techniques, such as CHIRPT and SPIFI, utilize spatiotemporally structured illumination patterns to enable rapid acquisition of multidimensional images with a single-pixel detector. CHIRPT in particular shows promise for enhancing image acquisition rates because it encodes the spatial phase difference between two interfering illumination beams into temporal modulations of the fluorescent light emitted from the specimen. Consequently, the complex-valued, 1D image measured with CHIRPT can be digitally propagated to recover a 2D image of the fluorophore distribution in the specimen. Moreover, the depth-of-field (DOF) in CHIRPT with planar illumination approaches 100x the conventional limit and thus allows for large volumes of the specimen to be imaged simultaneously. Unfortunately, all configurations of CHIRPT reported to date require the use of focused light sheets to form 2D and 3D images – thereby restricting the effective DOF provided by CHIRPT to the conventional limit. In this work, we show that the addition of a confocal slit to the CHIRPT microscope allows one to control the effective DOF with linearly-excited fluorescence. We present experimental data and a complete optical theory that describes the experimental results. Confocal CHIRPT may enable rapid imaging by dramatically reducing the number of axial translations required to form a complete 3D image, particularly when coupled with remote focusing of the confocal filter.

Paper Details

Date Published: 4 March 2019
Proc. SPIE 10883, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, 1088316 (4 March 2019); doi: 10.1117/12.2509842
Show Author Affiliations
Jeffrey J. Field, Colorado State Univ. (United States)
Randy A. Bartels, Colorado State Univ. (United States)

Published in SPIE Proceedings Vol. 10883:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI
Thomas G. Brown; Tony Wilson, Editor(s)

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