Real-time, extended depth DIC microscopy

Ingeborg E. Beckers; Robert H. Cormack; Carol J. Cogswell

doi:10.1117/12.848972

24 February 2010 Real-time extended-depth DIC microscopy

Ingeborg E. Beckers, Robert H. Cormack, Carol J. Cogswell

Proceedings Volume 7570, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVII; 757013 (2010) https://doi.org/10.1117/12.848972
Event: SPIE BiOS, 2010, San Francisco, California, United States

Abstract

Real-time visualization of live-cell dynamic processes has been realized in differential interference contrast (DIC) microscopy, with an extended-depth-of-focus (EDF) increase of about one order of magnitude. In addition, the diffraction-limited lateral resolution of the microscope is preserved. Experimentally, a custom-designed waveplate inserted in the optical path of a microscope causes feature information, from within the entire 3D specimen volume, to be uniformly encoded into a single CCD image in a way that, after processing, defocus blur artifacts are removed. The result is that extended-depth feature information can be visualized at video rates during live-cell dynamics investigations because there is no longer the need to acquire multi-focus image stacks at each time point. Retrieving the encoded extended-depth information requires specialized digital image processing techniques. This work concentrates on digital filter design for the reconstruction of the waveplate-encoded images. As a measure of filter quality, the signal-to-noise ratio (SNR), the modulation transfer function and the least mean square values are evaluated. Obtaining a high SNR and a lateral resolution comparable to those in conventional single-focus-plane microscopy images at the same time is a challenging goal in EDF microscopy. Filters are created in the frequency domain on the basis of the measured waveplate-encoded point spread functions. Results show that it is possible to produce video-rate, extended-depth-offocus images that have low noise levels and diffraction-limited resolution. This is illustrated by movies of fluorescent beads and of cytoplasmic streaming in live stamen hair cells from the spiderwort plant, Tradescantia, using extendeddepth DIC microscopy.

Citation Download Citation

Ingeborg E. Beckers, Robert H. Cormack, and Carol J. Cogswell "Real-time extended-depth DIC microscopy", Proc. SPIE 7570, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVII, 757013 (24 February 2010); https://doi.org/10.1117/12.848972

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