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.
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