Holographic reconstruction algorithms based on wave propagation require the object’s Z-plane location. The location is determined manually by selecting an image from a set of reconstructed images over a range of Z-planes. We evaluate five autofocus metrics; the standard deviation of Laplacian and Sobel edge detectors, sum of darkest 2% of pixels, sum of the difference of adjacent reconstructed images (DAMP method), and product of the variance of two orthogonal Gabor filters. The metrics were tested on ten classes of plankton collected from field deployments of a submersible digital holographic imaging system (HOLOCAM). Our results indicate that Gabor filters provide the best focus metric performance, correctly predicting focus distance with +/- 100 um for 78% of the images (n=687). The performance of each metric is significantly dependent on the plankton class, from 46% for the round Coscinodiscus class to 100% for the Thalassionema nitzschoid class using the Gabor focus metric. Focus metric waveform analysis provides a prediction confidence to eliminate images likely to produce erroneous Z predictions. Applying focus metrics to reconstructed image segments substantially containing the object greatly improves the performance of the DAMP method. While Gabor filters are the most computationally intensive focus metric evaluated, the Gabor focus metric curves are relatively smooth and unimodal, enabling iterative search methods to reduce the number of reconstructions required to determine focus.
Optical microscopy allows noninvasive imaging of biological tissues at a subcellular level. However, the optimal performance of the microscope is hard to achieve because of aberrations induced from tissues. The shallow penetration depth and degraded resolution provide a limited degree of information for biologists. In order to compensate for aberrations, adaptive optics with direct wavefront sensing, where guide-stars are used for wavefront measurement, has been applied in microscopy. The scattering effect limits the intensity of a guide-star and hence reduces the signal to noise ratio of the wavefront measurement. In this paper, we propose to use interferometric focusing of excitation light onto a guide-star embedded deeply in tissue to increase its fluorescence intensity, thus overcoming the signal loss caused by scattering. With interferometric focusing of light, we increase the signal to noise ratio of the laser guide-star through scattering tissue by more than two times as well as potentially extending the thickness of tissue that can be corrected using AO microscopy.
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