Autofocusing is widely used in microscopy since it provides sample details with high resolution and contrast. However, massive image recording along optical axis is often indispensable in classical autofocusing tactics, obviously decreasing autofocusing speed. To increase processing efficiency, we propose numerical wavefront propagation based autofocusing with high speed and large effective range. Firstly, quantitative sample phase is retrieved from multi-focal images according to transport of intensity equation with Gerchberg-Saxton algorithm. Then, various intensities along optical axis are numerically computed via wavefront propagation, thus focal position can be determined according to in-focus criterion. Finally, in-focus image is recorded via sample stage shifting. Since the proposed autofocusing approach can be easily integrated into commercial microscopes, additionally, it considerably reduces image captures as well as time consuming, we believe it can be adopted for rapid autofocusing in microscope.
KEYWORDS: Digital imaging, Live cell imaging, Numerical simulations, Microscopy, Real time imaging, Phase retrieval, Microscopes, Image retrieval, Phase imaging, Digital image correlation
As an ideal way for quantitative live cell imaging, dual view transport of intensity equation (TIE) method can provide both real time imaging, multi-mode observations, simple setup and large field of view (FoV). However, the image recorder installation error reduces the accuracy in both amplitude and phase retrievals, because of the inevitable FoV mismatch between the captured under- and over-focus intensities. In order to obtain higher accuracy amplitude and phase retrievals, the phase correlation based digital FoV correction is introduced into our method, rotation, scale and translation between the under- and over-focus images are compensated by the phase correlation based digital FoV correction. Measurements are implemented using standard sample detection and quantitative live cell imaging, proving that the proposed method can improve the accurate of the amplitude and phase computations.
KEYWORDS: Digital imaging, Live cell imaging, Digital image correlation, Numerical simulations, Real time imaging, Phase retrieval, Optical engineering, Image processing, Microscopy, Blood
Dual-view transport of intensity equation (TIE) method is an ideal way for quantitative live cell imaging as it has advantages such as real-time imaging, multimode observations, compact setup, and large field of view (FoV). However, due to the image recorder installation error, the inevitable FoV mismatch between the captured under- and over-focus intensities reduces the accuracy in both amplitude and phase retrievals. Here, to eliminate this undesired FoV mismatch, the phase correlation-based digital FoV correction is adopted to recognize and compensate the rotation, scale, and translation between the under- and over-focus images. Both the numerical simulations as well as the experiments in standard sample detection and quantitative live cell imaging prove that the digital FoV correction combined dual-view TIE method can maintain the consistence of the dual FoVs, thus guaranteeing the high-accurate amplitude and phase computations, proving the proposed method is a promising quantitative live cell imaging tool in various applications such as biological observations and medical diagnostics.
Massive image acquisition is required along the optical axis in the classical image-analysis-based autofocus method, which significantly decreases autofocus efficiency. A wavefront-sensing-based autofocus technique is proposed to increase the speed of autofocusing and obtain high localization accuracy. Intensities at different planes along the optical axis can be computed numerically after extracting the wavefront at defocus position with the help of the transport-of-intensity equation method. According to the focus criterion, the focal plane can then be determined, and after sample shifting to this plane, the in-focus image can be recorded. The proposed approach allows for fast, precise focus detection with fewer image acquisitions compared to classical image-analysis-based autofocus techniques, and it can be applied in commercial microscopes only with an extra illumination filter.
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