Bilateral prediction and intersection calculation autofocus method for automated microscopy

Abstract

SummaryIn this paper, a bilateral prediction and intersection calculation autofocus method for automated microscopy, which obtains the in-focus position by calculating the intersection of the predicted left and right focus measure curves located respectively in the left and right sides of the peak position of the focus measure curve, is proposed and performed. According to the autofocus method, the area including the peak position of the focus measure curve and its left and right neighbourhoods should be determined firstly, and the left and right neighbourhoods are considered as the left and right sampling areas. The left and right focus measure curves are predicted by appropriate predicting models according to the two sample sequences, which comprise the focus values by evaluating the sampled images in the left sampling area and right sampling area, respectively and their corresponding sampling positions. The intersection of the predicted left and right focus measure curves is calculated and can be considered as the in-focus position. The autofocus can be realized by moving the focus plane of the microscope to the intersection of the predicted left and right focus measure curves. The proposed bilateral prediction and intersection calculation autofocus method is experimentally verified in an automated light microscopy for implementing microassembly and micromanipulation. The theoretical analyses have shown that the proposed bilateral prediction and intersection calculation autofocus method can not only effectively avoid the principle error caused by assuming the symmetrical focus measure curve in the autofocus methods based on curve fitting, but also eliminate the possible waver search near the peak position in the modified fast climbing servo method. The experimental results have shown that the proposed bilateral prediction and intersection calculation autofocus method possesses the merits as follows: (1) the focusing accuracy is high and slightly affected by the sampling step size and (2) the focusing speed is higher than those of the 7-point hill-climbing search method and the quadratic curve fitting method with a determinate focusing accuracy.

Combining gradient ascent search and support vector machines for effective autofocus of a field emission–scanning electron microscope

“…The last benchmark was the solution of Wu et al . (). They used hill climbing search for initial guess.…”

“…SVMs are chosen for fitting search. Its advantages include accuracy like least squares (Nicolls et al ., ; Rudnaya et al ., ; Wu et al ., ; Nishi et al ., ), but it overtakes the latter in term of robustness to outliers coming from noise. Indeed, it uses a regularization parameter C that limits the values of the model parameters and more particularly the slack parameter ϵ that tunes the acceptable variations of the data (Bishop, ).…”

“…Wu et al . () used the standard hill climbing search to get the initial estimation of the peak. They assumed an exponential model of the focus left and right of that point (quasi‐Laplace model), and used linear least squares regression to find the both models.…”

“…It overtakes the methods presented above (Ong et al . a,b; Batten, ; Wu et al ., ) in term of speed, and of starting point that can be far from the peak. SVMs are chosen for fitting search.…”

Optical microscopy with flexible axial capabilities using a vari‐focus liquid lens

“…Active AF utilizes an active distance measuring element to measure the objective–sample distance, then adjusts the distance to acquire a clear image. Passive AF utilizes an image‐processing algorithm (Liang & Qu, ; Wu et al ., ) to evaluate the focus level of images, which is then fed back to a motion control system to adjust the optical components and acquire a clear image. EDOF has been a research focus for the past few years and many strategies (Qu et al ., ) have been proposed such as those incorporating multifocus detectors, moving detectors, variable apertures, mask or a phase plate, spherical aberration or chromatic aberration, volumetric sampling methods (Liu & Hua, ), etc.…”