Design and implementation of algorithms for focus automation in digital imaging time‐lapse microscopy

Abstract

Background: Digital time-lapse microscopy using Nomarski-DIC requires that an autofocusing system adapt to changes in cell shape, size, and position while contending with drift, noise, and hysteresis in the microscope and imager. We have designed and implemented an autofocusing system that tracks subjects under dynamic conditions and maintains focus within a threshold of discriminability. Methods: With the use of proven and novel algorithms for autofocusing in Nomarski, we performed "virtual" experiments on recorded image stacks to simulate drift and sudden displacements and test the search algorithm response. Results: We found that combining a simple [1, Ϫ1] contrast function with an adaptive "warmer-colder" focusing

Controller design and optimization for large-delays image processing in visual closed-loop systems

“…Much work has been done on algorithms to compute image quality [3,4]. The optimization algorithm to maximize the quality has also been the subject of research [5,6] in order to improve the speed of the systems. These techniques have been applied to the automation of focusing systems in both optical [7] and electron [8] microscopy.…”

A microfluidic positioning chamber for long‐term live‐cell imaging

“…In addition, days-long imaging experiment using an environmental chamber will fully occupy the microscope, making the expensive setup inaccessible for other users during the period. Finally, stage drift and focus drift induce systematic errors in long-term time-lapse imaging (Burglin, 2000; LeSage and Kron, 2002). …”

Focus‐Drift Correction in Time‐Lapse Confocal Imaging

“…3 Even a completely sta-ble microscope would be susceptible to movements caused by cell growth and locomotion or by changes in the volume of the recording chamber. 4 Software and hardware solutions may be introduced to correct for the mechanical drift. Autofocusing is not only required for focus-drift correction, but is also frequently employed in automatic routine cell inspection in medicine and biology.…”

“…9 Many such techniques have been investigated in the literature. 2,4,[9][10][11][12][13][14][15][16][17] In these techniques, the focus measure is an extreme for the focused image and it decreases or increases by defocusing. The best focus point is found by comparing focus measures in a series of images acquired at different z-positions.…”