Autofocusing in microscopy based on the OTF and sampling

The Weibull manifold in low-level image processing: An application to automatic image focusing

“…The simplest strategy [4], involves capturing a small number of images at some distance apart, thereby coarsely sampling the SF. This process is repeated at finer scales, around the location of the current optimum, until convergence.…”

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

“…To establish an accuracy standard and to estimate the threshold of focus discriminability for the operator, a psychophysical experiment was performed similar to that described briefly by Boddeke et al (7). A computer program was coded to behave like a "virtual microscope" system, serving images to a monitor from stored data rather than to the microscope; subjects attempted to focus this microscope, and their responses were compared with a fourth-order least-squares fitting of the focus function data.…”

“…One search algorithm performed a least-squares polynomial fit of the focus function, with sampling positions above and below the initial position selectively. Boddeke et al (7) applied this fit in three phases by using successively smaller intervals to find focus, with the last a "refine" phase in the region where the focus function closely approximates the parabolic ideal. With the consideration that practical time-lapse experiments begin with the subject in focus, we examined whether a practical autofocus could be implemented when using a single polynomial fit equivalent to the "refine" phase.…”

“…There is a good deal of literature on the subject of autofocus for microscopy and robotics in general (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Fitted with a video or digital camera and a motorized stage under computer control, the typical autofocus microscope system uses two complementary algorithms to achieve focus.…”

“…For brightfield imaging, this focus function establishes a roughly parabolic relation between focus score and position. Acceptable focus functions have been described for brightfield (7), phase-contrast (8), and fluorescence (5,6,8) imaging, and a general method has been described (3); however, Groen et al (11) found that focus functions optimized for one imaging mode may not perform well in another. This may be particularly relevant to Nomarski-DIC because optical sectioning may resolve detail in multiple planes within a subject, potentially resulting in a multimodal focus function.…”