Evaluation of autofocus functions in molecular cytogenetic analysis

Abstract

SummaryThis work describes a systematic evaluation of several autofocus functions used for analytical fluorescent image cytometry studies of counterstained nuclei. Focusing is the first step in the automatic fluorescence in situ hybridization analysis of cells. Thirteen functions have been evaluated using qualitative and quantitative procedures. For the last of these procedures a figure-of-merit (FOM) is defined and proposed. This new FOM takes into account five important features of the focusing function. Our results show that functions based on correlation measures have the best performance for this type of image.

A Bayes-spectral-entropy-based measure of camera focus using a discrete cosine transform

“…Numerous focus measures have been presented in the literature (Krotkov, 1987;Gillespie et al, 1989;Firestone et al, 1991;Yeo et al, 1993;Santos et al, 1997;Choi et al, 1999;Subbarao and Tyan, 1998;Widjaja and Jutamulia, 1998;Geusebroek et al, 2000;Chern et al, 2001;Lin et al, 2004;Yap and Raveendran, 2004). From among these we selected four well-known measures to compare against our measure.…”

“…The fourth measure, known as Vollath's F 4 , was reported by Santos et al (1997) to achieve good focusing, even in low-information-content images, and is defined as…”

“…Passive focusing based on image quality is a common tool in general-purpose CCD cameras and consists of three major parts: the region being focused (the focusing window); a measure of focus, which yields a maximum at the focused position; and an optimization algorithm, which searches for a global maximum of the focus measure. Many types of focus measure have been investigated in microscopy (Firestone et al, 1991;Yeo et al, 1993;Santos et al, 1997;Geusebroek et al, 2000), digital holography (Gillespie et al, 1989) and digital photography (Krotkov, 1987;Subbarao and Tyan, 1998;Widjaja and Jutamulia, 1998;Choi et al, 1999;Chern et al, 2001;Lin et al, 2004;Yap and Raveendran, 2004). Usually, these measures are calculated using certain norms of the image gradients or derivatives (Krotkov, 1987;Subbarao and Tyan, 1998;Chern et al, 2001), manipulation of gray level histograms (Krotkov, 1987;Chern et al, 2001) or operations on the longest lines in the images (Lin et al, 2004).…”

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

“…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.…”