Automated feature detection and imaging for high-resolution screening of zebrafish embryos

Abstract

The development of automated microscopy platforms has enabled large-scale observation of biological processes, thereby complementing genome scale biochemical techniques. However, commercially available systems are restricted either by fixed-field-of-views, leading to potential omission of features of interest, or by low-resolution data of whole objects lacking cellular detail. This limits the efficiency of high-content screening assays, especially when large complex objects are used as in whole-organism screening. Here we demonstrate a toolset for automated intelligent high-content screening of whole zebrafish embryos at cellular resolution on a standard wide-field screening microscope. Using custom-developed algorithms, predefined regions of interest-such as the brain-are automatically detected. The regions of interest are subsequently imaged automatically at high magnification, enabling rapid capture of cellular resolution data. We utilize this approach for acquiring 3-D datasets of embryonic brains of transgenic zebrafish. Moreover, we report the development of a mold design for accurate orientation of zebrafish embryos for dorsal imaging, thereby facilitating standardized imaging of internal organs and cellular structures. The toolset is flexible and can be readily applied for the imaging of different specimens in various applications.

Automatic Zebrafish Heartbeat Detection and Analysis for Zebrafish Embryos

“…7 Existing software for semiautomated analysis of heartbeats [7][8][9][10][11][12][13] has several limitations. One is that the embryos have to be anesthetized by adding Tricaine 11,14 or MS-222 12 to avoid movement of zebrafish embryos during videoing. Adding compounds that prevent embryo movement such as anesthetics, sedatives, or neuromuscular junction blockers can alter the heart rate.…”

“…Adding compounds that prevent embryo movement such as anesthetics, sedatives, or neuromuscular junction blockers can alter the heart rate. 8 A second limitation is that the embryos have to be fixed and oriented manually in an agarosegel before the video recording, 11,14,15 which is a timeconsuming and tedious task. Additionally, the oxygen supply in agarose can differ from the one in water.…”

In Vivo Chemical Screen in Zebrafish Embryos Identifies Regulators of Hematopoiesis Using a Semiautomated Imaging Assay

“…[22][23][24] Screening in 384-well plates at higher magnification with automated image analysis is rare and particularly challenging. 25,26 First, as zebrafish are nonadherent, robotic wash steps (e.g., after fixation) are at risk of washing the embryos away. Second, given their small size relative to the well, determining the location of the embryos for imaging is challenging.…”

Extracting information from imaging cytometry: a review

“…The colony has features that are associated not only with itself, e.g., area and perimeter, but also features that are measured from the cells within the colony; therefore, measurements such as the number of cells/colony and how many are positive for a certain marker protein, can be obtained (Barbaric 2011). Another example of higher-order structures linked to single cell information follows from the recent development of tools that allow zebrafish embryos to be imaged and analyzed quantitatively in high-throughput drug discovery investigations (Peravali 2011).…”