SCORE imaging: specimen in a corrected optical rotational enclosure

Abstract

Visual data collection is paramount for the majority of scientific research. The added transparency of the zebrafish (Danio rerio) allows for a greater detail of complex biological research that accompanies seemingly simple observational tools. We developed a visual data analysis and collection approach that takes advantage of the cylindrical nature of the zebrafish allowing for an efficient and effective method for image capture that we call Specimen in a Corrected Optical Rotational Enclosure imaging. To achieve a nondistorted image, zebrafish were placed in a fluorinated ethylene propylene tube with a surrounding optically corrected imaging solution (water). By similarly matching the refractive index of the housing (fluorinated ethylene propylene tubing) to that of the inner liquid and outer liquid (water), distortion was markedly reduced, producing a crisp imagable specimen that is able to be fully rotated 360 degrees. A similar procedure was established for fixed zebrafish embryos using convenient, readily available borosilicate capillaries surrounded by 75% glycerol. The method described here could be applied to chemical genetic screening and other related high-throughput methods within the fish community and among other scientific fields.

FIG. 1.

SCORE tools. (A) Anesthetized larval zebrafish are placed into a methylcellulose (pictured) or low-melting-point agarose solution before being loaded into the capillary housing. (B) A pipette pump with capillary adaptor is used to facilitate loading of zebrafish into the capillary housing. (C) Multiple larvae (arrows) can be imaged in a single capillary, allowing for rapid screening of mutants. (D) A corrective solution (water pictured) is placed upon the capillary to reduce distortion. Foam barriers, marked by arrows, are placed upon the glass slide to help contain corrective solution and to provide a friction surface for ease of rotation.

FIG. 2.

Specimen in a Corrected Optical Rotational Enclosure (SCORE) imaging of living animals. Zebrafish imaging is facilitated by use of a fluoro-carbon tube for housing. (A) Zebrafish images taken in polymer tubing produce a distorted image (arrows) due to the change in refractive index between the tubing and the air. Cartoon images representing a fish immobilized using traditional techniques on a skewed plane (B), a larval fish within a polymer tube without a corrective solution producing distortion (C), and within a corrective solution to produce a clear image (D). Fluorinated ethylene propylene (FEP) tubing produces a clear image at 50 × (E ) (also see Supplemental Movie S1, available online at www.liebertonline.com), 100 × (F), and 200 × (G) of a 5 day postfertilization (dpf ) larvae. A 5 dpf Tg(gata-1:DsRed)sd2 larvae in FEP tubing produces a clear background-free image at 50 × (H) (also see Supplemental Movie S2, available online at www.liebertonline.com) and 100 × (I). A 5 dpf Tg( fli:eGFP)y1 in FEP tubing produces a clear background-free image at 50 × (J) (also see Supplemental Movie S3, available online at www.liebertonline.com) and 100 × (K).

FIG. 3.

SCORE imaging of a fixed specimen. Imaging of a pax2a whole-mount in situ hybridization embryo and capillary housing in 75% glycerol. (A) Cartoon depicting the use of glycerol both inside and outside of the glass tube producing an undistorted image of a fixed embryo. (B) Sagittal image of embryo with pax2 staining at 50 × magnification shows no distortion (also see Supplemental Movie S4, available online at www.liebertonline.com). Note the edges of the capillary (arrow) that can be readily cropped for publication presentation. Sagittal embryo image of pax2 staining at 100 × (C) or 200 × (D) magnification shows no distortion. (E) Coronal image of embryo at 50 × magnification. (F) Angled image of pax2 staining at 50 × magnification. Rotation is angled slightly (∼30°) to show a more detailed view of kidney tubule staining (arrows).

FIG. 4.

SCORE imaging of fluorescent, living zebrafish. Imaging SEC0124 of a 5 dpf mRFP-labeled protein trap zebrafish housed in FEP tubing. (A) Sagittal image of an mRFP-labeled protein trap zebrafish shows enhancement in specific locations in the neural region as well as a bright kidney (Arrow). (B) Coronal image of an mRFP-labeled protein trap zebrafish includes ubiquitous neural expression with enhancement in specific locations. (C) Sagittal image of an mRFP-labeled protein trap zebrafish shows a general neural haze with enhancement in specific locations at 100 × magnification (also see Supplemental Movie S5 [showing the spatial location of RFP patterning], available online at www.liebertonline.com).

FIG. 5.

Ease-of-use microscope—the “headless” microscope setup. (A) A Sony HDR-HC9 high-definition video camera is attached to the camera port of the (B) Zeiss inverted microscope to facilitate a greater ability to view, record, and present visual data. (C) A high-definition monitor allows for reduced eyestrain and ease of presentation to other workers. (D) Attaching the video camera to a computer allows for direct recording of the sample in a purely digital format. (E) X-Cite light source allows for fluorescent microscopy. (F) Removal of the eye-pieces of the Zeiss inverted microscope allows for a reduction in the light bleaching of the sample and provides a less cluttered working area with no obstructions to the stage. (G) The microscope has been placed on a BostonTec adjustable lab table (controls indicated with arrows) to facilitate the ease of movement of the entire scope system for greater ability of access.