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High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

Nature Protocols volume 6, pages 229–241 (2011)Cite this article

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Abstract

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis.

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Figure 1: The LED fluorescence macroscope.
Figure 2: The LED fluorescence macroscope can distinguish five fluorescent proteins in adult transgenic zebrafish.
Figure 3: Multispectral imaging of adult transgenic zebrafish using the upright LED fluorescence macroscope.
Figure 4: Still images from a live video of swimming transgenic zebrafish using the upright macroscope.
Figure 5: The macroscope differentiates between zebrafish tagged with four fluorescent elastomers.
Figure 6: The LED fluorescent macroscope facilitates rapid screening of tumor-bearing zebrafish.

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Acknowledgements

J.S.B. is supported by National Institutes of Health (NIH) grant 5T32CA09216-26. C.D.S. is supported by the Dev and Linda Gupta Fund. D.M.L. is supported by NIH grants K01 AR055619-01A1 and 3 K01 AR055619-03S1, the Alex's Lemonade Stand Foundation, the Sarcoma Foundation of America, the Hollis Brownstein Research Grant from the Leukemia Research Foundation and by a grant from the Harvard Stem Cell Institute. We thank Northwest Marine Technology for expert technical advice and for providing visible implant elastomers for our pilot experiments.

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Authors and Affiliations

  1. Department of Molecular Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Jessica S Blackburn, Sali Liu, Aubrey R Raimondi, Myron S Ignatius & David M Langenau

  2. Massachusetts General Cancer Center, Boston, Massachusetts, USA

    Jessica S Blackburn, Sali Liu, Aubrey R Raimondi, Myron S Ignatius & David M Langenau

  3. Harvard Stem Cell Institute, Boston, Massachusetts, USA

    Jessica S Blackburn, Sali Liu, Aubrey R Raimondi, Myron S Ignatius & David M Langenau

  4. Department of Electrical and Computer Engineering, University of Massachusetts Amherst, Amherst, Massachusetts, USA

    Christopher D Salthouse

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  1. Jessica S Blackburn
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Contributions

S.L., A.R.R., M.S.I. and C.D.S. conducted the experiments; J.S.B. and D.M.L. conducted the experiments and wrote the manuscript.

Corresponding author

Correspondence to David M Langenau.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Basic construction of the inverted LED fluorescence macroscope. Measured photographs of the Inverted LED fluorescence macroscope highlighting the basic construction of the external shell. Right side view (A), front/top down view (B), top view (C), bottom view (D, the final 1/4” plywood cover is not shown),Top view after lights fixtures have been added (E), and bottom view after light fixtures have been added (F). Red arrows in D denote the attachments for securing the bottom of the macroscope to the wood frame. Blue arrow in F shows where the 1” hole should be drilled that receives wires to be connected to the switch box. (JPG 942 kb)

Supplementary Figure 2

Attaching the switch box and lights to the inverted LED fluorescence macroscope. Measured photograph of the inverted LED fluorescence macroscope highlighting attachment of the switch box, lights, and top of the macroscope. The switch box is attached to the macroscope by glue (A). Wooden 5/8” square dowel side supports are shown in B,C. Front/top view of the macroscope imaged with the wood top cover (D). Arrows in D denote placement of screws to support the bottom of themacroscope. Top view of macroscope (E,F), with the front side noted. (JPG 918 kb)

Supplementary Figure 3

Wiring the inverted LED fluorescence macroscope. The correct wiring technique is shown in A-D. Wire connections are shown for the black wires only (E). Circuit diagram of how to wire the macroscope (F). (JPG 748 kb)

Supplementary Figure 4

Preparing the electric switch box for the inverted LED fluorescence macroscope. The two tabs shown in A are removed using a saw (A, red lines) and the plastic tab is removed on the switch box (B, asterisk) to receive wires. (JPG 599 kb)

Supplementary Figure 5

Wiring the switches for the inverted LED fluorescence macroscope. The wires are connected to the switch (A), then the switches are attached to the blue switch box (B), and the switch plate cover is installed C). (JPG 371 kb)

Supplementary Figure 6

Setting up the camera for the inverted LED fluorescence macroscope. The camera is connected to a mounting bracket and wood insert (A-E). A ring of PVC is glued to the camera to house the optical filter (D). Multiple cameras can be attached to the wood insert for multispectral imaging. (F,G). The wood insert with cameras is placed within the inverted macroscope (H). (JPG 722 kb)

Supplementary Figure 7

Photographs of the upright LED fluorescence macroscope. Upright LED fluorescence macroscope imaged from above (A), below (B), the front (C), the back (D), the left side (E), and the right side (F). (JPG 838 kb)

Supplementary Figure 8

Measurements of the upright LED fluorescence macroscope. The upright LED fluorescence macroscope imaged from above (A), below (B), the front (C), the back (D), the left side (E), and the right side (F). Lengths of PVC tubing used in each connection are shown (A-D). The green box in panel A shows the corner connection, which is depicted in detail in Supplementary Figure 9. (JPG 1077 kb)

Supplementary Figure 9

Creating corner connectors for the upright LED fluorescence macroscope. PVC tubing and connectors used in creating the upright LED fluorescence macroscope (A, some connectors not shown), and parts used to create the corner connector (B-D). (JPG 687 kb)

Supplementary Figure 10

Wiring the upright LED fluorescence macroscope. Lights are attached to a T-type PVC connector that has a center threaded 1/2” PVC opening. The light is screwed into place and wires are connected through the inside of the PVC tubing (A). Once completed, the lights can be attached to the macroscope (B). A waterproof switch plate cover is used(C), and end cap with hole drilled through allows the wire to be plugged in to the wall outlet (D). (JPG 808 kb)

Supplementary Figure 11

Wiring diagram for the upright LED fluorescence macroscope. The negative wire is denoted by red and positive by black. All wiring is contained within the PVC piping, which is water-tight. (JPG 277 kb)

Supplementary Figure 12

Setting up the camera for the LED fluorescence macroscope. To attach the camera, holes are drilled in the center PVC pipe (A,B). Cameras are glued onto a mounting bracket as in Supplementary Figure 6, then the camera is attached to the PVC pipe using a wing nut (C,D). (JPG 698 kb)

Supplementary Movie 1

Multispectral video of swimming transgenic zebrafish. mylz-mCherry and mylz-GFP fish were imaged using a blue light with 640/35nm and 535/45nm filters. The capture rate is 60ms. (MOV 9697 kb)

Supplementary Manual 1

Instructions for building the inverted and upright macroscopes. (DOCX 30 kb)

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Blackburn, J., Liu, S., Raimondi, A. et al. High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope. Nat Protoc 6, 229–241 (2011). https://doi.org/10.1038/nprot.2010.170

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