Zebrafish-embryo image captured using a THUNDER Imager Tissue and live instant computational clearing.

Improving Zebrafish-Embryo Screening with Fast, High-Contrast Imaging

Enhancing clarity and speed of zebrafish-embryo analysis with advanced imaging technology

Zebrafish-embryo image captured using a THUNDER Imager Tissue and live instant computational clearing. Zebrafish_Embryo_THUNDER_Imager_Tissue__live_ICC_magenta.jpg

This article explains how the DM6 B microscope with THUNDER streamlines the screening of transgenic zebrafish embryos, addressing the common issues of low resolution and out-of-focus blur with traditional microscopy. High-speed imaging, enhanced contrast, and sensitivity to image low protein expression ensure accurate and efficient screening. Key features include instant computational clearing, high sensitivity cameras, and targeted illumination, making it ideal for developmental biology.

Screening of transgenic zebrafish embryos

Transgenic zebrafish embryos are common model organisms used in developmental biology [1]. These samples are often engineered for in vivo volumetric imaging using systems requiring specific sample preparation including light-sheet microscopy [2]. Therefore, a rapid and accurate screening technique is required to determine if appropriate protein expression is present for zebrafish downstream imaging with light sheet microscopy.

Zebrafish screening is often performed using stereomicroscopes with fluorescence imaging capabilities. However, a limitation of stereomicroscopy is reduced resolution and sensitivity, which becomes particularly important in samples where expression is low. Conventional widefield microscopy is a high-speed imaging technique, but also suffers from out-of-focus blur, particularly for thicker samples including zebrafish embryos. This out-of-focus blur reduces contrast which is crucial to visualizing the expression in zebrafish. 

The DM6 B microscope equipped with THUNDER is a user-friendly system that facilitates high-speed screening through:

  • High-speed triggering with the New SynapseTM electronics;
  • Gentle and targeted illumination using the 8-line LED source;
  • Detection of low expression proteins thanks to the high sensitivity K8 camera;
  • Fast overview scans to locate optimal transgenic zebrafish via the LAS X Navigator spiral scan;
  • Improved contrast and clearly visualized fluorescent proteins due to THUNDER Live Instant Computational Clearing. 
THUNDER Imager Tissue which was used for screening transgenic zebrafish. Image courtesy of Laura Kreplin PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.
Figure 1: THUNDER Imager Tissue which was used for screening transgenic zebrafish. Image courtesy of Laura Kreplin PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.

Materials and methods

  1. In this workflow, transgenic zebrafish are transferred into a 35 mm petri dish. However, other mounting platforms also exist that are compatible with the THUNDER Imager Tissue.
  2. Low magnification objective is used to locate zebrafish embryos in a petri dish (Figure 2).
  3. LAS-X Navigator is used to mark regions of interest for Zebrafish (Figure 3).
  4. A higher resolution objective is used to determine if expression is sufficient for downstream imaging. Such objectives include HC FLUOTAR L 16x/0,60 IMM CORR VISIR (506533), HC APO L 63x/0,90 W U-V-I (506148), and HC FLUOTAR L 25x/0,95 W VISIR (506374). The combination of low LED light dosage, 95% peak QE of the K8 Scientific CMOS camera, and real time triggering ensures endogenous level protein signals are preserved during screening (Figure 4).
Brightfield image of zebrafish embryos captured using a 1.25x/0.04 DRY Objective on the THUNDER Imager Tissue. Scale bar is 1500 µm. Image courtesy of Dr. Senthil Arumugam and Laura Kreplin PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.
Figure 2: Brightfield image of zebrafish embryos captured using a 1.25x/0.04 DRY Objective on the THUNDER Imager Tissue. Scale bar is 1500 µm. Image courtesy of Dr. Senthil Arumugam and Laura Kreplin PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.
Zebrafish embryos found with LASX Navigator and marked for screening with fluorescence. Image courtesy of Dr. Senthil Arumugam and Laura Kreplin PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.
Figure 3: Zebrafish embryos found with LASX Navigator and marked for screening with fluorescence. Image courtesy of Dr. Senthil Arumugam and Laura Kreplin PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.
A) THUNDER Imager Tissue configuration used with 16x/0.6 IMM dipping objective. B) Zebrafish embryo image captured using THUNDER live instant computational clearing. Specifically, LED 8 excitation line 555 nm was used at 2% excitation with exposure time of 150 ms to image mKate expression. Scale bar is 300 µm. Image courtesy of Dr. Senthil Arumugam and Laura Kreplin, PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.
Figure 4: A) THUNDER Imager Tissue configuration used with 16x/0.6 IMM dipping objective. B) Zebrafish embryo image captured using THUNDER live instant computational clearing. Specifically, LED 8 excitation line 555 nm was used at 2% excitation with exposure time of 150 ms to image mKate expression. Scale bar is 300 µm. Image courtesy of Dr. Senthil Arumugam and Laura Kreplin, PhD Student, Monash Biomedicine Discovery Institute, Monash University, Clayton/Melbourne, Australia.

Conclusion

Zebrafish embryos with appropriate transgenesis are selected for downstream experiments including light-sheet imaging. The high-speed imaging capabilities of the DM6 B equipped with THUNDER technology and combined with enhanced contrast and sensitivity for imaging low protein expression, help ensure the accurate and efficient screening results. This study demonstrates that the DM6 B and THUNDER technology can help improve the understanding of phenomena in developmental biology and other related fields.

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