All light-sheet imaging was performed using a Leica TCS SP8 DLS Microscope. GFP-expressing MDA-MB-231 aggregates were individually mounted into Leica capillary 2.5 mm sample holders (Leica Code No. 158007060) by embedding them in 1% low melting point agarose (IBI, Cat. No. IB70050). The light sheet microscope utilized a 488nm laser for GFP excitation, and a 505–540nm filter for emission.
Tcs sp8 dls microscope
Manufactured by Leica
The Leica TCS SP8 DLS Microscope is a confocal laser scanning microscope designed for high-resolution imaging. It features a dedicated deconvolution laser scanning (DLS) module that enhances image quality and resolution. The instrument is capable of acquiring 3D and 4D images with high temporal and spatial resolution.
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4 protocols using tcs sp8 dls microscope
1
GFP-Expressing MDA-MB-231 Aggregate Imaging
2
Microangiography for Vascular Leakage Imaging
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Microangiography was performed following a previously published protocol (Arnold et al., 2022 (link); Shin et al., 2019 (link)). Embryos were anaesthetised with Tricane and injected with 1 nl of Qtracker™ 655 Vascular labels (Thermo Fisher Scientific) at 7 dpf in the LFL for valve leakage experiments and imaged on a Leica TCS SP8 DLS microscope at ∼5 min post-injection.
3
Light-sheet Imaging of Zebrafish Xenografts
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We obtained fluorescence images of the zebrafish xenografts with a light-sheet microscope (Leica TCS SP8 DLS or Zeiss Z.1) at 5 dpf (invasion characterization) or 2 and 5 dpf (volumetric measurements). For Zeiss Z.1, we embedded whole fish in 1% low-melting agarose in a glass capillary. For Leica TCS SP8 DLS, we mounted whole zebrafish in 1% low-melting agarose within a 1.5 mm U-shaped glass capillary, glued to the center of a glass bottom dish (35 mm, Greiner Bio-One). Imaging chambers were filled with E3/H containing 0.6 mM tricane. We imaged whole zebrafish heads with a 10x/0.3W DLS objective (Carl Zeiss) plus DLS TwinFlect 5 mm water mirror cap on a Leica TCS SP8 DLS microscope (Leica Microsystems) by z-stack across the full depth of the head. Identical image acquisition settings were applied for all zebrafish, including controls. The light-sheet thickness was 4.8 μm, the z-step size was set to 3.7 μm, with system-optimized settings for 3D-merging for a total of 100-150 images (=slices) per z-stack.
4
Immunostaining and Fluorescence Imaging
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Cells were plated in chamber slides (ThermoFisher Scientifi™, Nunc™ Lab-Tek™ II Chamber Slide™ System, Catalogue number 154461PK). Immunostainings were performed as previously described using antibodies listed in Supplementary Table 3 . Images were acquired with the Leica TCS SP8-DLS microscope (Leica Microsystems).
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