Images were acquired by lightsheet microscopy, as described previously 37 (link). Brielfy, specimens were warmed and transferred to 2% low melting point agarose (Fisher BP165-25) in PBS at 37°C, then embedded in glass capillary tubes with paired pistons (Sigma Z328510 paired with BR701938, or Sigma Z328502 paired with BR701934) for embryos, or tip-truncated 1mL syringes (Becton Dickinson) for hearts. After the gel solidified, the capillaries or syringes were suspended specimen-down from 14mL polystyrene tubes sealed with Parafilm. Columns containing the specimen were partially extended into an ample volume optical clearing solution (OCS, EasyIndex EI-Z1001, LifeCanvas). Following overnight incubation in OCS, specimen capillaries were retracted and were brought to a Light Sheet Z.1 microscope coupled with ZEN software (Carl Zeiss Imaging). With immersion in OCS, specimens were rotated and imaged from multi-view whole-volume approach, to improve fluorescence signal intensity and resolution captured throughout the entire heart volume. One of two objective setups was used: EC Plan Neofluar 5X/0.16 with 5X/0.1 pair (hearts), or Clr Plan Neofluar 20X/1.0 paired with 10X/0.2 clearing pair (embryos). Z-stacks were collected at each view angle at the optimal slice thickness determined by Zeiss' ZEN software, ranging from 1.42 to 4.95 µm.
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