software for controlling the two-photon microscope and pre-processing of the calcium imaging data  (SourceForge net)

Journal: Development (Cambridge, England)

Article Title: Imaging the onset of oscillatory signaling dynamics during mouse embryo gastrulation

doi: 10.1242/dev.200083

Figure Lengend Snippet: Light-sheet microscopy for multi-sample live imaging of mouse embryos from gastrulation to organogenesis. (A) Imaging chamber and objective configuration of the Lightsheet Z.1 (Zeiss) with two illumination light-sheets (ill.) and one detection light path (det.). The sample-containing capillary (1) is suspended from the top into the imaging chamber (2) and orientated with respect to the illumination (3) and detection (4) objectives (degrees of freedom indicated by red arrows; XYZ, translation; R, rotation). (B) Vertical section view through the sample chamber with inserted embryo culture chamber (5). The embryo culture chamber consists of a chamber lid (dark blue) and the chamber body (light blue) with membrane-covered windows for the light paths. It is connected via tubes (6) to the closed-cycle perfusion system of the microscope ( Fig. S1 ). (C) For mounting, embryos were first agarose embedded in a capillary (left), then the agarose was peeled off around the embryo, leaving only the ectoplacental cone embedded to allow growth during culture (right). (D) Bright-field images of an embryo developing on the customized light-sheet microscope (anterior towards the left). The experiment starts at mid-streak stage at E6.5. Morphological features are the allantois (a), the head fold (hf), somites (s) and foregut pocket (fg). Scale bar: 500 μm. (E) Examples of embryos after culture on the microscope: (i) LuVeLu reporter imaging ( z -stacks with 7.5 μm spacing, 100 slices per sample, 300 ms exposure time with 2.0% 50 mW 514 nm laser and 10 min imaging interval. n =6); (ii) R26-H2BmCherry reporter imaging ( z -stacks with 7.5 μm spacing, 120 slices per sample, 200 ms exposure time with 1.2% 20 mW 561 nm laser and 10 min imaging interval. n =4); (iii) cultured on microscope without imaging ( n =9); (iv) roller culture ( n =29); and (v) in utero developed control embryos dissected at E8.5 ( n =12). For all embryos, a hybridization-chain reaction (HCR)-based in situ mRNA hybridization was performed for following genes: Msgn1 (cyan), a marker for presomitic mesoderm; Shh (yellow), expressed in notochord and the floorplate of the neural tube; and Uncx4.1 (magenta), marking the anterior-posterior subdivisions of the somites. Scale bar: 200 μm. Brightness and contrast were set identically for samples i, iii, iv and v, but adjusted separately for ii. (F) SPIM-for-4 multi-sample holder. Four sample capillaries are positioned in the sample holder (7), which is mounted on the Z.1 stage using the standard sample holder disc for syringes (8). A capillary cap is glued to the top of each capillary (9; in top view on the right). With a hex key, each capillary can be turned individually around its long axis (small R), while the rotation drive of the microscope stage allows for switching between samples (large R). (G) Horizontal section view through the imaging chamber in the plane of the light path. Sample capillaries (red circles) in the SPIM-for-4 multi-sample holder do not interfere with the light paths during imaging and the holder is compatible with the embryo culture chamber. CAD drawings of Z.1 microscope parts are courtesy of Carl Zeiss Microscopy.

Article Snippet: During acquisition, both images were combined using a mean fusion algorithm provided with the microscope control and image processing software ZEN (Carl Zeiss).

Techniques: Microscopy, Imaging, Embryo Culture, Membrane, Cell Culture, In Utero, Control, Hybridization, In Situ, Marker