Fig. 2: Conical cannula increases collected fluorescence and allows imaging of GCaMP6f expression in dorsal CA1.

A Ray tracing diagrams for the conventional cylindrical non-reflective cannula (i) and the conical reflective cannula (ii). In both panels the fluorescent source is illustrated at the center of the cannula, below the cover glass, in water. On the left-hand side of the diagram the two-photon excitation light rays are shown for the 0.6NA air objective. On the right-hand side, the fluorescent emission rays are shown. Only rays that escape the glass-air interface are extended. Compared to a standard cylindrical cannula, the conical reflective cannula redirects emitted fluorescence that would otherwise be lost towards the front aperture of the objective, where it may be recorded (providing they do not exceed the spatial-angular acceptance of the collection optics). B Change in measured fluorescence at 150 µm below the cover glass (bottom of cannula, this corresponds to the typical depth of the CA1 pyramidal layer). The conical cannula provides up to 1.5× increased fluorescence collection compared to a standard conical cannula. The greatest improvement was measured towards the edge of the cannula. The fall off of the fluorescence for the cylindrical cannula is due to partial obscuration of excitation beam, which is also a factor for the conical cannula since they have a common top diameter. The hatched area indicates an invalid region of measurement for the conical cannula since it has a smaller bottom diameter than the cylindrical cannula. C Photograph of the cannulas. The inner surface of the conical cannula is polished to a mirror finish to improve collection efficiency. D Installation of the conical cannula over dorsal CA1. Schematic, left; two-photon images from an anaesthetized animal showing expression of GCaMP6f in the pyramidal cell layer at different depths below the bottom of the cover glass, right. Expression was similar to other animals (n = 7).