Supplementary Figure 2: Overview of the modular configurations of NeuBtracker as an imaging and interrogation platform. | Nature Methods

Supplementary Figure 2: Overview of the modular configurations of NeuBtracker as an imaging and interrogation platform.

From: NeuBtracker—imaging neurobehavioral dynamics in freely behaving fish

Supplementary Figure 2

(a) Schematic of the combinations of the components used in the two configurations reported in this manuscript (‘MacroZoom’ and ‘MicroFixed’) and possible add-ons. The insets (upper left corner) gives a photographic overview of the alternative MicroFixed configuration which uses a microscope objective and fixed magnification. Several additional features were also tested such as coupling of additional lasers (either illuminating the whole arena or providing localized photostimulation via the galvanometric mirrors), or the use of motorized stages for high-throughput screening applications. (b-d) Different types of image contrasts that can be obtained by alternative configurations of NeuBtracker. As an example, the IR-illumination ring can either be positioned on top of the sample (IR-EPI inlet in (b)) or underneath it (IR-TRANS) to achieve epi- or transillumination. Furthermore, the addition of extra optical elements to the path can be achieved conveniently. A diffuser can be added for a projector to be placed under the sample (c- upper right). In this case, the noise level of the fluorescence channel is increased due to the bleed-through of backscattered excitation photons ((d)-middle right, images from MicroFixed configuration). By using a rotating holder, a dichroic can be positioned to deflect the excitation light after it passes through the sample. This results in images such as shown in c -top right. The presence of a sliding holder (or potentially a filter-wheel), enabled us to create the overlay in (bottom of c) were the same sensor captured fluorescence (green overlay) and directly afterwards, a brightfield image (gray) using an autofluorescent plastic plate as a 510 nm light source to create transillumination contrast utilizing the high dynamic range of the camera sensor. (d) To showcase the features that can be differentiated in the 7x magnified fluorescence channel of NeuBtracker MicroFixed, we show how the 7x channel may also be used to obtain anatomical details in reflection images with fast temporal sampling (5 ms exposure time) when an autofluorescent plastic slide (Chroma) is placed underneath the arena. The three frames on the right show movements of the fins and the heart displayed on a difference map (red for positive, blue for negative intensities with respect to the previous frame)

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