Dual excitation band fiber photometry combined with optogenetics use a Fluorescence Mini Cube with 5 ports: one for 460-490 nm GCaMP excitation, one for 405, 415, 425 or 440 nm isosbestic excitation, one for 500-540 nm window of isosbestic and functional fluorescence detection, one for 580-680 nm opsin silencing light, and one for the sample under the test. The cube has dichroic mirrors to combine isosbestic and fluorescence excitations, isolate the fluorescence emission, and let opsin stimulation light through. At the same time, narrow bandpass filters limit the excitation fluorescence spectrum.

E stands for fluorescence excitation, IE for isosbestic excitation, F for fluorescence, O for opsin stimulation light, and S for the sample port on the cube's schematic.

The separation of functional and isosbestic fluorescence signals of GCaMP is possible with modulated light sources. The Intensity Adjustment Ring on the 460-490 nm LED light source reduces its power below the LED driver's range if needed for low-power fiber photometry experiments.

Compared to Fluorescence Mini Cubes with only FC connectors on all ports, the ones with integrated photodetectors provide a 100% better signal-to-noise ratio (30% signal increase and a 40% noise reduction). Moreover, in the latest version of Mini Cubes, LEDs and LED drivers are integrated as well to reduce the number of connections and the system’s overall footprint. In his version, it is also possible to replace the excitation filter (to adjust the isosbestic excitation from 405 nm to 410 nm for instance) and decrease the excitation power by adding attenuating filters (for low power photometry).