TITLE: Targeted optogenetic stimulations
AUTHORS: Mai-Anh Vu, mwhowe

[DESCRIPTION]
We have developed a new micro-fiber array approach capable of chronically measuring and optogenetically manipulating local dynamics across over 100 targeted locations simultaneously in head-fixed and freely moving mice, enabling investigation of cell-type and neurotransmitter-specific signals over arbitrary 3-D volumes . This protocol includes the steps for targeted optogenetic manipulation. Please contact us (mwhowe@bu.edu) if you are interested in using this technique. 

[STEPS]
SECTION: Targeted optogenetic stimulations
1. To couple light into individual optical fibers in our array for targeted optogenetic manipulations, simultaneously with imaging, we integrated a programmable digital mirror device (DMD, Mightex Polygon1000 Pattern Illuminator DSI-K3-L20) into the light path of our imaging microscope (see Multi-color fiber array imaging protocol) as detailed via the substeps below:
SECTION: Targeted optogenetic stimulations
1.1. Excitation light was provided by a 3.2W, 465nm laser (Mightex, LSR-040-0465), which was coupled to the DMD with an optical fiber.
SECTION: Targeted optogenetic stimulations
1.2. Light from the DMD was coupled into the objective path by a dichroic (Chroma 570lpxr).
SECTION: Targeted optogenetic stimulations
1.3. 570nm excitation and emission filters (see Multi-color fiber array imaging protocol) enabled simultaneous imaging of red fluorophores during stimulation.
SECTION: Targeted optogenetic stimulations
2. Control of light patterning and stimulation parameters was achieved with PolyScan2 control software (Mightex) and custom MATLAB functions.
SECTION: Targeted optogenetic stimulations
3. A calibration step was performed prior to stimulations to align the camera view with the PolyScan2 software, allowing us to design patterns of circular light (~40μm diameter for each spot) to target individual fibers.
SECTION: Targeted optogenetic stimulations
4. Transmission efficiency through 50μm diameter fibers in our arrays was calculated at approximately 39% based on comparisons between power at the objective and transmitted light through individual fibers.
SECTION: Targeted optogenetic stimulations
4.1. For stimulations of dopamine release, a light spot of ~750 μW (measured at the objective) was used, resulting in an estimated power density of 175 mW/mm2 (0.29 mW total power) at each implanted fiber tip.
SECTION: Targeted optogenetic stimulations
4.2. For stimulations of D1 expressing neurons, a light spot of ~600 μW at the objective was used, resulting in an estimated power density of 141 mW/mm2 (0.23 mW total power) at the fiber tip.
SECTION: Targeted optogenetic stimulations
5. We estimated relative excitation light intensity and excitation area as a function of distance from the fiber tip by applying a light scattering model developed by Yona et. al 2016 with a scattering coefficient of 140 cm-1 approximated for the striatum based on Azimipour et al 2014 and Al-Juboori et al. 2013 and an activation threshold for ChR2 of 1 mW/mm^2 70,71.
SECTION: Targeted optogenetic stimulations
6. Light pulse trains for stimulation (30Hz, 4ms pulse width, 1s or 5/10s durations for DA and D1 neuron stimulation respectively) were programmed in PolyScan2 and triggered with 5V digital pulses controlled via MATLAB and sent from a NIDAQ board (National Instruments PCIe 6343) to the Polygon. Stimulations were triggered randomly (inter-stimulation interval 30-60 seconds) for 5-10 minutes per session for all stimulation experiments.