Orca flash 4 v3

In the prepared setup, the cortex was illuminated by light emitting diodes (LEDs). To provide stabilized radiation, a large battery together with a 10,000 µF capacitor and voltage regulator were used to supply power for 6 parallel 1w power LEDs. The LED’s intensity fluctuations were stabilized to about 10⁻⁴–10⁻⁵. To have uniform illumination on the cortex, a circular holder was used containing 9 LED including 3 green LEDs and 6 red LEDs (with wavelength about 620–625 nm and with intensity about 50-60 lumen). The camera used for OISi setup was a 16 bit sCMOS with a well-capacity of about 30,000 electrons (Hamamatsu Orca Flash 4, v3), located vertically above the cortex. Also, an extension tube attached to a Nikon lens (Sigma 105 mm f/2.8 EX DG OS HSM Macro Lens) was applied to reduce the depth of field of the imaging system to about 300 µm, which is critical for reducing the surface vascularity artifacts in OISi¹ . For stimulating the rat’s whiskers, a metal rod attached to a stepper motor was used. Also, the head of the anesthetized rat was stabilized relative to the camera using a stereotaxic frame. Ultimately, the entire imaging setup was enclosed in a dark box. The schematic view and the assembled setup for OISi experiments are shown in Supplementary Fig. S1a,b respectively.

The components used in the experimental setup are: Laser 1: Cobolt 06-01 series with wavelength of 647 nm and maximum power of 130 mW, Laser 2: Cobolt 08-01 series with wavelength of 561 nm and maximum power of 100 mW, Laser 3: Cobolt 06-01 series with wavelength of 488 nm and maximum power of 120 mW, Dichroic 1: Semrock Di03-R561-t3-25x36, Dichroic 2: Semrock’s Di03-R488-t3-25x36, Dichroic 3: Semrock’s Di03-R405/488/561/635-t3-25x36, Microscope objective: Leica HC PL APO 100x magnification with Numerical Aperture adjustable between 0.7 to 1.4, focal length of Lens 1: 150 mm, focal length of Lens 2, Lens 3 and Lens 4: 200 mm, Rectangular Aperture from Ealing (Hyland Optical Technologies), Linear Polizer: model LPVISE100-A from Thorlabs, Camera: Orca Flash 4 V3 from Hamamatsu. Holoeye’s Pluto-VIS-056 was used as the SLM in this microscope, which had a pixel size of 8 $\mu m$ . The Multi-bandpass filter deployed was custom-made by Alluxa having the central wavelengths ltocated at 460 nm, 512 nm, 610 nm and 671 nm, with corresponding bandwidths of 21 nm, 15 nm, 16 nm and 20 nm, respectively. The Rectangular Aperture was controlled such that the total field of view per subimage was set to $\sim$ 27 $\mu m^2$ .