An analogous holographic photostimulation path was coupled with widefield epifluorescence imaging on a second system, here denoted as setup 2 (see Supplementary Fig. S6 ).
This system was built around an Olympus BX51WI upright microscope, capable of widefield epifluorescence imaging using illumination with an arc lamp, (OptoSource Illuminator, Cairn Research, coupled with a monochromator, Optoscan Monochromator, Cairn Research), and an Orca Flash 4.0 Hamamatsu CCD camera for epifluorescence imaging. The native infrared differential-interference contrast (DIC) path of the Olympus microscope allowed DIC imaging on the CCD.
The holographic photoactivation laser source consisted of a conventional pulsed Ti:Sapphire laser, used at 920 nm (pulse width: 100 fs, repetition rate: 80 MHz, Mai-Tai, Deep-See, Spectra Physics).
The holographic path was analogous to the one described for setup 1: a beam expander enlarged the beam in front of the spatial light modulator (LCOS-SLM X10468-02), whose plane was projected at the back focal plane of a 40×-NA 0.8 objective (LUM PLAN FI/IR, Olympus) by an afocal telescope (f=750mm, Thorlabs #AC508-750-B and f=500mm Thorlabs #AC508-500-B). The holographic beam was coupled to the optical axis of the microscope by a dichroic mirror (FF670, SDi01, 25×36 mm, Semrock). Photostimulation light pulses were generated by a Pockels cell (350-80, Conoptics).
This system was built around an Olympus BX51WI upright microscope, capable of widefield epifluorescence imaging using illumination with an arc lamp, (OptoSource Illuminator, Cairn Research, coupled with a monochromator, Optoscan Monochromator, Cairn Research), and an Orca Flash 4.0 Hamamatsu CCD camera for epifluorescence imaging. The native infrared differential-interference contrast (DIC) path of the Olympus microscope allowed DIC imaging on the CCD.
The holographic photoactivation laser source consisted of a conventional pulsed Ti:Sapphire laser, used at 920 nm (pulse width: 100 fs, repetition rate: 80 MHz, Mai-Tai, Deep-See, Spectra Physics).
The holographic path was analogous to the one described for setup 1: a beam expander enlarged the beam in front of the spatial light modulator (LCOS-SLM X10468-02), whose plane was projected at the back focal plane of a 40×-NA 0.8 objective (LUM PLAN FI/IR, Olympus) by an afocal telescope (f=750mm, Thorlabs #AC508-750-B and f=500mm Thorlabs #AC508-500-B). The holographic beam was coupled to the optical axis of the microscope by a dichroic mirror (FF670, SDi01, 25×36 mm, Semrock). Photostimulation light pulses were generated by a Pockels cell (350-80, Conoptics).