Optical stimulation and fluorescence detection.
Olfactory bulb, hippocampal and cortical experiments. Blue light
stimulation at 470 nm (430-495 nm) was performed using a Dual Port
OptoLED (CAIRN, UK) dichroic mirror 495 nm at a power of 13, 5 or 1 mW
measured at the output of the x40 objective. Power loss at the working
distance (2.5 mm from the objective), due to the presence of ACSF, was
empirically estimated at 13%. The average power density in the tissue
was estimated by dividing the power at the working distance by the
empirically measured illumination area (~ 7
mm2 ) which gives 1.61 mW/mm2 for a
output power of 13 mW , 0.62 mW/mm2 for 5 mW and 0.12
mW/mm2 for 1 mW. The excitation light for the detection of tomato
expressed in cortical parvalbumin interneurons was produced by a white
light LED (Dual Port OptoLED, CAIRN, UK) filtered at 545/25 nm and
reflected on the sample by a dichroic mirror of 570 mn (Zeiss). The
emission light was filtered at 605/70 nm (Zeiss filter). To avoid an
effect of the light for tomato detection on neuronal activity, only
short light pulses (<100 ms) at power < 0.2 mW were
used. The illumination frequency was lower than 0.5 Hz, the total time
of green led illumination was lower than 2s and a minimum of 3 min was
applied between green and blue light stimulation.
Striatal experiments. Light was delivered under the control of
the acquisition software via the x40 microscope objective lens using
wide-field 475 nm LED illumination (Spectra Light Engine, Lumencor,
Optoprim).