The holographic stimulation is an extremely flexible method which produces simultaneous illumination in variable shape and size of multiple regions in the three-dimensional sample. It is extremely useful for optogenetics, uncaging and other methods that are based on simultaneous activation of various regions.
The simultaneous illumination of different regions is established by diffractive Spatial Light Modulator (SLM). The SLM consists of a parallel-aligned nematic liquid crystal layer which in the crystals disperse and shape the incoming laser beam and change the phase of light. This converted holographic beam excites multiple spots with arbitrary shapes in pre-determined depths simultaneously. The holographic unit is controlled by our MES software, which calculates the corresponding phase-hologram and addresses the pattern to the crystal layer. The holographic unit can be built to Femto2D microscope family.
- 3D pattern activation in a 350x400x400 µm3 stimulating volume (Olympus 20x, NA1)
- simultaneous excitation in several depths with many ROIs (~20-30 cells in 3-5 planes)
- ROIs are defined as polygons with arbitrary shape and size
- lateral resolution less than 3 µm, axial resolution less than 10 µm
- activation time down to 10 µs or 5 ms depending on the shutter device
- repetition rate of SLM: 60 Hz
- large illumination region allows activation of sufficient number of photoactive molecules
- wide laser wavelength range from the visible to the infrared regime
- illumination does not depend on the scanning parameters
- holographic system does not contain mechanically moving parts
Holographic illumination system is developed for photostimulation of neurons by activating light-sensitive ion channels such as channelrhodopsin or halorhodopsin and inducing membrane hiper- or depolarization (optogenetics). This could help to explore fundamental questions in neural connections and networks expanding in the three-dimensional tissue. The large illumination region established by holographic beam allows activation of sufficient number of photoactive molecules. The evoked events can be followed by Femto2D-Galvo.