Introduction

Optogenetics is a technique which uses light activated recombinant ion-channels such as channelrhodopsin (ChR2) or halorhodopsin (NpHR) introducing into excitable cells. Light activation of these molecules leads to influx of ions which induces turning neurons on or off selectively overcoming the membrane potential. Halorhodopsin and channelrhodopsin together enable multiple-color optical activation, silencing, and desynchronization of neural activity, creating a powerful neuro engineering toolbox.

STIMULATION AND IMAGING

For the stimulation of these molecules we offer LED light source illuminating the entire FOV, continuous wavelength (CW) and IR laser illuminating along various patterns. While the LED is built above the objective to the external light path, the laser beam(s) is coupled to the existing internal light path with a dichroic mirror. The optomechanical design enables the light path to be optimized for all wavelengths. All types of stimulation lights are used simultaneously with the imaging femtosecond IR laser too. The time of switching between the stimulation and imaging moves on sub-millisecond scale, and the PMT protection during the stimulation is established by a built-in shutter system (gated PMT).

Microscope solutions for optogenetics studies

FemtoS-Resonant equipped with LED light source module

For full field illumination

With the highly flexible X and Y mirrors of the galvanometric scanners coupled with the special electronic boards of Femtonics it became possible to scan along any arbitrary scanning pattern. Thus this panel can precisely follow the dendritic arbor of interest using i.e. multiple line scanning method. By limiting the scanning to these regions of high information content while omitting the space between the scanning regions and the background area, both the scanning speed (up to 2 kHz) and the SNR can be increased. See more at FemtoS-Galvo.

FemtoS-Galvo equipped with Multiple beam module

Stimulation along ROIs

To stimulate cells or subcellular components selectively, the best solution is to steer the laser beam rapidly through optimised scanning patterns, such as point, line, spiral, zigzag, etc. by FemtoS-Galvo. Beside of the scanning along points and lines, which allows stimulation on in precise locations on spines or dendrites, the spiral and zigzag patterns covering larger region enable enough molecules to stimulate on the soma. As Multiple beam module, we offer continuous laser tuned 473 or 561 nm for ChR2/NpHR activation. Photostimulation with LED light source and precise two-photon activation of these molecules is also a viable option with FemtoS-Galvo. The multiple beam module is available to Femto2D-Galvo too.

FemtoS-Dual

Simultaneous photoactivation and imaging

3D trajectory scanning is a spatial extension of galvanometric line scanning mode for which the microscope has been equipped with Piezo objective positioner and controlled Roller coaster software module. This advanced scanning mode enables collection of signals from i.e. 25 µm range with 150 Hz which is fast enough to follow changes in the Ca2+-level and to resolve biological functionality through the 3D space.

Holographic stimulation

Stimulation in 3D

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. Our Holographic module can be equipped to Femto2D-Galvo.

The simultaneous illumination is established by diffractive Spatial Light Modulator (SLM). The SLM has a parallel-aligned nematic liquid crystal layer in which 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.

Features

  • 3D pattern activation in a 350 x 400 x 400 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

References

An optogenetic toolbox for unbiased discovery of functionally connected cells in neural circuits. Dominique Förster, Marco Dal Maschio, Eva Laurell, and Herwig Baier, Nature Communication (2017)