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

An enlarged surface or the entire FOV can be stimulated with LED light source. LEDs are available with different wavelengths exciting ChR2 at 473 nm or NpHR at 561 nm. The impulses of the light are precisely timed and highly repeatable fitting to optogenetics studies. We offer FemtoS-Resonant microscope to follow the changes on the whole field of view with high speed. FemtoS-Resonant can be equipped with CW and IR laser too for photostimulation. The LED light source is available to Femto2D-Resonant too.

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

FemtoS-Dual microscope contains a galvo and resonant scanners in tandem combined the advantages of galvo and resonant microscopes. It offers possibility to simultaneous photostimulation and high speed imaging during optogenetics studies.

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