How to Use 3D Photostimulation for Optogenetics

In essence, the process of optogenetics refers to the introduction of light-activated recombinant ion channels, like channelrhodopsin (ChR2) or halorhodopsin (NpHR), into excitable cells. Once these molecues are light-activated, an influx of ions occurs, which therefore induces the selective turning of neurons on or off.

Once together, halorhodopsin and channelrhodopsin facilitate multicolor optical activation, silencing, and desynchronization of neural activity. This effectively generates a powerful neuroengineering toolbox. Visible or infrared light can be used to induce the photostimulation, whilst a femtosecond IR laser performs the imaging.

Switching between imaging and stimulation happens at a sub-millisecond scale. A significant element to note of this process is that a built-in gating system protects the detectors during the simulation.

 

Femto3D Atlas

 

3D Photostimulation and Imaging

By selecting a scan area covering one or more somata situated anywhere in the 3D field of view by the FEMTO3D Atlas Dichro extension, photostimulation can be interlaced with imaging. Indeed, the 3D chessboard scanning mode can perform stimulation: an advanced experimental approach, the 3D chessboard scanning mode extends 3D random-access point scanning into small local planes.

Maximum stimulation efficiency is allowed by distributing the stimulation energy homogeneously onto the somata of the selected cells, which thereby minimizes the risk of photodamage to the targeted cells. The channelrhodopsin expressed in the cells is excited by the laser beam used for photostimulation, which then elicits a controlled burst of action potentials.

A second scanning pattern with larger regions can be selected for calcium imaging, which incorporates all cell bodies with the surrounding areas. Once this is done, the evoked activity can even be followed during sample movements.

The software GUI and its various protocols can precisely control the timing of the photostimulation and the imaging. Once undertaken like this, the connectivity of the network elements can be studied and correlated with the network function.

ChrimsonR-mRuby2 and GCaMP6f expressing cells were stimulated and imaged by the 3D chessboard scanning method in the cortex of a mouse in vivo. Red square: stimulation of a cell at 10 x 10 µm, green squares: imaging of cells at 25 x 25 µm/cell in the presented plane (continuous line) and at other Z depths (dotted line). Stimulation was performed with 10 x 1 ms pulses (10 Hz repetition rate) for higher efficiency, imaging was performed with 12 Hz.

ChrimsonR-mRuby2 and GCaMP6f expressing cells were stimulated and imaged by the 3D chessboard scanning method in the cortex of a mouse in vivo. Red square: stimulation of a cell at 10 x 10 µm, green squares: imaging of cells at 25 x 25 µm/cell in the presented plane (continuous line) and at other Z depths (dotted line). Stimulation was performed with 10 x 1 ms pulses (10 Hz repetition rate) for higher efficiency, imaging was performed with 12 Hz.

Full-Field Illumination

How to Use 3D Photostimulation for Optogenetics

 

FEMTOSmart Resonant equipped with LED light source

The LED source can stimulate the entire FOV above the objective. LEDs are available at many different wavelengths, exciting ChR2 at 473 nm or NpHR at 561 nm. Homogenous illumination is provided by the light source, and the light impulses are both highly repeatable and precisely timed.

The changes over the whole field of view are followed by the FEMTOSmart Resonant microscope at a resolution of 31 frames per second.

FEMTOSmart Resonant microscope

 

Stimulation Along ROIs

FEMTOSmart Resonant microscope

 

FEMTOSmart Galvo equipped with Multiple beam path extension

The FEMTOSmart Galvo, equipped with a secondary laser (Multiple Beam Path) is the best solution to the problem of stimulating cells or subcellular components; here, it is used to steer the laser beam rapidly through optimized scanning patterns including point, line, spiral, zigzag.

This scanning along points and lines therefore facilitates stimulation on exact locations on spines or dendrites, whilst the zigzag and spiral patterns can cover larger regions, thus enabling a greater number of molecules to be stimulated simultaneously on the soma, resulting in activation.

Femtonics offers a continuous laser, which is tuned to 473 or 561 nm for ChR2 or NpHR activation. Another viable option is the precise two-photon activation of these molecules.

Concurrent Photoactivation and Imaging

FEMTOSmart Dual equipped with Multiple beam path extension

Both a galvo and a resonant scanner are incorporated in the FEMTOSmart Dual microscope, which can then work together to combine the advantages of both tools. The FEMTOSmart Dual is therefore the most applicable solution for photostimulation and high-speed imaging.

FEMTOSmart Dual

 

References and Further Reading

About Femtonics Ltd.

Femtonics focuses on the research and development of two-photon laser scanning microscopes for the booming area of cutting-edge brain research and pharmaceutical development. Our specialty is represented by the acousto-optical scanner-based Femto3D Atlas microscope which takes the ability to scan the three-dimensional sample with astonishing speed and thereby it is unique on the market.

In the field of traditional galvanometric and resonant scanner-based systems, we present our customers the flexibility and freedom to customize their own products according to their vision and objective.

The high-valuable measurement and analysis solutions of our MES control software enable scientists to perform a wide variety of experiments. A well-selected microscope working together with the appropriate software modules shapes the customer’s idea into a remarkable product.

Our technology is pioneering and innovative in the field of microscopic imaging. It has been awarded at high levels namely by winning a number of grants and professional prizes and confirmed by our publications in the most prestigious international scientific journals and our presentations at the major conferences.

However, the main achievements for us are customer satisfaction and the scientific results and breakthroughs produced by our microscopes. We are proud of the fact that many results on the field of neuroscience have been published in the highest quality international scientific journals with the tools that we have designed.


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Last updated: Feb 7, 2020 at 7:40 AM

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