Introduction

To truly understand the intricacies of how cells carry out processes requires the ability to precisely alter the activity of specific cells at specific times. This precise control is now possible by combining optics and genetics, also known as optogenetics.

To alter cellular behavior, genes that code for light-responsive proteins known as opsins are inserted into cells. Photo-excitation or photo-inhibition of these proteins causes them to alter cell function in specific ways, allowing scientists to observe the effects that such changes have on cellular activity.

The light source used for photoexcitation or photo-inhibition is a key part of the optical setup for optogenetic studies. Well-defined spectral, temporal, and spatial control is important as well as homogenous and constant illumination. In experiments involving multiple opsins, narrow emission spectrum is important to selectivity activate each opsin. In some experiments illumination stability is very important because any fluctuations or “hot spots” may cause inconsistent protein activation in the cells under illumination.

Optogenetics illumination sources include lasers and LEDs, and photoactivation can be done under a microscope or via a fiber for in vivo applications. Using a Prizmatix LED illumination system for optogenetics studies provides many advantages compared to laser-based systems including:

• Illumination Homogeneity and Stability
• Opsin Selectivity
• Light Switching
• Illumination Intensity Control
• Versatility

Illumination Homogeneity and Stability

Prizmatix Microscope LED , and Ultra High Power Microscope LED (UHP-Microscope-LED) systems feature high-end LED drivers that ensure stable output from the LED. All driver circuits are current sources that provide stable current for LED operation. The power is stable over time because the LED thermal management features high-end heat sinks and even fans if necessary.

Because LED systems don’t have a resonant cavity like lasers, they don’t exhibit the noise related to emission modes or instability from back reflections that can be found with diode lasers.

Opsin Selectivity

In experiments involving multiple opsins a narrow emission spectrum from the illumination source is important to achieve selective photoactivation. The emission spectrum of most LEDs is from 10 to 30 nm, a spectral width that is ideal for selective activation of multiple opsins. Interference filters can be used to achieve a narrower spectrum.

Light Switching

Optogenetics studies require well-defined temporal control, in other words the light source must be turned off and on in a very fast and precise manner. Most DPSS laser systems with TTL modulation input become instable at fast switching rates, so mechanical fast shutters are necessary.

Prizmatix LED current drivers feature a direct TTL input for fast switching with a rise/fall time of microseconds, much faster than millisecond pulses required for optogenetics applications. The Prizmatix VHP-Microscope-LED and UHP-Microscope-LED series feature a standard fast opto-isolator at the TTL input that ensures complete isolation of the sensitive electrophysiology electronics from the LED driver electronics.

Illumination Intensity Control

The output of LEDs is directly dependent on current, yet most LEDs do not perform well at low currents. To precisely control illumination levels most commercial LED systems use pulse width modulation (PWM). However, PWM is not suitable for most scientific applications such as fluorescence microscopy or the fast switching used for photoactivation in optogenetics. For optogenetics experiments, the LED must be operated with a stable current and the ON/OFF controlled through the TTL input. All Prizmatix LED current controllers feature a constant current operation mode and provide direct TTL modulation. The LED current can manually adjusted using a precise 10-turn potentiometer with a locking dial, or it can be set using a computer through the optional analog modulation input.

Versatility

The Prizmatix Microscope-LED and UHP-Microscope-LED series can be combined in many different configurations to enable multiple or single wavelength outputs for various flexible connection ports, adaptors, and couplers. They can be directly connected to a microscope via epi-fluorescence port adaptors or a Liquid Light Guide (LLG).

The UHP-Microscope-LED-White with the optional Filter Wheel is very versatile because it allows one light source to be connected to a fiber coupler for single wavelength illumination or through a single fiber or multiple fibers for photoactivation with multiple wavelengths.

An OptiBlock Beam-Switcher can be added to a LED system attached to microscope for additional versatility. It enables simple manual switching between two illumination modes. For example, it can be used to switch between direct epifluorescence illumination and fiber optic illumination without disconnecting the LED system from the epifluorescence system.

Fiber optic Rotary Joint is another useful accessory for Optogenetics in vivo studies. It enables coupling of an optical fiber to a freely moving preparation. The Rotary Joint stator is affixed to a cage or above a maze while the rotor side can rotate freely while maintaining unchanged light transmittance.