Introduction

To truly understand the intricacies of how cells carry out processes one needs 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 such changes have on cellular activity.

The light source used for photo-excitation 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 selectivily activate each opsin. In some experiments illumination stability is vital 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 Ultra High Power Microscope LED systems (UHP-Microscope-LED) are equipped with high-end LED drivers that guarantee stable output from the LED. All driver circuits are current sources that provide stable current for LED operation. Power is stable over time because LED thermal management offers high-end heat sinks and even fans if needed.

Because LED systems do not have a resonant cavity like lasers, they do not show the noise related to emission or instability modes of posterior reflexes that can be found with diode lasers.

Opsin selectivity

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

Light switching

Optogenetic studies require a well-defined temporal control, in other words the light source must be turned off and turned on very quickly and precisely. Most DPSS laser systems with TTL modulation input become unstable at high switching speeds, so fast mechanical fillings are required.

Prizmatix LED drivers feature a direct TTL input for fast switching with a microsecond rise / fall time, much faster than the millisecond pulses required for optogenetics applications. The Prizmatix UHP-Microscope-LED and UHP-Microscope LEDs are equipped with a standard rapid optocoupler at the TTL input which guarantees complete isolation of the sensitive electrophysiology electronics from the LED driver electronics.

Illumination Intensity Control

The LED output depends directly on the current, however most LEDs do not work well at low currents. To accurately control lighting levels, most commercial LED systems use pulse width modulation (PWM). However, PWM is not suitable for most scientific applications such as fluorescence microscopy or rapid switching used for optogenetic photoactivation.

For optogenetic 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 mode of operation and provide direct TTL modulation. The LED current can be adjusted manually using a precise 10-turn potentiometer with a locking dial, or it can be set using a computer via the optional analogue modulation input.

Versatility

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

The UHP-Microscopio-LED-White. with the optional filter wheel is very versatile because it allows you to connect a light source to a fiber coupler for single wavelength lighting or through a single fiber or more fibers for photoactivation with more wavelengths.

An OptiBlock Beam-Switcher can be added to a LED system connected to the microscope for greater versatility. It allows easy manual switching between two lighting modes. For example, it can be used to switch from direct epifluorescence illumination to fiber optic illumination without disconnecting the LED system from the epifluorescence system.

The optical fiber rotary joint is another useful accessory for in vivo studies of Optogenetics. It allows the coupling of an optical fiber with a preparation in motion. The stator of the rotary joint is affixed to a cage or over a labyrinth, while the rotor side can rotate freely while maintaining the light transmission unchanged. See more on Prizmatix Optogenetics Toolbox page.