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
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.
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.
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 unstable 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 UHP-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.
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 photo-activation 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 permits 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 maintaining unchanged light transmittance.
Read more on Prizmatix Optogenetics Toolbox page.