When selecting an optogenetic light source, you will consider two key factors: the required light wavelength and intensity. 

First, the opsin you select has a specific activation spectrum, such that an opsin will only be activated by certain wavelengths of light. An opsin usually has a peak wavelength, at which it  will require the least power to elicit a response. Wavelengths farther away from the peak wavelength  will require a more powerful output to elicit a response. Therefore, it’s best to choose a light source closer to the peak wavelength to achieve optimal activation. For example, most researchers will use wavelengths close to 470nm for optogenetic activation of ChR2.

Second, the required light intensity for your experiment is dependent on the field of view and the opsin you select. The field of view, which is dependent on the optical fiber (for in vivo experiments) or the microscope objective, will determine how large of an area you need to cover with your light source. Opsins have an intensity threshold for eliciting a response resulting from illumination. These two variables are directly coupled, such that optimal illumination power is required to elicit a response and increasing the field of view requires more power as you have to cover a larger area with a given minimum intensity.

Now that you understand the requirements for an optogenetic light source, let’s discuss the two most commonly used light sources for this application: LED light sources and laser sources. You may be wondering, what are the differences between these light sources? Which light source is best for my optogenetics experiment?

LED Light Source

LEDs are a prime choice for optogenetic experiments for a multitude of reasons. To start, LEDs are low cost, eye safe and have a long lifetime, making them a great choice for any lab looking to perform optogenetics.

In addition, LEDs come in a wide-range of wavelengths (UV to NIR), and hence scientists can usually find the appropriate LED that best fit their choice of opsins. Depending on the wavelength, the available output power can vary significantly between LEDs. In the visible range, LEDs can produce a large amount of power, making them an ideal solution for most optogenetic experiments. 

Compared to other typical microscopy light sources, such as halogen or xenon lamps, LEDs have a low operating temperature and do not require any heat up or cool down time. This allows LEDs to be easily turned on and off for easy use during experiments, and scientists can easily modulate the intensity or pulse the LEDs without worry about their stability or causing damage. In addition, LEDs have a much longer lifetime than traditional light sources, and they do not require frequent replacement of bulbs, making them a more sound investment in the long-term.

Importantly for optogenetic experiments, LEDs can be switched on and off with microsecond timing for pulsing protocols. LEDs are easily triggered via TTL to synchronize pulsing with external equipment.

Compared to a laser source, LEDs have relatively high divergence, causing the power of the LED to be spread across the field of view, although on this front LEDs are far better than the traditional light sources such as a mercury lamp or a halogen lamp. As a result, LEDs are not ideal for applications such as laser-scanning systems in which light must be tightly focused into a tiny spot in order to provide the necessary spatial resolution for point-by-point scanning across the field of view. Secondly, LEDs, compared to lasers, also have a wider spectral bandwidth. This may or may not have a negative impact on your experiments, as you can easily block off the tails of the LED using optical filters. However, using optical filters could lead to a lower overall output power from the LED, as the output power is based on the full spectrum of the LED light source.

LEDs are suitable for a wide-range of optogenetic experiments. A popular use of LEDs is for widefield optogenetics on a microscope. LEDs are easily integrated into upright and inverted microscopes, and they provide sufficient output power for stimulation of all optogenetic probes. In addition, LEDs are easily synchronized with electrophysiological equipment. 

LEDs are also ideal for freely-behaving optogenetics experiments, as they can be coupled with an optical fiber while providing sufficient power for such experiments, long lifetime, and low cost. 

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Optogenetic Stimulation: The Ultimate Guide

Laser Source

Lasers are commonly used for optogenetic experiments that require higher intensity and/or tighter focus of light. Lasers provide very high intensity in a single spot, and hence they are often used for laser-scanning applications. Compared to LEDs, lasers have a very narrow spectral width, enabling users to gain high intensity at a specific wavelength. In addition, lasers can be well collimated to fit on a microscope, or can be  efficiently coupled to an optical fiber for optogenetic experiments that require high optical power.

Although lasers provide high intensity for optogenetics experiments, there is a list of potential drawbacks. In itself, high output power may be a pitfall for specific optogenetic experiments. In particular for standard microscopy experiments, lasers may provide too much output power and cause tissue damage. In addition, for microscopy, the beam must be expanded to cover across the field of view and must be corrected for laser speckle. 

Unlike LEDs, lasers have a limited availability of wavelengths; they are also much more expensive than LEDs, and may require eye safety measures such as interlock etc.

Due to a higher optical coupling efficiency, lasers are particularly useful for performing optogenetics across a large area, such as a macroscope. Lasers can also be used for in vivo optogenetic applications where high intensity may be needed; however, most LEDs are suitable for in vivo optogenetics applications. 

Where lasers can outperform LEDs is for laser-scanning and two-photon microscopy optogenetic experiments, as they can provide high-intensity in a small spot at longer wavelengths.

Next Post

Widefield vs. Cellular-Resolution Optogenetics: What’s the Difference?

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Free eBook Download
Optogenetic Stimulation: The Ultimate Guide