Featured Article: Spectral, Spatial, and Temporal Output Characteristics of Light Sources for Optogenetic Stimulation

Spectral, Spatial, and Temporal Output Characteristics of Light Sources for Optogenetic Stimulation

Iain Johnson of Lumencor explains Optogenetic techniques developed over the past 15 years, that have served neuroscientists well.

They provide spectrally and spatially resolved data of the functional complexity of neural networks, while avoiding direct physical interrogation using historically ubiquitous microelectrodes1. Optogenetics addresses cells and cellular function using non-destructive illumination, rather than invasive electrodes. ‘Genetics’  refers to transgenic expression of photoactivatable ion channel proteins, required to transduce light input into electrical activity in the cells of interest. 

Figure 1: Oscilloscope recording of alternating 485nm (~0.5ms width) and 560nm (~3 ms width) output pulses generated by TTL triggering of a Spectra X light engine (Lumencor, USA). Two superimposed oscilloscope traces are shown in which the 485nm intensity is adjusted from 100 to 55 per cent via RS232 serial commands while the 560nm intensity remains constant. Temporal separation of the 485 and 560nm pulses is ~0.25ms.

Illumination sources used for optogenetic stimulation must meet certain requirements in terms of spectral, spatial, and temporal output characteristics. This article considers these characteristics in relation to the performance of solid-state LED- and laser-based light engines. 

The primary spectral output requirement is maximal intersection of the light engine  spectral output with the action spectra of photoactivatable ion channel proteins.  Currently, the most commonly used spectral outputs are 475nm for stimulation with  channelrhodopsin (ChR2) and 575nm for inhibition with halorhodopsin (NpHR). LED-based light engines provide spectral outputs that are temporally discrete (figure 1) but spatially coincident. Additional spectral outputs facilitate use of novel photoproteins, and provide for excitation for fluorescent markers and voltage sensitive dyes, in parallel with optogenetic stimulation or inhibition.

For example, deeper tissue penetration can be attained by using near-infrared (>700nm) light for optogenetic  stimulation2. It is common practice to use co-expression of yellow fluorescent protein (YFP) to enable localization of ChR2 by fluorescence microscopy3. The choice of YFP is dictated by the fact that its fluorescence excitation (~510nm) is sufficiently wavelength-separated from photo-stimulation of ChR2 (~475nm) that the two processes can be independently initiated. 

For in vitro experimentation on brain slice or cultured neuron preparations, LED-based light engines coupled to fluorescence microscopes provide wide-field illumination  suitable for simultaneous stimulation of thousands of neurons. For stimulation of  ChR2, an irradiance threshold of ~1mW/mm2 at the sample plane is required, a level which is towards the lower end of the range used for conventional fluorescence microscopy. Spatially selective illumination can be generated by addition of a digital micro-mirror device (DMD)4

For selective stimulation of individual cells, lasers that can be focused to diffraction limited spot sizes <10µm are required. In vivo experiments provide the added capacity to observe complex behavioral outcomes elicited by optogenetic stimulation. Light is typically delivered through a 200µm diameter multimode optical fibre, connected to a cannula implanted in the brain of an animal subject. Lasers provide better coupling efficiency into these optical fibres than LEDs, and are therefore the preferred light sources for in vivo optogenetics. 

The electrical activity of neurons is modulated on the millisecond timescale. Therefore, illumination sources used for optogenetic stimulation must have the capacity for optical modulation on the same timescale (figure 1). A recent publication by Kubota and co-workers3 takes full advantage of the temporal and output power control provided by the Spectra X Light Engine (Lumencor, USA) to characterize photo-stimulation of ChR2-expressing rat dorsal root ganglion (DRG) neurons. Effects of varying light pulse durations (0.1 to 10ms) at constant intensity and varying stimulation intensities (2 to 78mW) at constant pulse width were recorded. 

In addition, stimulation by pairs of 0.5ms pulses separated by varying intervals from 5 to 20ms was used to assess the effects of ChR2 desensitization. 

Quantitative comparisons of optogenetic and conventional electrical stimulation are included, providing a useful point of reference for readers previously unacquainted with the capabilities of optogenetic techniques. To find out more please visit www.lumencor.com.

References

1 ES Boyden F1000 Biol Rep (2011) 3:11

2 S Chen, AZ Weitemier, TJ McHugh et al. Science (2018)

3 59:679–684 3 S Kubota, W Sidikejiang, K Seki et al. J Physiol (2019) 597:5025–5040

4 Zhu, O Fajardo, RW Friedrich et al. Nat Protoc (2012) 7:1410–1425


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Light BYTES – March 2021: MAGMA Light Engine®: Solid State Illumination for Solar Test Platforms

MAGMA Light Engine®: Solid State Illumination for Solar Test Platforms

Artificial light sources are essential for performance validation in photovoltaic device manufacturing and for characterization of properties such as photoconductivity and quantum efficiency in the development of new photovoltaic materials. Traditionally, characterization of photovoltaic devices has employed xenon arc or halogen lamps to approximate the solar spectrum. However, their spectral output is not readily amenable to controlled adjustment, and long duration (weeks to months) tests are limited by their relatively short operating lifetimes. Lumencor’s MAGMA Light Engine employs modern solid state illumination technology to overcome these limitations. Within a compact 15 cm x 35 cm footprint, the MAGMA Light Engine incorporates 21 individually addressable LED light sources, ranging from 365 nm to 1050 nm, under the control of an onboard microprocessor. The LED outputs are merged into a common optical train directed to the light output port on the front panel. Adjustment of the relative output intensities of the 21 elements of the LED array enables synthesis of user-specified spectral distributions, such as the AM1.5G solar spectrum.


Download the PDF of Lumencor lightBYTES: March 2021

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