SPONSORED: OPTOGENETICS


Spectral, spatial and temporal output characteristics of light sources for optogenetic stimulation


O


ptogenetic techniques developed over the past 15 years 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. 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


www.electrooptics.com | @electrooptics


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 localisation of ChR2 by fluorescence microscopy3


. The choice of


YFP is dictated by the fact that its fluorescence excitation (~510nm) is sufficiently wavelength-separated from photostimulation 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 widefield illumination suitable for simultaneous stimulation of thousands of neurons. For stimulation of


“Illumination sources used for optogenetic stimulation must have the capacity for optical modulation on the same timescale”


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 micromirror 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 behavioural outcomes elicited by optogenetic stimulation. Light is typically delivered through a 200µm


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.


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


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


characterise photostimulation 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 desensitisation. 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. EO https://lumencor.com


March 2021 Electro Optics 19


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50