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: May 2019- See the MIRA light engine in action at VT’s School of Neuroscience!

MIRA on the Brain

At Virginia Tech’s School of Neuroscience, Professor Ian Kimbrough provides students with hands-on experience of real-world neuroscience research techniques including immunohistochemistry, neuroanatomy, and microsurgery, just to name a few. Fluorescence microscopy is the fundamental and essential tool underpinning these techniques. Professor Kimbrough’s teaching laboratory has a fleet of ~20 Nikon Eclipse E200 fluorescence microscopes equipped with Lumencor MIRA light engines®. The MIRA light engine provides powerful, stable, and responsive fluorescence excitation for visualization of coronal mouse slices to elucidate cellular definition of mouse brain anatomy. The images below are representative of many images obtained by the 2018 cohort of Professor Kimbrough’s neuroscience laboratory class using the MIRA light engine. A video presentation including more images can be viewed here. The coronal mouse brain sections were stained using immunohistochemical techniques to fluorescently label various brain cell types and structures: neuronal nuclei (blue), neurons (red), astrocytes (green), and NISSL bodies (yellow).


Submit your best microscopy images in Lumencor’s 2019 Earth Day Light Microscopy Competition. Winning submissions have the potential to earn you up to $10,000 worth of high tech, solid state lighting.

Download the PDF of Light BYTES: May 2019

LOOK ON THE BRIGHT SIDE AT ASCB | EMBO, DECEMBER 8-12, 2018

Lumencor’s best-in-class, solid-state illuminators for imaging cell structure and function are better than ever. Head to BOOTH 521 in the San Diego Convention Center to learn how broad spectral content, brightness and precise electronics are illuminating even the most demanding applications.

Here’s a product preview of Lumencor illuminators at the show:


NEW CELESTA LIGHT ENGINE®

  • Seven independent lasers: 408, 445, 473, 518, 545, 635, 750 nm
  • ~1 watt per output, 7 watts total
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  • Well suited for spinning disk confocal microscopy, photoactivation, optogenetics and super resolution microscopy

NEW SPECTRA III LIGHT ENGINE®

  • Eight independent solid-state light sources: Optimized excitation for DAPI, CFP, GFP, YFP, Cy5, mCherry, Cy5, Cy7
  • As much as 8X more power per color than predecessor product
  • Optical power stabilization for exceptional reproducibility and quantitation

NEW SOLA SE U-nIR LIGHT ENGINE®
NOW WITH ADDED CY7 EXCITATION

  • UV-visible-nIR light output: 350–760 nm
  • ~4.0 W white light through 3 mm dia liquid light guide
  • On/off and intensity control via hand-held control pod or PC
  • Stable, quiet, cool, long-lived lamp replacement
  • No maintenance, no consumables, mercury–free
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