Fabrication of Biomimetic Structures by Patterned Ultraviolet Photopolymerization Using the SOLA Light Engine®
Solid state light sources are ideal for controlled initiation of photopolymerization reactions1, which are the basis of widely used techniques for non-contact, in situ fabrication and molding of microscopic structures.For example, a recent publication from a research team at the University of Birmingham2 described the use of photopolymerization of polyethyleneglycol diacrylate (PEGDA) to fabricate in vitro models of bicuspid venous valves inside microfluidic channels.The technique is outlined in Figure 1.Ultraviolet light from a SOLA Light Enginewas passed through a mask that defines the contours of the valve.The extent of PEGDA polymerization and therefore the elasticity of the model valves was controlled by varying the UV exposure time or the concentration of photoinitiator.Ghost particle velocimetry (GPV), a bright field light scattering technique, was used to map the flow of aqueous nanoparticle suspensions in the proximity of the flexible valves.The findings provided insights into the effects of flow patterns around the valves on the pathogenesis of deep vein thrombosis (DVT).Notably, the capability to fabricate valves with leaflets of different elasticity revealed how asymmetrical stiffness links to the aggregation of particles behind the valve leaflet, replicating the association typically found in vivo.
Figure 1. (A) Fabrication of model venous valves by patterned photopolymerization of PEGDA. Controlled polymerization is driven by UV light from a SOLA light engine in the presence of the photoinitator 2-hydroxy-2-methyl propiophenone. (B) Image of a model bicuspid valve. Red arrow indicates the direction of fluid flow. Scale bar = 100 μm. Reproduced from Schofield et al. (2020)2 under the terms of the Creative Commons Attribution License.
1 CB Jaffe, SM Jaffe, GS Tylinski; Solid state light source for photocuring. US Patent 9,217,562; European Patent 2,861,342.
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.
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
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.
Method of the Year: MERFISH Imaging using the CELESTA Light Engine®
Nature Methods journal recently selected spatially resolved transcriptomics as Method of the Year for 2020.Spatially resolved transcriptomics is a collective term for a set of techniques directed towards a single goal – molecular level characterization of single cells within the spatial context of the tissues that they inhabit. This maintenance of spatial context is crucial for understanding key aspects of cell biology, developmental biology, neurobiology and tumor biology.MERFISH (multiplex error robust fluorescence in situ hybridization) is one of the most prominent spatially resolved transcriptomics techniques.MERFISH is an imaging technique that profiles cell populations based on identification of thousands of RNA transcripts per cell.The CELESTA Light Engine is an ideal and widely-adopted illumination source for this application.In a recent paper published in Cell , Xiaowei Zhuang and co-workers at Harvard University used MERFISH and a CELESTA Light Engine to simultaneously image more than 1000 genomic loci with nascent RNA transcripts of more than 1000 genes residing in these loci and landmark nuclear structures, including nuclear speckles and nucleoli. This approach allows exploration of the relationship between chromatin organization, transcriptional activity, and nuclear structures in single cells.The paper also provides detailed descriptions of the procedures for performing the multiple cycles of probe hybridization and imaging required for multiplexed detection of thousands of DNA and RNA sequences.