Light BYTES: June 2020

Dichroic Mirrors and Filters for SPECTRA, CELESTA, and ZIVA Light Engines®

In fluorescence microscopy, the filter set, consisting of excitation and emission bandpass filters and a dichroic beamsplitter, plays a critical role. It performs the essential functions of directing excitation light from the light source to the sample and then separating it on the basis of wavelength, from fluorescence emitted from the specimen. The filter set consists of excitation and emission bandpass filters and a dichroic beamsplitter. Optimized filter sets are critical because fluorescence emission from a microscopic specimen is many orders of magnitude (>106) weaker than the excitation from the light source. Recognizing optimized filter set specifications are critical for obtaining images with high signal:background contrast. Lumencor has developed uniquely high performance, multiband dichroic beamsplitters and multiband and single band emission filters for use with our SPECTRA, CELESTA, and ZIVA light engines®. These light engines are setting the standard for high performance, high brightness, turn-key solutions in solid-state lighting for life and industrial sciences.

All emission filters and dichroics have standard dimensions and are supplied in an unmounted form. Installation in filter cubes, filter wheels, or other instrumentation-specific mountings is required before use.

The multiband emitters and dichroics are optimized for compatibility with the electronically selectable excitation outputs of SPECTRA, CELESTA, and ZIVA light engines® (Figure 1). This enables fast multicolor imaging without the need for filter wheels or other positioning devices to execute filter interchanges. Single bandpass filters are offered for use in situations where fluorescence crosstalk (e.g. detection of FITC fluorescence derived from violet (DAPI) excitation) confounds identification of fluorescently labeled components of the specimen.    

Figure 1. Transmission spectra of CELESTA/ZIVA VCGRnIR pentaband dichroic and emitter superimposed on the violet, cyan, green, red, and near-infrared output lines of the CELESTA and ZIVA light engines®.


Lumencor’s Earth Day Light Microscopy Imaging Competition is in full swing, offering the opportunity to win up to $10,000 worth of state-of-the-art, solid-state lighting! Submit your images today and help us celebrate and promote a brighter, greener planet through the use of mercury-free illumination. Qualifying images must be acquired using Lumencor light engines.

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Light Bytes: Application SpotLIGHT

Multiplexed imaging of gene expression using the CELESTA light engine

MERFISH (multiplex error robust fluorescence in situ hybridization) is an imaging technique that profiles cell populations based on the identification of thousands oRNA transcripts per cell.  The CELESTA light engine is an ideal and widely-adopted illumination source for this application.  In a recent paper published in Nature [1], Wheeler and co-workers used MERFISH imaging with a CELESTA light engine to quantify the expression of nine specific astrocyte and T-cell markers.  Five of the CELESTA light engine’s seven laser lines were used in the highly multiplexed MERFISH imaging protocol.  The overall objective of the research described in the paper was to characterize astrocyte populations that contribute to pathogenesis in a preclinical model of multiple sclerosis.

Reference
[1]
MA Wheeler, JR Moffitt, IC Clark, EC Tjon, Z Li, SE J Zandee, CP Couturier, BR Watson, G Scalisi, S Alkwai, V Rothhammer, A Rotem, JA Heyman, S Thaploo, LM Sanmarco, J Ragoussis, DA Weitz, K Petrecca, JR Moffitt, B Becher, JP Antel, A Prat, FJ Quintana, Nature (2020) 578:593–5990

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Light BYTES: May 2020

Independent Intensity and Pulse Width Control for Stroboscopic Illumination

Evaluation of photo-stimulation intensity dependence is often a necessary part of neuromodulation experimentation utilized in optogenetics studies [1]. The inherent stability and quantitative nature of Lumencor’s SPECTRA X light engine make it particularly well suited as the pulsed light source of choice for studies requiring pulse width and frequency of stroboscopic illumination analyses. Find more detail regarding this extremely stable, reproducible, and well-behaved data, as well as a specific reference in a recent Journal of Physiology publication by authors Kubota, Sidikejiang, and Seki, on Lumencor’s website.

Figure Description: Alternating cyan (485/25 nm, ~0.5 ms) and green (560/32 nm, ~3 ms) output pulses generated by TTL triggering of a SPECTRA X light engine. Two superimposed oscilloscope traces are shown in which the cyan intensity is adjusted from 100% to 55% via RS232 serial commands while the green intensity remains constant. Separation of the cyan and green pulses is ~0.25 ms.

Reference

[1] S Kubota, W Sidikejiang, K Seki et al. J Physiol(2019) 597:5025–5040

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Light BYTES: March 2020

Lumencor’s Best Practices
During COVID-19

Lumencor’s manufacturing operations remain healthy and active. While daily operations are inevitably constrained by measures taken to foster good health and minimize the transmission of COVID-19, productivity remains high. Minimal component shortages have developed as shelter-in-place practices have dampened our suppliers’ activities. However, we expect our supply chain will likely suffer additional shortages. Lumencor’s practices now include the quarantine of all receivables as our first commitment is to ensure the safety of our employees while safeguarding their work. In some cases, that may mean that our deliveries to customers will be delayed. We will do our best to inform all customers with pending orders of changes to anticipated shipment dates. Please notify us of any urgency around your order and we will do our very best to assist.

Please be assured that we are watching and managing your business with great care during the current emergency. Please contact to our sales, customer service or technical support representatives if you have any questions or wish to discuss deliverables for your organization’s needs.


A Spectacular Triple Play: LIDA Light Engine®, NIS Elements and sCMOS Cameras for Color Light Microscopy

For histologists, clinical pathologists and anyone seeking improvements in the speed, sensitivity and precision of transmitted light microscopy, Lumencor’s LIDA light engine in combination with Nikon NIS Elements software enable high-speed color imaging data without the need for a dedicated color camera. Instead, monochrome images generated by sequential triggering of the LIDA’s red, green and blue light sources by a sCMOS camera are processed by NIS Elements, delivering video-rate color output. These capabilities allow rapid and fully automated imaging of large tissue sections, as illustrated below. The software also enables convenient switching between camera-synchronized RGB illumination and white-light illumination for ocular viewing. Our application note RGB Color Imaging using the LIDA Light Engine and NIS Elements outlines hardware set-up for Hamamatsu ORCA-Flash4.0 and Andor Zyla sCMOS cameras and provides instructions for image acquisition control using NIS Elements software.

Color image of a 1.5 cm x 1 cm section of adenocarcinoma from human breast acquired using Lumencor’s LIDA light engine and NIS Elements software. Image courtesy of Dr. Michael Weber (Harvard Medical School).

Lumencor’s LIDA light engine mounted on the transillumination port of a Nikon Ti2 microscope with Andor Zyla 5.5 megapixel sCMOS camera


Submit your best microscopy images in Lumencor’s 2020 Earth Day Light Microscopy Competition

Winning submissions have the potential to earn you up to $10,000 worth of high tech, solid state lighting.



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Light BYTES: February 2020

Precise power regulation on each of
Lumencor’s newest and brightest light engines:
AURA®, SPECTRA®, CELESTA®, and ZIVA light engines®

In addition to high power and intense brightness, output power regulation is one of the many advanced control features incorporated in Lumencor’s next generation products: SPECTRA, AURA, CELESTA, and ZIVA light engines. To use power regulation, a desired power reference value in milliwatts is entered in the onboard control GUI, as shown in the attached link. To activate power regulation, click the padlock icon next to the reference power value. Gray shading of the padlock icon and the reference power value shows that power regulation is active for the selected output channel. When power regulation is active, the intensity setting for the channel is controlled by the onboard microprocessor, based on feedback from the light engine’s reference photodiode array. The microprocessor continuously adjusts the intensity setting so that the output power matches the power reference value set by the user.

Output power regulation settings in the CELESTA light engine control GUI

Performance of the output power regulation feedback system of a SPECTRA light engine is illustrated below. The response time of the feedback system is approximately one second. Output power regulation allows users to eliminate variations in light output due to temperature fluctuations and other environmental factors in photometric and quantitative imaging applications where reproducibility and accuracy are essential.

Teal (510/25 nm) channel output from a SPECTRA light engine with and without power regulation. Power output from the light guide was monitored with a Coherent PowerMax II-TO power meter with model PM3 thermopile detector.


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Light BYTES: November 2019

ZIVA® versus CELESTA®: It’s all about the fiber

In 2018, Lumencor introduced the CELESTA light engine incorporating 7 individually addressable laser light sources. The ZIVA light engine, introduced at the 2019 Society for Neuroscience meeting in Chicago, while similar in some respects, is distinctive in being designed to couple into smaller bore optical fibers.

The output of the ZIVA light engine is suitable for structured illumination microscopy (SIM) and other super-resolution microscopy techniques. In these applications, it provides an alternative to more costly and hard-to-align single mode laser sources. The larger illumination field of the CELESTA light engine is preferred for applications such as spinning disk confocal microscopy, MERFISH or smFISH.


Common Features of ZIVA and CELESTA Light Engines

  • Same 7 laser lines (405 nm, 446 nm, 477 nm, 520 nm, 546 nm, 638 nm, 749 nm)
  • Same compact 15 cm x 35 cm footprint
  • Same onboard microprocessor-based control and feedback interface
  • Same laser safety interlock configuration

100X widefield (a) and structured illumination microscopy (b) images of actin (green) and mitochondria (orange) in fixed bovine pulmonary endothelial cells. From Pospíšil et al, GigaScience (2018) 8:1–12


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