SPECTRA Light Engine® Takes the Win Highlighting smFISH
In celebration of Earth Day Lumencor launched its annual Light Microscopy Imaging Competition to highlight Lumencor’s commitment to manufacturing bright, clean, and mercury-free light engines. The tallies are in and we are happy to announce the winners. As always, we are impressed with the breadth and skill it took to capture each of the images submitted in this years competition. For more information on how Lumencor light engines can help you in your research, please contact us.
1st Place – George McNamara and Lauren Blake
Johns Hopkins School of Medicine, Baltimore, MD Light Engine:SPECTRA light engine®
5-plex mouse embryonic fibroblasts expressing MS2-tagged beta-actin mRNA. Red Halo-JF549-NLS-MCP, Green Atto594 POLR2A mRNA FISH, Blue DAPI, Cyan Cy5 beta- actin-MS2 mRNA FISH, Magenta Alexa Fluor 488-anti-DDX6 immunofluorescence. Microscope details at http://confocal.jhu.edu/ current-equipment/fishscope. Specimen preparation by Lauren Blake, Prof. Bin Wu’s lab (JHU Biophysics). Imaging by George McNamara, Ross Fluorescence Imaging Center.
A nostoc discovered in Antarctica autofluorescing under TRITC excitation. This nostoc was found inside a microbial mat in the Dry Valleys of Antarctica. A nostoc is a genus of cyanobacteria with beaded filaments intricately woven inside a gelatinous pouch. This image highlights the structure of the beaded filaments and their scaffolding within the gelatinous pouch.
Cos-7 cells spread on a tension gauge tether (TGT) surface and imaged on a Nikon Ti2 eclipse microscope using a Lumencor light engine and a turret wheel with different excitation and emission filter cubes – The RICM image (panel 1) was taken by removing the emission filter from the path. Cell tension indicated by TGT probe opening (panel 2) and paxillin staining (panel 3).
Control latency limits the response time of all USB-connected microscope accessories, including cameras, stage controllers, filter wheels, and Lumencor light engines. Latency originates from the fact that the supervising PC operating system (usually Microsoft Windows) must allocate time to many competing tasks. As shown in the adjacent data plots, the impact on the user is that the duration of light output on the specimen is longer, and sometimes much longer, than the exposure time set in the acquisition software.
Duration of light output produced by a CELESTA light engine®, and detected by an external analog photodiode, in response to various exposure times set for a Hamamatsu ORCAFlash 4.0 camera controlled by MicroManager (v1.4.23) image acquisition software. Note that the discrepancies between set exposure time and light output duration are not speciﬁc (except in minor details) to any particular image acquisition software or light engine or camera model. Panel A shows data for light engine control via LEGACY mode USB communication. Panel B shows the same nominal exposure sequence controlled via STANDARD mode USB communication.
Two scenarios are shown, one using LEGACY mode USB communication (as implemented on the SPECTRA X and SOLA SE light engines), and the other using STANDARD mode communication (as implemented on the AURA III, SPECTRA III and CELESTA light engines). Because the USB data transmission rate in STANDARD mode is faster than that of LEGACY mode (115,200 vs 9,600 baud), it provides a signiﬁcant reduction of latency for exposure times on the order of 100 ms (and above). However at short exposure times (5–10 ms), the impact of the faster communication speed in STANDARD mode diminishes, as the response is dominated by the software processing speed.
To obtain light output durations less than about 50 ms, timing must be derived from a hardware controller instead of the PC operating system. The hardware controller supplies TTL timing signals to the light engine via a breakout cable (Table 1). Examples of millisecond-duration light pulses generated in this way can be found in the Performance section of our website. At the present time, the capacity to acquire time-lapse sequences of short duration exposures is limited by the camera (modern sCMOS cameras typically have a maximum frame rate of around 100/second), rather than by the light source.
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 ismany 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.
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 . 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.
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
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.
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.