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.
The Relevance of Time-lapse Imaging of GFP Expression Using the SOLA Light Engine® to COVID-19 vaccine Efficacy
The delivery of mRNA through lipid-based transfection has been a longstanding challenge for the development of RNA therapeutics. Moreover, it has acquired a new and urgent prominence from the development of COVID-19 vaccines consisting of mRNAs encapsulated in lipid nanoparticles by Pfizer/BioNTech and Moderna. It is clearly important to understand the effects of mRNA-lipid complex formulation and extracellular medium composition on downstream expression of the protein immunogen that in turn determines vaccine efficacy.In 2019, before the start of the COVID-19 pandemic, a team of researchers from Ludwig-Maximilians-University in Munich and Stony Brook University, New York described the use of live-cell imaging on single-cell arrays (LISCA) to monitor the onset and rate of GFP expression following mRNA lipoplex transfection .Single cells are arrayed on a micropatterned fibronectin substrate (Figure 1A), incubated with mRNA-lipid complexes for 1 hour and then monitored by time-lapse fluorescence microscopy for 20 hours (Figure 1B).For GFP fluorescence to give an authentic representation of protein expression levels, stable and reproducible excitation is essential, making the SOLA Light Engine the ideal illumination source for this application.As well as characterizing the pronounced cell-to-cell variability in onset times and rates of protein expression (Figure 1), LISCA was used to determine the effect of serum proteins on the cellular uptake of different mRNA-lipid complex formulations.
Figure 1. (A) Single GFP-expressing HuH7 cells arrayed on micropatterned fibronectin. (B) Single cell fluorescence trajectories representing GFP expression. The gray-shaded area represents the initial 1-hour period of incubation with mRNA-lipid complexes. (C) Enlarged region of (B) showing cell-to-cell variation in onset of protein expression. Reproduced from Reiser et al. (2019)  under the terms of the Creative Commons Attribution License.
You asked and we delivered! Lumencor is proud to introduce the newly updated family of SOLA light engines that will continue to set the standards for performance and reliability in white light illumination. This product line now includes 4 standard models that are differentiated by the number of sources and spectral output: SOLA, SOLA FISH, SOLA U-nIR and SOLA V-nIR light engines. The SOLA light engine provides white light output for excitation of DAPI, GFP/FITC, YFP, Cy3, mCherry, Cy5 and spectrally similar fluorophores. In the SOLA FISH light engine, output in the 475–600 nm region is red-shifted to provide optimal excitation for SpectrumGreenTM, SpectrumRedTM and other fluorophores commonly used for fluorescence in situ hybridization (FISH) analysis in cytogenetic testing laboratories. The SOLA V-nIR and U-nIR light engines offer the broadest spectral coverage, including near infrared (nIR) output for excitation of fluorophores such as Cy7 and ICG, and for other applications that benefit from the enhanced tissue penetration of nIR light.
The 3 mm liquid light guide required for delivering the SOLA light engine output to microscopes or other bioanalytical instruments is now automatically included with all purchases. To request a sales quotation for any (or all) of the four new SOLA light engine models, please submit our online quote request form today!
Lumencor’s new Generation of Light Engines: Intensity Control linearity
AURA, RETRA, SPECTRA, CELESTA and ZIVA light engines, as well as the newly refreshed SOLA light engine for 2021, incorporate on-board microprocessors, providing impressive advances in control and monitoring capabilities. One such advancement is linear intensity control. Each of these illuminators generate optical power that has a precisely linear relationship to intensity settings across all colors, providing more quantitive and predictable responses for users. In the case of white light, constant color temperature is achievable regardless of output power. Just another reason customers who care about performance come to Lumencor for solid state illumination.
Parallel Light Engine Performance monitoring Using the Onboard Control GUI
AURA, CELESTA, RETRA, SPECTRA, and ZIVA Light Engines incorporate a control GUI accessed through a web browser via anethernet connection.Image acquisition applications used to control the light engine though connection of either the USB or RS232 serial ports can be run in parallel with ethernet-connected control GUI to aid in trouble-shooting.As shown in Figure 1, this allows the light engine to be controlled by the image acquisition software, while the GUI serves as a passive monitor of the light engine status.
Figure 1. Screenshots of parallel operation of image acquisition software (NIS Elements, left) and CELESTA Light Engine Control GUI (right). A. NIS Elements command to turn on red light output at 21.7% is inoperative. Examination of the GUI display reveals that this is due to an open interlock condition (e.g. no optical fiber connected to the light engine output port). B. After closing the interlock, the same NIS Elements command results in light output, indicated by the filled red channel radio button and non-zero output power reading in the GUI.
The control GUI displays many types of information pertinent to the performance of the light engine that are not accessible in current releases of most image acquisition software packages. These include:
Real-time light output power readouts
Standby mode status
Light engine operating software error messages
Humidity/dew point data
Serial port configuration
TTL port configuration
Cumulative run time data
In cases where the PC being used for image acquisition control has a single ethernet port that is dedicated to internet access, a USB-to-ethernet adapter (Figure 2) can be used for connection to the light engine control GUI.USB-to-ethernet adapters are readily available from online vendors for less than $20.