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.
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.
Lumencor, Inc. has been named 2020 Product Innovation of the Year honoree by the Portland Business Journal for our next generation SOLA light engine®. Each year, the PBJ honors the region’s top manufacturing companies who drive the economy with innovation, excellence and productivity. The new generation SOLA features increased power, longevity, stability and robustness over the projected 15 year life time with no replacement parts. Lumencor’s SOLA light engine is used in fluorescence microscopy for life science and materials science applications… Read Press Release
To support the long-term stability of the laser light sources in CELESTA, SPECTRA and ZIVA light engines it is recommended that they should be operated only in environments where the dew point is below 15ºC. For reference, at a typical room temperature of 24ºC, a dew point of 15ºC corresponds to 57% humidity. The current dew point inside the light engine, calculated from onboard temperature and humidity sensors, is displayed on the settings page of the onboard control GUI.
Standby Mode, previously described in the October 2019 issue of Light Reading, is another control system designed to support the long-term stability of the laser light sources. Consequently, users writing their own light engine control software are strongly advised to NOT programmatically disable standby mode.
If operational situations arise where it is necessary to avoid the onset of standby mode during a data acquisition process, please contact email@example.com. We will be happy to work with you and our software engineering team to devise appropriate solutions.
Lumencor can support your laboratory as you return to work. The challenges are numerous for researchers and instrument manufacturers alike- maintain best lab practices, manage constrained budgets, keep staff safe and productive. Let Lumencor help with illumination tools designed for the best data quality and best lab safety!