When using techniques like qPCR to analyze gene expression, some cell types exhibit substantial heterogeneity, which might restrict resolution. The advent of single-molecule RNA FISH in recent years has allowed for the detection of RNA expression at the single-cell level, yet classical confocal imaging lacks the throughput required to explore smaller fractions of cell populations.
RNA FISH method also allows immunofluorescence to visualize the expression of targets that are hard to stain at the protein level.
The Hermes high content wide field microscope allows for the examination of subcellular features at the single cell level while working with large sample sizes.
This method generates a vast number of photos, necessitating the use of automated computational analysis. Quantification of characteristics per cell can reveal new information about cell biology.
In this study, researchers used the WiScan® Hermes high content microscope to look at immunological responses to stimuli in primary human monocyte-derived macrophages, which have a lot of variation in gene expression.
Materials and methods
Human monocyte-derived macrophages (MDMs) were activated for 4 hours with the innate immune agonist’s Zymosan, LPS, or Poly I: C on Day 7 before being fixed (Figure 1). Tumor Necrosis Factor Alpha (TNF-α) and Interleukin-10 (IL-10) mRNA were stained with ViewRNA FISH kits (Thermo Fisher) as indicators of pro-inflammatory and regulatory signaling, respectively.
Figure 1. Outline of the experiment. Image Credit: IDEA Bio-Medical Ltd.
Before quantification with Metamorph 7 (Molecular Devices) and R, cells were observed using a Hermes Wiscan® (IDEA Bio-Medical) high content microscope.
As RNA expression in individual cells was frequently too high to allow for individual spot counting, mean fluorescence intensity (MFI) was measured in a zone surrounding each cell’s nucleus (Figure 2).
Figure 2. Image analysis strategy. A ring-shaped zone of interest was generated around each nucleus. Mean fluorescence intensity inside each zone was determined for each RNA stain. Image Credit: IDEA Bio-Medical Ltd.
This allowed for a representative sample of RNA expression without the use of a secondary cell mask or individual spot segmentation. Since single monocyte-derived macrophages are highly heterogeneous, at least 5,000 cells were examined for each condition to evaluate the total population.
Results
The Hermes system quickly and simply supplied a sufficient sample size for analysis, scanning 1116 pictures in 11 minutes (captured 31 images in each of the 12 wells at 20× magnification, three fluorescence channels per field). In the absence of stimulation, MDMs expressed few copies of IL-10 and TNF mRNA.
Based on the stimulation, the cytokine response varied greatly. There was a lot of variation in the levels of expression among individual cells (Figure 3).
Figure 3. Representative images from Hermes automated wide field microscope for RNA FISH (20× magnification) in macrophages stimulated with innate immune agonists. Nuclei were stained with DAPI (blue) while IL-10 (green) and TNFα (red) were stained with ViewRNA FISH. Image Credit: IDEA Bio-Medical Ltd.
While many cells did not express IL-10 or TNFα, RNA labeling in each cell indicated that a minority of cells produced substantial amounts of cytokine RNA (Figure 4). Poly I: C generated a mild TNFα response with low IL-10 expression, but LPS and Zymosan induced expression of both.
Figure 4. Quantitation of staining for IL-10 and TNF at the single-cell level. Density represents the proportion of cells with a specific MFI. Image Credit: IDEA Bio-Medical Ltd.
Zymosan elicited significantly more TNFα than LPS, although LPS produced a greater level of IL-10 induction. These discrepancies could be due to the distinct pattern recognition receptors that initiate responses to each ligand (TLR4 for LPS, TLR2/Dectin-1 for Zymosan, and TLR3 for Poly I:C), but they could also be due to dose-dependent expression.
Within each cell, the expression of the two cytokines could also be directly compared (Figure 5). This demonstrated a preference for expressing TNFα and IL-10 together instead of IL-10 separately, which could be due to IL-10’s role as an immunomodulatory cytokine produced during the inflammatory response.
Figure 5. Scatter/density plot of expression of IL-10 RNA against TNF RNA. Percentages of events within each segment of a plot are indicated. Image Credit: IDEA Bio-Medical Ltd.
Conclusion
The Hermes system allowed for high-throughput imaging of RNA FISH labeling, allowing for single-cell quantification of cytokine expression. The cytokine response varied depending on the stimulus, with a tendency for IL-10 and TNF to co-express.
Future research should look at the impact of stimulus dose on the ratio of inflammatory and regulatory cytokine signaling, as well as expand the assay to include other markers.
Data courtesy of David Stirling and Matthew Solomons (Noursadeghi Lab, Division of Infection and Immunity, University College London).
About IDEA Bio-Medical Ltd.
IDEA Bio-Medical is founded in 2007 through a partnership between YEDA (the Weizmann Institute’s commercialization arm) and IDEA Machine Development (an innovation hub).
We specialize in automated imaging systems and image analysis software, offering a broad range of biological applications based on the company’s unique algorithms library. The company is developing novel image-based screening platforms for the pharmaceutical industry and medical centers, dedicated to broadening the scope of personalized medicine.
Our WiScan Hermes system incorporates the most advanced technologies currently available in the machine vision field, integrated with engineering methodologies of high reliability and quality at the level of semi-conductors and digital printing industries, which are the specialty of our mother company, IDEA Machine Development Design and Production Ltd.
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