Widefield microscopy is characterized by the illumination of the complete specimen and differs from confocal microscopy where only a small spot is illuminated. Its variants, especially widefield fluorescence microscopy, are among the most applied techniques in life sciences. For example, the detection of various fluorescence markers in a specimen—fixed or alive— can deliver insight into many biological processes such as the human immune system.
The human body exhibits both innate and adaptive immune responses upon microbial invasion. Whereas the adaptive response develops a highly specific reaction over time, the innate response immediately reacts on special structures that are found on pathogens. They are called pathogen-associated molecular patterns (PAMPs). Lipopolysaccharides (LPS), which are specific to the cell walls of gram-negative bacteria, are a typical example of such a pattern. LPS binds to a special receptor on the surface of human cells—the toll-like receptor 4 (TLR4). Once bound, it can trigger an intracellular process ending up with the production of pro-inflammatory proteins called cytokines (Figure 1).
One step during this intracellular process is the translocation of the protein NF-κB from the cytoplasm into the cell nucleus. In its role as a transcription factor, NF-κB supports the transcription of the relevant cytokine genes. The produced cytokines afterward prompt white blood cells (leukocytes) to release reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can kill bacteria and viruses. Unfortunately, ROS/RNS also cause collateral damage and can have negative effects on the cells themselves. Injured by ROS/ RNS, tissue cells release so-called damage-associated molecular patterns (DAMPs), which themselves can trigger TLRs, starting the whole process again and leading to chronic inflammation. This auto-amplifying toll-like receptor radical cycle (TLR radical cycle) can be associated with a number of diseases such as Parkinson’s disease, stroke, depression, (auto)immune disorders, or chronic obstructive pulmonary disease (COPD), to name but a few [1,2].
Besides the abovementioned intrinsic activation by DAMPs, there is also potential for substances originating from other processes to start the cycle. Examples are pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), ROS such as H2O2 produced by further cellular processes, and radical inducing radiation.
Untangling the molecular processes of inflammation can be supported using an inverted research microscope. This article describes the relevance of widefield microscopy for our team. Initially we took the purchase of a confocal laser scanning microscope into consideration. Instead, a widefield fluorescence microscope in combination with deconvolution was chosen for practical reasons, such as short training time for students who join the group for only a limited period of time. Moreover, widefield fluorescence microscopy has very good live-cell imaging capabilities and applies relatively low light intensities, resulting in reduced cellular stress.
A widefield microscope equipped with various fluorescence filter cubes, dedicated objectives, fast filter wheels, climate control, and convenient imaging software can shed light on many facets of the TLR radical cycle. Starting from plain brightfield microscopy through to immunofluorescence experiments, and live-cell and FURA imaging, we can investigate important phenomena, such as the fate of affected tissues in animals, the triggering of TLR4 with LPS and subsequent translocation of NF-κB to the nucleus, and the Ca2+ reaction upon inflammation.
Materials and Methods
Cell culture. For immunofluorescence imaging of NF-κB, HeLa cells stably transfected with human TLR4, MD-2 and CD14 were used. Cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10%