Extending fluorescence microscopy into anaerobic environments
Graphical abstract
Introduction
Fluorescent proteins (FPs) are among the most widely used tools to investigate a range of in vitro and in vivo biological phenomena. These probes remain one of the most prominent and robust tools in biology due to the simplicity of genetically encoding FPs, which is accomplished by appending an FP gene to a target gene. Both the discoveries of new FPs and protein engineering efforts have generated extensive libraries of FPs with wide ranges of excitation and emission spectra, enhanced stokes shifts and brightness; other FPs have been developed to have useful properties such as photo-conversion [1] and photo-activation [2,3]. FPs have been reviewed extensively in the literature [4, 5, 6, 7].
However, the use of fluorescence microscopy is limited in anaerobic conditions; conventional FPs such as the blue-green GFP [8], the red DsRed [9], and the far-red mKate [10] families of enzymes require oxygen to generate a mature fluorescent chromophore [11]. This essential step precludes the use of a wide range of established FPs in obligate anaerobes. Developing robust fluorescent probes suited for anaerobic imaging would allow biological exploration of anaerobic systems and extend live-cell fluorescence imaging to medically important organisms and microbial communities, including members of gut and soil microbiomes.
This review provides an overview of alternative strategies that have been, or could be, employed to label bacterial cells: these tools include oxygen-independent fluorescent proteins, bioconjugation techniques, and target-based approaches (Figure 1). We discuss the contexts in which these strategies are likely to succeed, and we describe future efforts that could make an approach more robust and easier to employ.
Section snippets
Flavin-mononucleotide-based fluorescent proteins
Flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) rely on the photoactive light-oxygen-voltage (LOV) domain to produce blue fluorescence. Native LOV proteins covalently bind the FMN cofactor and are found in bacterial and plant photosensors. While native LOV proteins are typically non-fluorescent, FbFPs have been engineered to fluoresce [12]. FbFPs have been used in many biological conditions, including as fluorescent markers for labeling anaerobic gut bacteria [13] and hypoxically
Biarsenical–tetracysteine tags
Whereas fusions to traditional FPs may disturb a biological system due to steric bulk, a smaller peptide tag that reacts with a substrate can be used for targeted labeling [28]. For instance, the tetracysteine peptide tag binds to biarsenical substrates [29,30]. This system uses a short sequence of 6–20 amino acid residues that includes a CCXXCC motif, in which four reactive cysteine residues flank two other canonical amino acids. These cysteines can covalently react with either a green
Nanobodies
While antibodies have long been used as important diagnostic and imaging tools in research, nanobodies are an alternative to bulky full-length antibodies and can be used for the same in vitro applications. Nanobodies contain only a single variable domain from heavy chain antibodies (12–15 kDa) and are generated to directly target proteins or nucleic acids or to recognize generalized peptide tags [49•]. Nanobodies must be modified for live-cell imaging in two key ways: (1) they must be
Conclusions
When choosing fluorescence probes, one must consider the intrinsic advantages and disadvantages of each labeling strategy. An ideal probe should balance brightness (for high contrast), minimal biological disturbance (to not affect the interrogated system), specificity (to provide an accurate image) and ease of implementation (such that the highest possible proportion of target molecules are labeled).
Fluorescence microscopy has allowed us to visualize biological phenomena in living systems, and
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by Army Research Office grant W911NF-18-1-0339 to JSB; Army Research Office grant W911NF-16-1-0147, Defense Threat Reduction Agency grant HDTRA1-16-1-0004, and National Institutes of Health grant GM 093088 to ENGM. HEC was also supported by the National Science Foundation Graduate Research Fellowship Program (grant DGE-1256260).
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