MICROSCOPY & IMAGING
bioluminescence imaging (BLI) should be viewed as complementary to fluorescence microscopy. Although it cannot provide the same spatial or temporal resolution, it offers the opportunity to make full use of the toolbox of bioluminescence technologies and assays for functional imaging. It is also anticipated that advances in detector technologies are poised to make BLI more accessible, as demonstrated by the recent development of photon counting gCMOS cameras. Te following examples illustrate how BLI can be applied in discovery and assay development.
Fig. 2. BLI of endogenous HiBiT-tagged proteins
55°C and shows no selective partitioning or post-translational modifications in mammalian cells. Although NanoLuc was originally conceived as an improved transcriptional reporter, it soon became evident that it provided the foundation for expanding bioluminescence into entirely new applications in life sciences. Building on the strength of the system, scientists further engineered two binary reporter systems, NanoBiT and HiBiT. Both systems consist of a small peptide (SmBiT or HiBiT) that bind to LgBiT, a truncated version of NanoLuc. NanoBiT, a low-affinity complementation system, has proved valuable for the analysis of protein-protein interactions (PPI). In contrast, HiBiT represents a high-affinity complementation system that allows sensitive detection of tagged proteins and is widely used in the field of targeted protein degradation. Furthermore, combining NanoLuc with suitable fluorophores enables development of bioluminescence resonance energy transfer (BRET)-based assay for quantitative analysis of PPI and protein- specific small molecule target engagement in living cells.
Te utility of NanoLuc technologies is illustrated here using imaging and protein translocation as examples.
BIOLUMINESCENCE IMAGING Bioluminescence is a well-established imaging modality for small animal imaging,
but it has not been used widely in the past for microscopy for several reasons. First and foremost, low light imaging requires long exposure time and/or acquisition strategies such as image stacking, which leads to comparatively poor temporal and spatial resolution, especially in living cells. In addition, the demands of low light detection require the use of specialised and costly equipment. On the other hand, using bioluminescence for microscopy provides significant benefits. Te absence of an exogenous light source eliminates issues with phototoxicity and photobleaching. Bioluminescence is also mostly devoid of sample- associated background, which translates into large dynamic range and high sensitivity. Tese are critical features for detection and analysis of low-expressing endogenously tagged proteins. In this context, it
should be emphasised that
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PROTEIN LOCALISATION CRISPR-mediated tagging of endogenous proteins with peptide tags (e.g. HiBiT) or reporters (e.g. NanoLuc) has proven extremely valuable for the interrogation of proteins in the appropriate physiological context. However, addition of peptide tags or reporters has the potential to impact protein function. Subcellular localisation is one of the indicators of protein function that can be rapidly assessed for HiBiT- or NanoLuc-tagged protein in living cells by BLI without the need for protein-specific antibodies and labour-intensive staining protocols. Furthermore, the ability to conduct BLI in living cells allows analysis
Fig. 3. BRET time-lapse imaging of interaction between beta-arrestin 2 tagged with NanoLuc and Vasopressin receptor 2 tagged with HaloTag
Fig. 4. Imaging with NanoBRET Caspase3 biosensor in HeLa cells (timelapse after addition of Staurosporin)
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