Abstract
GFP and its homologs are widely used as genetically encoded labels in many in vivo imaging applications. The fluorescent proteins emitting in the longer wavelength part of the spectrum provide even more opportunities, as they ensure less autofluorescence background, higher fluorescence resonance energy transfer efficiency and much more effective light penetration for deep imaging of animal tissues. Evrogen offers a set of exceptionally bright red and far-red fluorescent proteins optimized for different applications.
Main
Fluorescent proteins have proven to be indispensable tools for visualizing biological processes. Being genetically encoded, fluorescent proteins do not require additional chemicals to become fluorescent and can be precisely targeted to a specific tissue, cell or cell organelle. They can also be used for tagging proteins inside living cells, allowing in vivo studies of protein localization, movement and interactions.
Evrogen's fluorescent protein color palette covers the entire visible range from blue to far-red and consists of two main groups: TagFPs and TurboFPs. TagFPs are monomeric fluorescent proteins optimized for work with fusions. TurboFPs are dimeric fluorescent proteins that are not recommended for tagging proteins, but often perform better in other applications, such as labeling cells and cell organelles, tracking promoter activity, and whole-body imaging. In the long-wavelength part of the spectrum, monomeric TagRFP1 and mKate2 (ref. 2) and dimeric TurboRFP1 and TurboFP635 (Katushka3) have advantageous properties and excellent performance.
Main properties
One of the most important properties to consider when choosing a fluorescent protein for most applications is the brightness. Table 1 summarizes the brightness values for Evrogen red and far-red fluorescent proteins in comparison with their closest competitors.
All four Evrogen proteins are characterized by high resistance to low pH, allowing their use for labeling acidic organelles. Far-red TurboFP635 and mKate2 are extremely photostable under both wide-field and confocal illumination, which makes them ideally suited for long-term time-lapse imaging. The absence of cytotoxicity caused by overexpression of Evrogen's red and far-red fluorescent proteins has been confirmed in long-term experiments, including the generation of stable cell lines and transgenic animals.
All tags can be visualized using generally available light sources (either mercury arc lamp or appropriate laser lines) and most common filter sets. Among Evrogen fluorescent proteins, only TagRFP has been tested so far in two-photon laser scanning microscopy (TPLSM)4. This work showed that TagRFP has higher two-photon cross-section and brightness than all competitor proteins.
Use for protein labeling
The exceptional brightness of TagRFP and mKate2 enables their use for in vivo protein labeling both independently and as additional colors in multicolor applications. We provide examples of fluorescence imaging of TagRFP and mKate2 fusions (Fig. 1). Neither tag affects normal localization of the fused protein, even in quite sensitive systems such as α-tubulin. No visible aggregates, nonspecific localization or cytotoxic effects were observed in the tested cell lines.
FRET applications
Fluorescence resonance energy transfer (FRET) using a donor-acceptor pair of two fluorescent proteins fused to proteins of interest is a powerful technique that allows in vivo studies of protein interactions in living cells. The traditional cyan and yellow FRET partners have several substantial drawbacks limiting their utility for such application, such as relatively low dynamic range (donor/acceptor emission ratio change) and difficulties with spectral separation. Using TagRFP as an acceptor for the Evrogen green fluorescent protein TagGFP2 ensures higher FRET efficiency and more reliable spectral separation of the donor and acceptor fluorescence. Shifting the wavelengths toward the red part of the spectrum reduces input of cellular autofluorescence. The excellent performance of TagRFP in FRET applications had been demonstrated5 both in vitro and in vivo.
Whole-body imaging
Deep-tissue imaging using fluorescent proteins allows direct and noninvasive observation of the biological processes inside living organisms. The favorable 'optical window' for the visualization in living tissues is approximately 650–1,100 nm. Within this optical window, TurboFP635 and mKate2 are the brightest fluorescent proteins available so far (Table 1). An experimental study6 shows that the signal coming from TurboFP635 located deep inside tissue is about 45 times stronger than the signal from GFP and 2 times stronger than the signal from the closest far-red competitor protein tested (Product F in Table 1). Together with excellent photostability and fast maturation, this makes TurboFP635 and mKate2 the proteins of choice for whole body imaging (Fig. 2).
Conclusions
Using Evrogen red and far-red fluorescent proteins for in vivo imaging ensures bright signal and low background. Their excellent performance in protein labeling applications, FRET-based studies and whole-body imaging has been proven experimentally. Additional product information, images and movies are available on the Evrogen website, http://www.evrogen.com/.
References
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Kelmanson, I. Enhanced red and far-red fluorescent proteins for in vivo imaging. Nat Methods 6, iii–iv (2009). https://doi.org/10.1038/nmeth.f.249
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DOI: https://doi.org/10.1038/nmeth.f.249