Tool box of red fluorescent proteins for various imaging techniques: A comparative evaluation

Session

LSA.7 - Pathology, immunocytochemistry and biomolecular labelling

Authors

Dr. Ralph Palmisano (1), Dr. Philipp Tripal (1), Dr. Tina Fraass (1), Dr. Benjamin Schmid (1), Dr. Zoltan Winter (1)

Affiliations

1. Optical Imaging Centre Erlangen - FAU

Keywords

Fluorescent proteins, Intra-vital imaging, mCarmine, mScarlet, Multi-photon, Three dimensional imaging

Abstract text

With the advent of mScarlet¹ and mCarmine², novel bright monomeric red fluorescent proteins (FP), the potential for cellular imaging has been extended. Red and far red excitable fluorescent proteins have become the FPs of choice when it comes to questions that are in need off deep penetration of tissues, organoids or in intra-vital imaging. This is fostered by the availability of tunable multi-photon lasers delivering excitation wavelength’s up to 1500 nm. Further the red fluorescent variant mScarlet and the far red fluorophore mCarmine were constructed to reduce the limitations of the classical red FPs such as RFP or DsRed. 

We have investigated the localisation accuracy of novel red FPs on different intra-cellular structures such as cytoplasm, actin, a-tubulin, peroxisomes, H2A or mitochondria, compared to established sub-cellular stainings like SiR-DNA, Phalloidin, Mitotracker, Cellmask, anti-a-tubulin antibody, BacMam. Furthermore, we compared the performance of red FPs using different fixation methods (PFA, Methanol e.g.). Our observation showed no evidence of any unexpected localisation or unwanted artifacts on fixed or living cells, which were due to the labeling with FPs.

For a comparative study, all red FPs were fused to LifeAct and expressed under control of a CMV promotor. By use of these constructs, we were able to compare the following properties of novel red FPs. We investigated multiphoton excitation, STED applicability, stability and the impact of fixation on the fluorescent properties of respective fluorophores. Furthermore, we imaged LifeAct-red FPs in living cells over time to evaluate their impact on viability and bleaching characteristics. This evaluation enables the appropriate chose of red FPs for the respective experiment. As there is no red FP, exhibiting all the desired preferences, we offer a tool-box to choose the appropriate fluorescence label for the respective approach.

From our findings we conclude mScarlet to have the power to be the new standard red-fluorescent protein in particular for single or multi-photon live cell imaging and furthermore also to become extremely helpful for future in-vivo imaging on small animals like mouse or light-sheet based imaging technologies. In cell cultures, mCherry2 was well tolerated by the cells and the new far-red fluorescent protein mCarmine could have its advantage, when penetration depth is required. Our finding by investigation of the respective properties give evidence to make use of mScarlet and mCarmine in two colour deep penetration imaging either using single or multi-photon excitation as they can be well separated by their emission spectra.

References

1) Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M, Aumonier S, Gotthard G, Royant A, Hink MA, Gadella TW Jr.

mScarlet: a bright monomeric red fluorescent protein for cellular imaging

Nat Methods. 2017 Jan;14(1):53-56. doi: 10.1038/nmeth.4074.

 

2) Fabritius A, Ng D, Kist AM, Erdogan M, Portugues R, Griesbeck O. 

Imaging-Based Screening Platform Assists Protein Engineering

Cell Chem Biol. 2018 Dec 20;25(12):1554-1561.e8. doi: 10.1016/j.chembiol.2018.08.008.