Far-Red Fluorescent Proteins: Tools for Advancing In Vivo Imaging

Abstract

Far-red fluorescent proteins (FPs) have emerged as indispensable tools in in vivo imaging, playing a pivotal role in elucidating fundamental mechanisms and addressing application issues in biotechnology and biomedical fields. Their ability for deep penetration, coupled with reduced light scattering and absorption, robust resistance to autofluorescence, and diminished phototoxicity, has positioned far-red biosensors at the forefront of non-invasive visualization techniques for observing intracellular activities and intercellular behaviors. In this review, far-red FPs and their applications in living systems are mainly discussed. Firstly, various far-red FPs, characterized by emission peaks spanning from 600 nm to 650 nm, are introduced. This is followed by a detailed presentation of the fundamental principles enabling far-red biosensors to detect biomolecules and environmental changes. Furthermore, the review accentuates the superiority of far-red FPs in multi-color imaging. In addition, significant emphasis is placed on the value of far-red FPs in improving imaging resolution, highlighting their great contribution to the advancement of in vivo imaging.

Keywords: far-red fluorescent proteins; fluorescence imaging; fluorescent biosensors.

Figure 1

(A) Time-lapse images of mCherry-tubulin in MCF-7 breast cancer cells 5 min before the launch (T-300 s) of the rocket and during the real microgravity (r-µg) phase (T + 177 s−T + 402 s). The green arrows indicate changes in α-tubulin [54] (reproduced with permission from Copyright 2019, Multidisciplinary Digital Publishing Institute). (B) Images of Glb1-2A-mCherry in indicated tissue sections from Glb1^+/m mice. The white boxes show cells with mutually exclusive signals for mCherry and Lamin B1 [55]. (C) Images of Glb1-2A-mCherry in a cohort of Glb1^+/m mice at indicated ages [55]. The images in (B,C) were reproduced with permission from Copyright 2022, Springer Nature.

Figure 2

Structure diagrams of two conformations of mPlum as indicated. The water molecule inside the red circle mediates the hydrogen bonding [57] (reproduced with permission from Copyright 2022, American Chemical Society).

Figure 3

(A) Structure diagrams of the eqFP611 chromophore [61]. (B) Structure diagrams of the chromophore within the β-can fold of eqFP611. The β-can fold starts from blue, increasing to green with increasing residue number. The eqFP611 side chains are in yellow and the chromophore is in magenta [61]. The images in (A,B) were reproduced with permission from Copyright 2003, Elsevier.

Figure 4

(A) Images of Katushka-labeled E. coli TOP10 bacteria in mice [16] (reproduced with permission from Copyright 2019, Springer Nature). (B) Time-lapse images of mito::mKate2 in Hela cells [67]. (C) Images of mito::mKate2 in hind paw of CAG-mito::mKate2⁺ transgenic founder progeny as compared to a negative littermate [67]. The images in (B,C) were reproduced with permission from Copyright 2018, Wiley.

Figure 5

Structure diagrams of Katushka in a cis fluorescent state at pH 8.5 and in a trans nonfluorescent state at pH 5.0. Hydrogen bonds are shown as blue dashed lines, water (W) is shown as red spheres, and van der Waals contacts are shown as black “eyelashes” [68] (reproduced with permission from Copyright 2011, Wiley).

Figure 6

(A) Structure diagrams of the Neptune chromophore. The conjugated π system of the chromophore and side chain changes are shown in stick representation with nitrogen in blue and oxygen in red, and the van der Waals surfaces of water oxygen atoms are depicted as dotted spheres colored light blue [30]. (B) Detailed model of the hydrogen-bonding network. Hydrogen atoms are attached to donors with solid lines and to acceptors with dotted lines [30]. The images in (A,B) were reproduced with permission from Copyright 2009, Elsevier.

Figure 7

(A) Scheme of FRET between EGFP-PTS1 and mCherry-PEX5 [93]. (B) Images of EGFP-PTS1 and mCherry-PEX5 in pex5^−/− cells [93]. The images in (A,B) were reproduced with permission from Copyright 2020, Multidisciplinary Digital Publishing Institute.

Figure 8

(A) Scheme of FRET-GFPRed [94]. (B) Images of FRET-GFPRed and FRET-CFPYPet in two Hela cells of one field before and after ionomycin stimulation [94]. The images in (A,B) were reproduced with permission from Copyright 2020, Multidisciplinary Digital Publishing Institute. (C) Scheme of Booster-PKA [95]. (D) Time-lapse images of Booster-PKA and EKAREV in Hela cells before and after treatments with indicated stimulants and inhibitors [95]. The images in (C,D) were reproduced with permission from Copyright 2020, American Chemical Society. (E) Scheme of mKate2-DEVD-iRFP. (F) Time-lapse images of the donor mKate2 before (0 h) and after indicated treatments [98] (reproduced with permission from Copyright 2022, Springer Nature), Individual cells in (F) are numbered.

Figure 9

(A) Scheme of the mLumin-BiFC system [43]. (B) Images of mLumin-BiFC signals in COS-7 cells with indicated proteins. Scale bar: 20 μm [43]. (C) Images of three pairs of protein interactions in the same living cells with indicated BiFC systems. Scale bar: 10 μm [43]. The images in (A–C) were reproduced with permission from Copyright 2009, Elsevier. (D) Scheme of the mNeptune-BiFC system [101]. (E) Images of mNeptune-BiFC signals in live cells with indicated proteins [101]. (F) Images of mNeptune-BiFC signals in live mice injected subcutaneously with indicated cells [101]. The images in (D–F) were reproduced with permission from Copyright 2014, Oxford University Press.

Figure 10

(A) Scheme of the cpFusionRed-based voltage sensor. S1–S4 are transmembrane voltage-sensitive domains (VSDs). The red barrel is FusionRed. The green and red arrows represent excitation and emission light, respectively [105]. (B) Images of fluorescence changes to single voltage steps and trains of 2.5 Hz and 5 Hz in voltage-clamped PC12 cells [105]. The images in (A,B) were reproduced with permission from Copyright 2017, Public Library of Science.

Figure 11

(A) Scheme of mRFP-eGFP-LC3 [51]. (B) Images of mRFP-eGFP-LC3 in primary neurons. APs (double arrowheads), ALs (arrowhead), and pa-ALs (asterisk) are shown as indicated [51]. The images in (A,B) were reproduced with permission from Copyright 2022, Springer Nature. (C) Scheme of mt-Keima [115]. (D) Images of mt-Keima and mito-QC in the mouse heart following the exhaustive exercise protocol. Mitolysosomes (white arrows) and mitochondria (white boxes) are shown as indicated. Scale bar: 20 μm [115]. The images in (C,D) were reproduced with permission from Copyright 2021, Taylor & Francis.

Figure 12

(A) Images of fluorescent signals in the mKate2, KO2, and AzG channels throughout the mice embryo. The higher magnification images within the white boxes are shown in the original article. Scale bar: 1 mm [67] (reproduced with permission from Copyright 2018, Wiley). (B) Images of fluorescent signals in the CFP, mMiCy, EGFP, YFP, dKEIMA570, and mKeima channels in one Vero cell. Scale bar: 10 μm [49] (reproduced with permission from Copyright 2006, Springer Nature).

Figure 13

(A) Images of NFA-labeled tumor cells in a nude mouse acquired at wavelengths between 585 and 620 nm [125] (reproduced with permission from Copyright 2020, Society of Photo-Optical Instrumentation Engineers). (B) Images of rsFusionRed2–F-Tractin in live U2OS cells with MoNaLISA nanoscopy [127] (reproduced with permission from Copyright 2018, Springer Nature).