Abstract
Fluorescence microscopy is advantageous for investigating biological processes and mechanisms in living cells. One of the most important considerations when designing an experiment is the selection of an appropriate fluorescent probe. Equally important is deciding how the probe will be attached to the protein of interest. The advantages and disadvantages of different fluorescent probe types and their respective labeling methods are discussed to provide an overview on selecting appropriate fluorophores and labeling systems for fluorescence-based assays. Protocols are outlined when appropriate.
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References
Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Kluwer Academic/Plenum, New York
Lippincott-Schwartz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91
Jonkman J, Brown CM (2015) Any way you slice it-a comparison of confocal microscopy techniques. J Biomol Tech 26(2):54–65
Vogel SS, Thaler C, Koushik SV (2006) Fanciful FRET. Sci STKE 2006(331):re2
Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120(Pt 24):4247–4260
Marks KM, Nolan GP (2006) Chemical labeling strategies for cell biology. Nat Methods 3(8):591–596
Schneider AFL, Hackenberger CPR (2017) Fluorescent labelling in living cells. Curr Opin Biotechnol 48:61–68
Chaiyen P, Scrutton NS (2015) Special issue: flavins and flavoproteins: introduction. FEBS J 282(16):3001–3002
Blacker TS, Duchen MR (2016) Investigating mitochondrial redox state using NADH and NADPH autofluorescence. Free Radic Biol Med 100:53–65
Shu X et al (2009) Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324(5928):804–807
Chalfie M et al (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805
Zhang J et al (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918
Toseland CP (2013) Fluorescent labeling and modification of proteins. J Chem Biol 6(3):85–95
Javois LC (1999) Direct immunofluorescent labeling of cells. Methods Mol Biol 115:107–111
Fili N, Toseland CP (2014) Fluorescence and labelling: how to choose and what to do. Exp Suppl 105:1–24
Wang Y et al (2015) Excited state structural events of a dual-emission fluorescent protein biosensor for Ca(2)(+) imaging studied by femtosecond stimulated Raman spectroscopy. J Phys Chem B 119(6):2204–2218
Tang L et al (2015) Unraveling ultrafast photoinduced proton transfer dynamics in a fluorescent protein biosensor for Ca(2+) imaging. Chemistry 21(17):6481–6490
Zhu J et al (2015) Ultrafast excited-state dynamics and fluorescence deactivation of near-infrared fluorescent proteins engineered from bacteriophytochromes. Sci Rep 5:12840
Marini A et al (2010) What is solvatochromism? J Phys Chem B 114(51):17128–17135
Ha T et al (1999) Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism. Proc Natl Acad Sci U S A 96(3):893–898
Thorn TLK. Fluorescent protein properties. www.fpvis.org/FP.html. Accessed July 2019
Eggeling C et al (1998) Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis. Anal Chem 70(13):2651–2659
Shaner NC et al (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22(12):1567–1572
Ono M et al (2001) Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy. J Histochem Cytochem 49(3):305–312
Cordes T et al (2011) Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy. Phys Chem Chem Phys 13(14):6699–6709
Henriques R et al (2011) PALM and STORM: unlocking live-cell super-resolution. Biopolymers 95(5):322–331
Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10):793–795
Heilemann M et al (2009) Super-resolution imaging with small organic fluorophores. Angew Chem Int Ed Engl 48(37):6903–6908
Betzig E et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645
Fernandez-Suarez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9(12):929–943
Kim Y et al (2008) Efficient site-specific labeling of proteins via cysteines. Bioconjug Chem 19(3):786–791
Martinez-Jothar L et al (2018) Insights into maleimide-thiol conjugation chemistry: conditions for efficient surface functionalization of nanoparticles for receptor targeting. J Control Release 282:101–109
Zhang Y, Yu LC (2008) Single-cell microinjection technology in cell biology. BioEssays 30(6):606–610
Zahid M, Robbins PD (2012) Protein transduction domains: applications for molecular medicine. Curr Gene Ther 12(5):374–380
Gautier A et al (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15(2):128–136
Los GV et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6):373–382
Sun X et al (2011) Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. Chembiochem 12(14):2217–2226
Keppler A et al (2004) Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro. Methods 32(4):437–444
Cole NB (2013) Site-specific protein labeling with SNAP-tags. Curr Protoc Protein Sci 73:30.1.1–30.1.16
Griffin BA et al (2000) Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol 327:565–578
Hoffmann C et al (2005) A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods 2(3):171–176
Michalet X et al (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307(5709):538–544
Gu W et al (2007) Measuring cell motility using quantum dot probes. Methods Mol Biol 374:125–131
Bruchez M Jr et al (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281(5385):2013–2016
Shcherbakova DM, Verkhusha VV (2013) Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat Methods 10(8):751–754
Stepanenko OV et al (2017) Interaction of biliverdin chromophore with near-infrared fluorescent protein BphP1-FP engineered from bacterial phytochrome. Int J Mol Sci 18(5)
Rodriguez EA et al (2016) A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods 13(9):763–769
Shemetov AA, Oliinyk OS, Verkhusha VV (2017) How to increase brightness of near-infrared fluorescent proteins in mammalian cells. Cell Chem Biol 24(6):758–766.e3
Ding WL et al (2017) Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2. Biochim Biophys Acta, Mol Cell Res 1864(10):1877–1886
Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182
Shimomura O (2005) The discovery of aequorin and green fluorescent protein. J Microsc 217(Pt 1):1–15
Patterson G et al (2010) Superresolution imaging using single-molecule localization. Annu Rev Phys Chem 61:345–367
Shen Y et al (2019) Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration. Commun Biol 2(1):18
Shaner NC et al (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5(6):545–551
Davidson MW, Campbell RE (2009) Engineered fluorescent proteins: innovations and applications. Nat Methods 6(10):713–717
Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38(10):2887–2921
Hoi H et al (2010) A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. J Mol Biol 401(5):776–791
Lam AJ et al (2012) Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9(10):1005–1012
Fouquet C et al (2015) Improving axial resolution in confocal microscopy with new high refractive index mounting media. PLoS One 10(3):e0121096
Kohl J et al (2014) Ultrafast tissue staining with chemical tags. Proc Natl Acad Sci U S A 111(36):E3805–E3814
Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21(11):1387–1395
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Jacoby-Morris, K., Patterson, G.H. (2021). Choosing Fluorescent Probes and Labeling Systems. In: Brzostowski, J., Sohn, H. (eds) Confocal Microscopy. Methods in Molecular Biology, vol 2304. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1402-0_2
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DOI: https://doi.org/10.1007/978-1-0716-1402-0_2
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