Focus on Fluorescence Imaging
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Focus on fluorescence imaging Principles and practice of microscope techniques hen Sir George Gabriel Stokes first described the phe- CONTENTS methods nomenon of fluorescence in 1852 it is doubtful many COMMENTARY people ever considered its potential as a tool for biolo- W 902 Fluorescence gists. As often happens with new discoveries, however, scientists microscopy today figured out a way to exploit this physical process and began to .com/nature Rafael Yuste e use fluorescent molecules as biological labels. In concert with microscopy this permitted previously undreamt-of possibilities PERSPECTIVE .natur w for detection and visualization. 905 A guide to choosing Central brain hemisphere and Notably, immunofluorescence techniques became powerful fluorescent proteins eye-antenna disc of Drosophila tools for inquisitive biologists. Recently, the description of new Nathan Shaner, Paul http://ww melanogaster at the third larval Steinbach & Roger Tsien imaging modalities such as confocal and two-photon micros- stage stained with three Alexa copy, the creation of new fluorescent molecules, and the discov- oup dyes and imaged on a Zeiss LSM r REVIEWS G 510 Meta. Image courtesy of ery and exploitation of fluorescent proteins have triggered an 910 Fluorescence Veta Trunova, The Johns Hopkins explosion in fluorescence microscopy techniques. This has also University. microscopy resulted in their use within living systems where they are revo- Jeff Lichtman & José- lishing b lutionizing biological imaging. A sampling of seminal papers Angel Conchello Pu documenting this advance is available in the ‘Classics Library’ 920 Optical sectioning on the Focus website. microscopy The application of fluorescence techniques to living speci- José-Angel Conchello & Nature Jeff Lichtman 5 mens presents many challenges to users. There is thus a greater 932 Deep tissue two- 200 need for users to understand the principles of fluorescence photon microscopy © and how different imaging systems work so biologists can fully Fritjof Helmchen & exploit this revolution. Determining the most suitable equip- Winfried Denk ment and reagents for an application, and properly designing 941 Fiber-optic fluorescence imaging and conducting experiments both require a clear understanding Benjamin Flusberg, of the basic principles involved. This Focus is intended to pres- Eric Cocker, Wibool ent this basic information as well as practical advice that biolo- Piyawattanametha, gists need to use these powerful techniques in their work. Juergen Jung, Eunice Cheung & Mark Schnitzer Fluorescent proteins. Image We are pleased to acknowledge the support of Carl Zeiss courtesy of Roger Tsien, University Microscopy as principal sponsor and Intelligent Imaging of California at San Diego. Innovations, Inc. as supporting sponsor in producing this focus. As always, Nature Methods carries sole responsibility for all editorial content and peer review. Daniel Evanko Editor, Nature Methods Senior Production Editor Design Erin Boyle Veronique Kiermer Renee Lucas Sponsorship Claire Hines Focus Editor Daniel Evanko Copy Editor Irene Kaganman Marketing Eric Claiborne NATURE METHODS | VOL.2 NO.12 | DECEMBER 2005 | 901 PERSPECTIVE A guide to choosing fluorescent proteins Nathan C Shaner1,2, Paul A Steinbach1,3 & Roger Y Tsien1,3,4 methods The recent explosion in the diversity of available fluorescent proteins (FPs)1–16 promises a wide variety of new tools for biological imaging. With no unified standard .com/nature for assessing these tools, however, a researcher is faced with difficult questions. e Which FPs are best for general use? Which are the brightest? What additional factors .natur w determine which are best for a given experiment? Although in many cases, a trial-and- error approach may still be necessary in determining the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing http://ww down the options. oup r G We can begin by stating several general requirements for For example, the newly released DsRed-Monomer the successful use of an FP in an imaging experiment. (Clontech) is described as “bright,” even though in fact, lishing First, the FP should express efficiently and without toxic- it is the dimmest monomeric red fluorescent protein b ity in the chosen system, and it should be bright enough (RFP) presently available. Pu to provide sufficient signal above autofluorescence to be The perceived brightness of an FP is determined by reliably detected and imaged. Second, the FP should have several highly variable factors, including the intrinsic Nature sufficient photostability to be imaged for the duration of brightness of the protein (determined by its maturation 5 the experiment. Third, if the FP is to be expressed as a speed and efficiency, extinction coefficient, quantum 200 fusion to another protein of interest, then the FP should yield and, in longer experiments, photostability), the © not oligomerize. Fourth, the FP should be insensitive optical properties of the imaging setup (illumination to environmental effects that could confound quanti- wavelength and intensity, spectra of filters and dichroic tative interpretation of experimental results. Finally, in mirrors), and camera or human eye sensitivity to the multiple-labeling experiments, the set of FPs used should emission spectrum. Although these factors make it have minimal crosstalk in their excitation and emission impossible to name any one FP as the brightest over- channels. For more complex imaging experiments, such all, it is possible to identify the brightest protein in each as those using fluorescence resonance energy transfer spectral class (when more than one protein is available), (FRET)17 or selective optical labeling using photocon- as this depends only on the intrinsic optical proper- vertible FPs12,15, additional considerations come into ties of the FP. The brightest proteins for each class are play. General recommendations to help determine listed in Table 1, with greater detail on the properties of the optimal set of FPs in each spectral class for a given each listed protein available in Supplementary Table 1 experiment are available in Box 1, along with more detail online. As discussed below in relation to photostability, on each issue discussed below. the choice of optimal filter sets is critical to obtaining the best performance from an FP. ‘Brightness’ and expression Generally, FPs that have been optimized for mam- FP vendors typically make optimistic but vague claims malian cells will express well at 37 °C, but some pro- as to the brightness of the proteins they promote. Purely teins may fold more or less efficiently. We have not qualitative brightness comparisons that do not pro- done extensive tests in mammalian cells to determine vide clear information on the extinction coefficient relative efficiency of folding and maturation at 37 °C and quantum yield should be viewed with skepticism. versus lower temperatures, but expression of proteins in 1Department of Pharmacology, 2Biomedical Sciences Graduate Program, 3Howard Hughes Medical Institute and 4Department of Chemistry and Biochemistry, 310 Cellular & Molecular Medicine West 0647, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Correspondence should be addressed to R.Y.T. ([email protected]). PUBLISHED ONLINE 18 NOVEMBER 2005; DOI:10.1038/NMETH819 NATURE METHODS | VOL.2 NO.12 | DECEMBER 2005 | 905 PERSPECTIVE bacteria at 37 °C versus 25 °C gives some indication of the relative Photostability efficiencies. These experiments suggest that there are several pro- All FPs eventually photobleach upon extended excitation, though teins that do not mature well at 37 °C. Indications of potential fold- at a much lower rate than many small-molecule dyes (Table 1). In ing inefficiency at 37 °C should not be taken with absolute certainty, addition, there is substantial variation in the rate of photobleaching however, as additional chaperones and other differences between between different FPs⎯even between FPs with otherwise very simi- mammalian cells and bacteria (and even variations between mam- lar optical properties. For experiments requiring a limited number malian cell lines) could have substantial influences on folding and of images (around 10 or fewer), photostability is generally not a maturation efficiency. major factor, but choosing the most photostable protein is critical Generally, modern Aequorea-derived fluorescent proteins (AFPs, to success in experiments requiring large numbers of images of the see Supplementary Table 2 online for mutations of common same cell or field. AFP variants relative to wild-type GFP) fold reasonably well at A unified characterization of FP photostability has until now been 37 °C⎯in fact, several recent variants have been specifically opti- lacking in the scientific literature. Although many descriptions of methods mized for 37 °C expression. The UV-excitable variant T-Sapphire6 new FP variants include some characterization of their photostability, and the yellow AFP (YFP) variant Venus1 are examples of these. the methods used for this characterization are highly variable and the The best green GFP variant, Emerald18, also folds very efficiently resulting data are impossible to compare directly. Because many FPs at 37 °C compared with its predecessor, enhanced GFP (EGFP). have complex photobleaching curves and require different excitation .com/nature e The only recently developed AFP that performed poorly in our intensities and