High-performance probes for light and electron microscopy

Journal name:
Nature Methods
Year published:
Published online


We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.

At a glance


  1. Probe development and preliminary characterization.
    Figure 1: Probe development and preliminary characterization.

    (a) Schematic of modular smFP construction. sfGFP is shown in cartoon; the chromophore is either left intact or rendered invisible (gray). Epitope tags are inserted at the N (Ins 1, blue sphere) and C (Ins 3, red) termini and into a loop (Ins 2, yellow). (b,c) Fluorescence correlation spectroscopy (FCS) measurements to quantify antibody binding to Flag epitopes of smFP on the basis of changes in diffusion with molecular weight (MW). (b) Calibration of solution-phase diffusion time τD and diffusion coefficient D versus MW measured by FCS. MW markers were hydrolyzed Alexa 488, 534 Da; hydrolyzed Alexa 546, 963 Da; epidermal growth factor–FITC, 6.5 kDa; EGFP, 32.7 kDa; smFP_Flag_bright, 42.3 kDa; bovine serum albumin–Alexa 488, 69 kDa; anti-Flag M2-FITC, 153 kDa. n = 5 experimental replicates; mean ± s.d. shown. Data were fit to a power law: τD = (0.072 ± 0.006) × MW(0.39 ± 0.02) (±s.e.m., R2 = 0.99). Diffusion time and diffusion coefficient are related by D = w02/(8τD), where w0 is the e−2 laser-beam radius; here, w0 = 430 nm at 940-nm excitation. Also shown are literature values of diffusion coefficients of proteins47 and of dyes and proteins measured using scanning FCS48. (c) FCS-determined diffusion times of 10 nM smFP_Flag_bright (with ten Flag copies per protein yielding 100 nM of Flag epitopes) titrated with monoclonal antibodies against GFP (Invitrogen rabbit monoclonal) or Flag (Sigma M2), which shows saturated binding above 100 nM antibody; and 10 nM smFP_3×Flag_bright (30 nM epitopes) with anti-Flag antibody, which shows saturated binding above 30 nM antibody. n = 5 experimental replicates for titrations; mean ± s.d. shown. Ladder at right is the calculated number of antibodies bound to smFP (42.3 kDa) for the corresponding τD on the basis of the calibration obtained in c). (d) Fly brains showing expression and staining of smFP probes. R59A05-GAL4 (ref. 49) was crossed with UAS-myr-smFP flies, with myr-sfGFP as a control. All probes, including sfGFP, lack chromophores. Dissected fly brains were stained with anti-tag antibodies or anti-GFP and Alexa 488–conjugated secondary antibodies, in small volumes (~10 μl); slight differences in staining are likely due to variation in antibody penetration. Scale bar, 50 μm.

  2. Multichannel projection labeling with smFP probes and FPs.
    Figure 2: Multichannel projection labeling with smFP probes and FPs.

    (a) Injection schematic. The right hemisphere was injected with smFP_Flag and the left hemisphere with smFP_Myc. smFP_Flag and smFP_Myc were detected with tag-specific primary antibodies and corresponding secondary antibodies conjugated with Alexa 488 (Myc) and Alexa 594 (Flag). (b) Representative (n = 2 replicates) injection-site image. (c) Confocal image showing a magnified view of the boxed area in b; long-range axonal projections from S1 in both hemispheres cross the corpus callosum. (d) Injection schematic for the four-color tracing. The left hemisphere of a Rbp4_KL100_Cre mouse was injected (all AAV-FLEX-CAG) with two FPs (tdTomato and EGFP) as well as two smFP constructs (smFP_Myc and Ruby2_Flag). Injections targeted four topographic areas: vibrissal sensory (vS1) and vibrissal motor cortex (vM1) as well as limb motor (fM1) and sensory areas (llS1) as indicated. (en) Schematics and images of 80-μm coronal sections. (e,f) Injection sites. Fluorescent neuronal somata in L5 at injection sites in fM1/vM1 (h) and vS1/llS1 (i) are clearly visible, as are long-range axonal projections. Schematics of long-range targets of L5 neurons are provided (g,j,m) alongside images of coronal sections from these planes. Red boxes indicate enlarged areas. (h,i) show corticortical and corticothalamic axons in ectorhinal (Ect) and perirhinal (PRh) cortex as well as thalamus (POm). These descending axons continue in adjacent tracts to the cortical peduncle (cp; hl) and target midbrain regions including substantia nigra reticulata (SNR) and superior colliculus (SC; k,l), as well as the spinal trigeminal nucleus (Sp5; m,n), >5 mm from the injection site. Intermingling of axons of all four colors in the pyramidal tract (py) results in the bright white color in n. Drawings are adapted from ref. 50, Elsevier.

  3. Improved labeling of cells and single-molecule tracking efficiency of proteins in fixed and live preparations.
    Figure 3: Improved labeling of cells and single-molecule tracking efficiency of proteins in fixed and live preparations.

    (ad) Comparison of smFP_Flag (a,b) and EGFP (c,d) labeling of CA1 hippocampal pyramidal cells. (a,c) Fields of view showing sparsely labeled cells, from apical dendrites through soma to distal dendrites. (b,d) Zoom of distal dendrite in boxed areas in a and c. All images were taken under identical confocal settings. Blue shows Hoechst nuclear counterstain. (eg) Images of CA3 'thorns' in brain slices from a 15-d-old mouse, expressing either smFP_Flag (e) or EGFP (f) or filled with Lucifer yellow (LY) (g). Samples were amplified with anti-Flag, anti-GFP or anti-LY primary antibodies and secondary antibodies conjugated to Alexa 488. (hk) Single dendrites of cultured rat hippocampal neurons coelectroporated with smFP_Myc and either N-cadherin–smFP_HA (h,j) or Ncadherin–HA (i,k). The smFP_Myc fills cells cytoplasmically and serves as a control for normalizing expression levels. Blue, anti-Myc primary + Alexa 488 secondary, pseudocolored blue; red, anti-HA primary + Alexa 555 secondary. h,i show combined imaging channels; j,k show the Ncadherin channel alone. (lo) HeLa cells with tracked labeled histone molecules. All images were acquired and displayed with identical parameters. (l) H2B-smFP_Flag–transfected cell with Alexa 488–labeled anti-Flag antibody fragments. (m) H2B-EGFP–transfected cell with Alexa 488–labeled anti-GFP antibody. (n) H2B-HaloTag–transfected cell with Alexa 488 Halo substrate added. (o) Background labeling of anti-Flag antibody. Alexa 488–labeled anti-Flag antibody was added to untransfected cells. (p) Histogram of the intensity of single-molecule tracks from EGFP (green), smFP_Flag (magenta) and HaloTag (black). Arrows point to the smFP_Flag histogram peaks (intensities at ~7K, 14K, 21K, 28K, 35K, 42K and 49K) that give an estimate of the number of bound Fab molecules per tag. The smFP_Flag histogram is fit by with a determined from the fit to the HaloTag histogram (n = 1), and with p = 0.2, consistent with stochastic antibody binding with an average Fab occupancy of 20% (dashed curve). The three solid curves depict the first three components of f(I). Movies corresponding to lo are provided in Supplementary Video 1.

  4. Utility of smFP_Flag for array tomography (AT).
    Figure 4: Utility of smFP_Flag for array tomography (AT).

    (a) Left, large-scale AT z projections showing smFP_Flag labeling of somatostatin-Cre interneurons with cell bodies in CA1 hippocampus stratum oriens (SO) and axonal projections in stratum lacunosum-moleculare (SLM). Center, EGFP+ pyramidal cell signal from the same volume. Right, merged projection. (b) A series of three consecutive ultrathin sections (100 nm) showing an smFP_Flag-labeled varicosity (magenta) forming a putative synapsin (gray) immunopositive synapse onto a distal pyramidal cell dendrite (green) in SLM.

  5. Utility of smFPs for STORM and immunoEM.
    Figure 5: Utility of smFPs for STORM and immunoEM.

    (a,b) Multicolor STORM imaging of smFPs in mouse brain slice. (a) STORM image of IHC-labeled neurites expressing tdTomato and smFP_Myc constructs from an ultracryosection of mouse cortex. (b) smFP_Myc and tdTomato yield comparable STORM images of individual dendritic spines on this L5 pyramidal neuron (bottom panels correspond to dashed boxed region in a. The diffraction-limited conventional image counterparts are shown for comparison (top). (c) Schematic of the double immunogold labeling experiments in mouse brain tissue. The confocal image shows a representative vibratome section expressing green smFP_Flag_bright fluorescence and immunolabeled with anti-Myc (red) to verify expression of both constructs. smFPs were double labeled with 6-nm (Myc) and 12-nm (Flag) colloidal gold particles, and labeling was followed by silver enhancement. HPF-FS, high-pressure freezing followed by freeze substitution; E16.5, embryonic day 16.5. (d,e) smFP_Flag immunogold labeling (yellow pseudocolor) and the resulting serial reconstruction (41 serial sections) of dendrites from an L2/3 cortical neuron. (f,g) Double immunoEM labeling and the resulting serial reconstruction of dendrites from two L2/3 cortical neurons (30 serial sections). smFP_Flag is pseudocolored yellow; smFP_Myc is pseudocolored red. Note the high density of stain in all images, even in spines, with minimal background labeling. (h,i) Silver-enhanced anti-HA immunogold labeling in HeLa cells with 1% OsO4 fixation (h) or no secondary fixation (i). Notice the improved structure preservation with 1% OsO4, with no observable difference in label density. Movies showing the 3D reconstruction of the data in dg are provided in Supplementary Videos 2–5. Scale bars: 5 μm (a), 500 nm (b), 20 μm (c) and 1 μm (dg).


  1. Supplementary Video 1
    Video 1: Supplementary Video 1
    Single-molecule tracking of H2B molecules labelled with EGFP, Halo-tag and Alexa488 substrate, and smFP_FLAG and anti-FLAG antibody
  2. Supplementary Video 2
    Video 2: Supplementary Video 2
    Movie showing 41 silver enhanced immunogold labelled sections expressing smFP_FLAG
  3. Supplementary Video 3
    Video 3: Supplementary Video 3
    3D reconstruction of smFP_FLAG immunogold labelled dendritic segment
  4. Supplementary Video 4
    Video 4: Supplementary Video 4
    Movie showing 30 silver enhanced immunogold labelled sections expressing smFP_FLAG and smFP_myc
  5. Supplementary Video 5
    Video 5: Supplementary Video 5
    3D reconstruction of smFP_FLAG and smFP_myc immunogold labelled dendritic segment

Accession codes

Primary accessions


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Author information

  1. Present addresses: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA (T.J.S.); Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA (B.M.H.); Advanced Microscopy Facility, University of Virginia School of Medicine, Charlottesville, Virginia, USA (Y.W.).

    • Timothy J Stasevich,
    • Bryan M Hooks &
    • Yalin Wang


  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.

    • Sarada Viswanathan,
    • Erik B Bloss,
    • Timothy J Stasevich,
    • Aljoscha Nern,
    • Barret D Pfeiffer,
    • Bryan M Hooks,
    • Wei-Ping Li,
    • Brian P English,
    • Teresa Tian,
    • Gilbert L Henry,
    • John J Macklin,
    • Ronak Patel,
    • Yalin Wang,
    • Gerald M Rubin &
    • Loren L Looger
  2. Department of Neurobiology & Anatomy, University of Utah, Salt Lake City, Utah, USA.

    • Megan E Williams
  3. Howard Hughes Medical Institute, Department of Chemistry & Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Colenso M Speer &
    • Xiaowei Zhuang
  4. Laboratory of Systems Neuroscience, National Institute of Mental Health, Bethesda, Maryland, USA.

    • Bryan M Hooks &
    • Charles R Gerfen
  5. Howard Hughes Medical Institute, Department of Physics, Harvard University, Cambridge, Massachusetts, USA.

    • Xiaowei Zhuang


S.V., G.M.R. and L.L.L. conceived of the project. L.L.L. performed molecular modeling and designed sequences. S.V. and B.D.P. constructed the clones. S.V. and M.E.W. performed experiments in cultured neurons. M.E.W. performed hippocampal neuron work. B.M.H. and C.R.G. performed four-color labeling experiments. E.B.B. performed AT experiments. C.M.S. and X.Z. designed STORM experiments, and C.M.S. performed STORM imaging and analyzed data. J.J.M. and R.P. performed biophysical characterization. A.N. performed fly experiments. W.-P.L. and Y.W. performed EM. T.J.S. and B.P.E. performed single-molecule imaging. T.T. and G.L.H. performed pulldown experiments. S.V. and L.L.L. led the project.

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The authors declare no competing financial interests.

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Supplementary information


  1. Video 1: Supplementary Video 1 (3.05 MB, Download)
    Single-molecule tracking of H2B molecules labelled with EGFP, Halo-tag and Alexa488 substrate, and smFP_FLAG and anti-FLAG antibody
  2. Video 2: Supplementary Video 2 (35.3 MB, Download)
    Movie showing 41 silver enhanced immunogold labelled sections expressing smFP_FLAG
  3. Video 3: Supplementary Video 3 (13.15 MB, Download)
    3D reconstruction of smFP_FLAG immunogold labelled dendritic segment
  4. Video 4: Supplementary Video 4 (42.52 MB, Download)
    Movie showing 30 silver enhanced immunogold labelled sections expressing smFP_FLAG and smFP_myc
  5. Video 5: Supplementary Video 5 (8.79 MB, Download)
    3D reconstruction of smFP_FLAG and smFP_myc immunogold labelled dendritic segment

PDF files

  1. Supplementary Text and Figures (3,205 KB)

    Supplementary Figures 1–18 and Supplementary Tables 1 and 2

Additional data