A Genetically Encoded Tag for Correlated Light andElectron Microscopy of Intact Cells, Tissues, andOrganisms
Xiaokun Shu1,2*¤, Varda Lev-Ram2, Thomas J. Deerinck3, Yingchuan Qi1,4, Ericka B. Ramko5, Michael W.
Davidson5, Yishi Jin1,4, Mark H. Ellisman3,6, Roger Y. Tsien1,2,7*
1 Howard Hughes Medical Institute, University of California at San Diego, La Jolla, California, United States of America, 2 Department of Pharmacology, University ofCalifornia at San Diego, La Jolla, California, United States of America, 3 National Center for Microscopy and Imaging Research, Center for Research on Biological Systems,
University of California at San Diego, La Jolla, California, United States of America, 4 Division of Biological Science, Section of Neurobiology, University of California at San
Diego, La Jolla, California, United States of America, 5 National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University,
Tallahassee, Florida, United States of America, 6 Department of Neurosciences, University of California at San Diego, La Jolla, California, United States of America,,
7 Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, United States of America

Abstract
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has
lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce‘‘miniSOG’’ (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2.
MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates
sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction productresolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact
nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde
fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits highquality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block
face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons,
whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could beheterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.
Citation: Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, et al. (2011) A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells,
Tissues, and Organisms. PLoS Biol 9(4): e1001041. doi:10.1371/journal.pbio.1001041
Academic Editor: J. Richard McIntosh, University of Colorado, United States of America
Received November 30, 2010; Accepted February 14, 2011; Published April 5, 2011
Copyright: ß 2011 Shu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by HHMI funding (R.Y.T. and Y.J.), NIH grants GM086197 to R.Y.T. and M.H.E., and NS035546 to Y.J. Microscopic analyses
and development of EM methods were conducted at the National Center for Microscopy and Imaging Research, supported by NIH P41-RR004050 (to M.H.E.). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: ADPA, anthracene-9,10-dipropionic acid; Cx43, connexin 43; DAB, diaminobenzidine; EM, electron microscopy; FA, focal adhesion; FAD, flavinadenine dinucleotide; FM, fluorescence microscopy; FMN, flavin mononucleotide; GFP, green fluorescent protein; H2B, histone 2B; HRP, horseradish peroxidase;
IFP, infrared fluorescent protein; LOV, light-oxygen-voltage; miniSOG, small singlet oxygen generator; NGL, netrin-G ligand; SBFSEM, serial block-face scanningelectron microscopy; SEM, scanning electron microscopy; SynCAM, synaptic cell-adhesion molecules* E-mail: rtsien@ucsd.edu (RYT); xiaokun.shu@ucsf.edu (XS)¤ Current address: Department of Pharmaceutical Chemistry and Cardiovascular Research Institute, University of California at San Francisco, San Francisco,California, United States of America

limited diffusibility and need detergent permeabilization to enter
cells [1]. Genetic labeling methods should overcome many of theseshortcomings, just as fluorescent proteins have revolutionized lightmicroscopic imaging in molecular and cell biology [2]. However,no analogous genetically encoded tag for EM contrast has yet
proven widely applicable. Metallothionein has been proposed as a
genetic tag that can noncatalytically incorporate cadmium or gold[3], but its main applications to intact cells have been to Escherichia
coli conditioned to tolerate 0.2 mM CdCl2 for 18 h [4] or 10 mM
AuCl for 3 h [4,5]. Such high concentrations of heavy metal salts
would not seem readily transferable to most multicellularorganisms or their cells. Also many higher organisms express
endogenous metallothionein, which would contribute backgroundsignals unless genetically deleted or knocked down [5]. Horserad-

Introduction
The most general techniques for imaging specific proteins
within cells and organisms rely either on antibodies or genetic tags.
EM is the standard technique for ultrastructural localization, butconventional EM immunolabeling remains challenging because of
the need to develop high-affinity, high-selectivity antibodies thatrecognize cross-linked antigens, and because optimal preservationof ultrastructure and visibility of cellular landmarks requires strong
fixation that hinders diffusibility of antibodies and gold particles.
Thus the target proteins most easily labeled are those exposed at
cut tissue surfaces. Replacement of bulky gold particles by eosin
enables catalytic amplification via photooxidation of diaminobenzidine (DAB), but eosin-conjugated macromolecules still have
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excited state energy of FMN is consumed to form a covalent bondwith a cysteine [20]. To divert this energy into 1O2 generation, we
carried out saturation mutagenesis of the relevant cysteine(Cys426) of the LOV2 domain of Arabidopsis thaliana phototropin
2 (AtPhot2). To screen for optimal 1O2 production, these sitespecific mutants were fused to an infrared fluorescent protein,
IFP1.4, which is readily bleached by 1O2 (Figure S2) [21].
Colonies of E. coli expressing the fusion proteins were imaged in
the IFP channel (ex 684/em708 nm) before and after blue light(488 nm) illumination (Figure 1A). Several colonies showed adecrease of IFP fluorescence from wild-type colonies and two withthe largest decrease (,70%) had the single site substitution of
Cys426 to Gly. The small side chain of the glycine residue may
provide space around the cofactor that would allow O2 close
apposition to FMN for efficient energy transfer. To increase thebrightness of the C426G mutant, we also performed saturation
mutagenesis of other residues surrounding the chromophorebinding site. DNA shuffling of the improved mutants plus randommutagenesis led to a new protein, miniSOG (106-residue)(Figure 1B and C, Figure S3), which absorbs maximally at
448 nm with a shoulder at 473 nm with extinction coefficients(16.760.7)6103 and (13.660.5)6103 M21cm21, respectively(Figure 1D). Excitation of miniSOG leads to green emission withtwo peaks at 500 and 528 nm (Figure 1D). The 1O2 quantum yieldof miniSOG (0.4760.05) was measured using anthracene-9,10dipropionic acid (ADPA) as 1O2 sensor (Figure 1E) [22]. Free
FMN was used as the standard for the measurement of 1O2
generation (quantum yield 0.51) [10].
MiniSOG was determined by light scattering to be monomericin solution, with a molecular weight of 13.960.4 kDa, close to thetheoretical value of 15.3 kDa. Absence of oligomerization was
further supported by the good separation by gel filtration of
miniSOG from its tandem dimer (td-miniSOG) (Figure S4). Mass
spectrometry confirmed that the flavin cofactor is FMN (FigureS5). Equilibrium dialysis reported a dissociation constant of
17068 pM (Table S2), similar to values for some flavoproteins(e.g. 260660 pM for a flavodoxin [23]) and consistent with the
crystal structures of LOV domains, which show FMN deeply
buried inside the protein core [24]. Furthermore, overexpressionof miniSOG in HEK293 cells caused the FMN content to increase,3-fold, presumably to keep miniSOG nearly saturated withFMN (Figures S6–S8), but caused no obvious toxicity in theabsence of light (Table S1). Feedback pathways involving enzymes
such as riboflavin kinase (EC 2.7.1.26) and FAD (flavin adenine
dinucleotide) diphosphatase (EC 3.6.1.18) probably regulate
intracellular FMN to titrate endogenous flavoproteins andminiSOG [25]. Riboflavin kinase phosphorylates riboflavin into
FMN, while FAD diphosphatase catalyzes the production of FMN
from FAD.

Author Summary
Electron microscopy (EM) once revolutionized cell biology
by revealing subcellular anatomy at resolutions of tens ofnanometers, well below the diffraction limit of lightmicroscopy. Over the past two decades, light microscopy
has been revitalized by the development of spontaneously
fluorescent proteins, which allow nearly any protein ofinterest to be specifically tagged by genetic fusion. EM has
lacked comparable genetic tags that are generally
applicable. Here, we introduce ‘‘miniSOG’’, a small (106residue) fluorescent flavoprotein that efficiently generates
singlet oxygen when illuminated by blue light. In fixedtissue, photogenerated singlet oxygen locally polymerizes
diaminobenzidine into a precipitate that is stainable with
osmium and therefore can be readily imaged at highresolution by EM. Thus miniSOG is a versatile label forcorrelated light and electron microscopy of geneticallytagged proteins in cells, tissues, and organisms including
intact nematodes and mice. As a demonstration of
miniSOG’s capabilities, controversies about the localizationof synaptic cell adhesion molecules are resolved by EM ofminiSOG fusions in neuronal culture and intact mousebrain.

ish peroxidase can be a genetic label in the secretory pathway but
is greatly limited by its requirements for tetramerization,
glycosylation, and high Ca2+, so that it is not functional when
expressed in the cytosol [6]. Furthermore, its DAB reaction
product tends to diffuse from sites of enzymatic generation,
resulting in poorer resolution than immunogold or the reaction
product of photogenerated singlet oxygen (1O2, the metastable
excited state of O2) with DAB [1,7,8]. The best previous
genetically targetable generator of 1O2 was the biarsenical dye
ReAsH, which binds to genetically appended or inserted
tetracysteine motifs [9]. However, ReAsH has modest 1O2
quantum yield (0.024) (Figure S1), requires antidotes to prevent
cell toxicity, needs careful precautions to reduce nonspecificbackground signal, and has been difficult to apply to multicellulartissues and organisms [10]. Although fluorescence photooxidation
using GFP has been reported [11,12], the 1O2 quantum yield of
the naked GFP chromophore is extremely low (0.004), and the 1O2
quantum yield of the intact protein was yet lower andunquantifiable [13], presumably because the beta-barrel of theprotein shields the chromophore from oxygen. The phototoxic
fluorescent protein ‘‘Killer Red’’ [14] is now acknowledged not towork through 1O2 [15], and we have confirmed that its 1O2
quantum yield is negligible (Figure S1).
Here, we introduce miniSOG, a small, genetically encodable
protein module that needs no exogenous cofactors to fluoresce andphotogenerate 1O2 with a substantial quantum yield. MiniSOG
provides major improvement in correlated light and electron
microscopy in cells and multicellular organisms via photooxidation
techniques.

Correct Localization of Well-Understood Proteins Taggedwith MiniSOG in Tissue Culture Cells
We used the fluorescence from miniSOG fusion proteins to
successfully localize a wide variety of proteins and organelles incultured mammalian cells (Figure 2). Its green fluorescence, whilemodest compared to GFP (quantum yield of 0.37 versus 0.6),
revealed that labeled components appeared to have correct
localizations (Figure 2A–H). Figure 2A shows ER-targeted
miniSOG, indicating that miniSOG can work within the secretory
pathway. Figure 2B–F show Rab5a, zyxin, tubulin, b-actin, and aactinin as examples of proteins tagged in cytosolic compartments.
Mitochondrial targeting and nuclear histone 2B-fusions(Figure 2G,H) show that miniSOG expresses within thoseorganelles. Using the fluorescence and photo-generated 1O2 from

Results
Structure-Based Design of MiniSOG
The LOV (light, oxygen, and voltage) domain of phototropin (ablue light photoreceptor) binds flavin mononucleotide (FMN)[16,17], which by itself is an efficient singlet oxygen photosensitizer [18]. FMN is ubiquitous in cells and performs indispensablebiological functions such as mitochondrial electron transport, fatty
acid oxidation, and vitamin metabolism [19]. In phototropin, the
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Figure 1. MiniSOG, a small and efficient singlet oxygen generator, is engineered from a blue light photoreceptor based on protein
crystal structure. (A) Infrared fluorescence of E. coli colonies expressing the fusion proteins before and after irradiation (480615 nm excitation). (B)Predicted structure of miniSOG by the Swiss-Model structure homology-modeling server [52]. (C) Mutations introduced into miniSOG compared to its
parent. Numbers in bracket are based on miniSOG protein sequence. (D) Normalized absorbance (blue) and emission (red) spectra. (E) Degradation of
ADPA by illumination of miniSOG (red) or free FMN (blue).
doi:10.1371/journal.pbio.1001041.g001

miniSOG for fluorescence photooxidation of DAB (Figure 3A),
correlated confocal and EM imaging could be performed withseveral miniSOG fusion proteins (Figure 3B–E), producing
excellent EM contrast, efficient labeling, and good preservationof ultrastructure.
a-Actinin. a-Actinin cross-links actin bundles and attaches
actin filaments to focal adhesions (FA) [26]. EM images of stained
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miniSOG fusion proteins expressed in HeLa cells contained
fibrous densities consistent with published observations associating
a-actinin with actin bundles in the cell cortex adjacent to theplasma membrane FA-like structures (Figures 3B–E, S9C–D). The
higher contrast between cells expressing miniSOG tagged aactinin versus non-expressing cells is clearly evident in the cytosolin these electron micrographs (Figure S9A).
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Figure 2. MiniSOG-labeled proteins and organelles exhibit correct localization at the light microscopic level. Confocal fluorescence
images of miniSOG-targeted endoplasmic reticulum (A), Rab5a (B), zyxin (C), tubulin (D), b-actin (E), a-actinin (F), mitochondria (G), and histone 2B (H)in HeLa cells; scale bars, 10 mm.
doi:10.1371/journal.pbio.1001041.g002

Histone 2B (H2B). MiniSOG-tagged H2B revealed largescale organizations of chromatin fibers in the perinucleolar andintranuclear regions [27] as imaged by confocal fluorescence,transmitted light after photooxidation, and correlated thin sectionand electron tomography (arrows, Figure 3F–H). The
tomographic slice demonstrates the utility of miniSOG labelingfor 3-dimensional EM analysis. Fibrillar chromatin structures near
the nuclear envelope and nuclear pores were also observable athigh resolution (arrows and arrowhead, respectively; Figure 3I).
The H2B fusion seemed to have no deleterious effects whenincorporated into chromosomes since H2B-miniSOG expressing
cells can be found in several stages of mitosis (Figure S10).
Mitochondrial matrix. Mitochondria containing cytochrome
C–targeted miniSOG fusions had well-preserved morphology ofouter and inner membranes and cristae with a strong EM signal
present in the mitochondrial matrix consistent with the targeting(Figure 3L and M). The contrast differential between mitochondria
in cells expressing targeted miniSOG and photooxidized comparedto adjacent cells not expressing miniSOG is apparent by both LM(Figure 2J and K) and EM (Figure S9B).
Connexin 43 (Cx43). Cx43 forms gap junction channels. EMof the Cx43-miniSOG fusion showed densely stained DAB
photooxidation reaction product outlining structures (Figure 4B)
roughly corresponding in size to gap junction channels eachcomposed of 12 connexins (six in each hemichannel). A cartoon(Figure 4E) based on the x-ray crystal structure of thetransmembrane and extracellular domains of Cx26, whichshares 46% sequence identity with that of Cx43 [28], and the
NMR structure of the carboxy-terminal domain of Cx43 [29] isshown for interpretation of the EM. Furthermore, we speculatethat the black dots studded on the outside of trafficking vesicles(black dots, Figure 4C) may represent single connexons [30–32].
As a comparison, EM of densely packed Cx43 gap junctions using
immunogold showed much sparser, more random labeling(Figure 4D).
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Localization of MiniSOG in Tissues of Multicellular
Organisms
C. elegans mitochondrial labeling. We expressed miniSOG
in the matrix of body wall muscle mitochondria using a cytochromec targeting sequence in C. elegans to explore the usefulness of
miniSOG for correlated fluorescence and EM in multicellularorganisms. In transgenic worms the green fluorescence of miniSOG
showed labeled mitochondria in body wall muscle cells (Figure 5A)
while EM revealed a subset of stained mitochondria with wellpreserved morphology (Figure 5B,C).
Pre- and post-synaptic localization of SynCAM1 and 2,
respectively. To ascertain if miniSOG could reveal newmolecular details of the organization of neuronal synapses, we
expressed miniSOG attached to two isoforms of SynCAM todetermine their locations in synapses of mouse neurons. SynCAMs
are cell-adhesion molecules involved in synapse formation,
maturation, and plasticity whose extensive expression throughout
the brain suggests important functions [33]. SynCAMs play animportant role in establishing and stabilizing synapses throughCa2+-independent interactions, in contrast to Ca2+-dependent
neurexin-neuroligin interactions [34]. In spite of their recognizedrole in synapse assembly, the specific localization of SynCAMs had
not been accomplished previously. A prior EM study suggested
both pre- and post-synaptic membrane localization of SynCAM1
using antibodies raised against its C-terminus, but ambiguity
remained because these antibodies cross-react with SynCAM2 andSynCAM3 [33,34]. To overcome this limitation, we separately
examined the synaptic distribution of SynCAM1 and SynCAM2
fusions to miniSOG, initially in cultured cortical neurons.
SynCAM1-miniSOG was found only at presynaptic terminals,
identified by the presence of synaptic vesicles, confirming apresynaptic localization (Figure 5A, Figure S10). This presynaptic
targeting of SynCAM1-miniSOG was also observed in transfected
single neurons forming synapses onto themselves in a micro-island
culture system (Figure S11) [35], ruling out the possibility that4

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Figure 3. MiniSOG produces correlated fluorescence and EM contrast with correct localization of labeled proteins and organelles.
(A) Schematic diagram of how miniSOG produces EM contrast upon blue-light illumination. Spin states are depicted by the arrows. ISC, intersystemcrossing. Correlated confocal fluorescence (B,F,J), transmitted light (C,G,K), and electron microscopic (D,E,H,I,L,M) imaging of a variety of proteins. (B–E) HeLa cells expressing miniSOG labeled a-actinin. Arrows denote correlated structures. (F–I) Histone 2B. Panel H is a 3 nm thick computed slice froman electron tomogram. Panel I is a high magnification thin section electron micrograph showing labeled chromatin fibers near the nuclear envelope(arrows) and a nuclear pore (arrowhead). (J–M) Mitochondrial targeted miniSOG. Panels J and K show a confocal image prior to photooxidation and atransmitted light image following photooxidation, respectively. The differential contrast generated between a transfected (arrows) and nontransfected cell (arrowheads) is evident. Bars B–D, 1 micron; E, 200 nm; F–H, 2 microns; I, 100 nm; J–L, 5 microns; M, 200 nm.
doi:10.1371/journal.pbio.1001041.g003

systematically find synapses from the few transfected neurons
within the brains of young adults. The instrument consists of an
ultramicrotome fitted within a backscatter-detector equipped
scanning electron microscope. In an automated process, theultramicrotome removes an ultra-thin section of tissue with an
oscillating diamond knife and the region of interest is imaged. Thissequence is repeated hundreds or thousands of times until thedesired volume of tissue is traversed. This method potentiallyenables the reconstruction of microns to tenths of millimeters ofvolumes of tissue at a level of resolution better than that obtainable
by light microscopy [36,37]. However, optimal backscatter signal

postsynaptic neurons are more difficult to identify or transfect. In
contrast, SynCAM2 localized to postsynaptic sites in cultured
cortical neurons, identified by postsynaptic densities and by the
opposition of these terminals to presynaptic boutons bearing
synaptic vesicles (Figure 5B, Figure S12).
Next, we introduced these fusion proteins into prenatal mousebrains by in utero electroporation in order to study theirlocalizations. Because neurons expressing miniSOG fusion proteins may be sparse, we turned to serial block-face scanningelectron microscopy (SBFSEM), a relatively new method thatfacilitates large-scale 3–D reconstruction of tissue to help
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Figure 4. MiniSOG-tagged Cx43 forms gap junctions. (A) The green fluorescence of miniSOG reveals gap junctions and transporting vesicles.
(B) Electron microscopy indicates negatively stained structures of appropriate size and spacing to be gap junction channels (arrows). (C) Studs on themembranes of trafficking vesicles suggest single connexons. The arrowhead points to two dots with a center-to-center distance ,14 nm. (D) A highquality immunogold image showing a randomly labeled fraction of densely packed Cx43 gap junctions. This figure is reproduced from Figure 4D ofGaietta et al. [9]. (E) A cartoon showing miniSOG-labeled Cx43 gap junctions. Bar A, 10 microns; B–D, 100 nm.
doi:10.1371/journal.pbio.1001041.g004

is dependent on very strong scattering from heavy metal stains.
The photooxidation of MiniSOG generated a strongly osmiophilic
reaction product that in combination with en bloc uranyl acetatestaining provided a specific and strong backscatter electron signal,
which confirmed that the fusion to SynCAM2 was postsynaptic inintact mouse brain (Figure 5). Thus, the combination of miniSOG
fusion proteins and SBFSEM provides a method to correlate thelocation of specific molecules throughout large 3–D volumes andwith good preservation of ultrastructure (Figure S13).

provide improved localizations, each landmark of interest would
need to be labeled with fluorophores emitting at different color.
MiniSOG probes have several advantages over other correlated
LM/EM probes. MiniSOG needs no exogenous cofactors andproduces 1O2 with about 20 times higher quantum efficiency than
ReAsH on a tetracysteine motif. Therefore, miniSOG photooxidation has considerably better sensitivity and lower backgroundthan ReAsH labeling. MiniSOG is much smaller than GFP, andunlike GFP can mature and become fluorescent in the absence of
O2. GFP-based photooxidation is very difficult due to its extremely
low 1O2 quantum yield [13]. Genetically encoded horseradish
peroxidase is tetrameric and far larger than GFP, only becomesfunctional inside the secretory pathway [6], and produces
relatively diffuse precipitates [1,7,8]. Metallothionein fusions
would seem most appropriate for purified macromolecules [3],because imaging of intact cells requires them to survive prolonged
incubation in high concentrations of Cd2+ or Au+ [4,5] and not to
express endogenous metallothionein.
Our results with miniSOG fusions demonstrate that SynCAM1
and SynCAM2 are localized to pre- and post-synaptic membranes,
respectively, and these observations are consistent with thereported strong heterophilic interaction between SynCAM1 andSynCAM2 in the formation of trans-synaptic structures [41]. The
presynaptic membrane localization of SynCAM1 is also consistent
with the recent report that SynCAM1 is expressed in growth conesin the early developmental stages of mouse brain and is involved in
shaping the growth cones and the assembly of axo-dendritic
contact [41]. Analogous trans-synaptic pairs include neurexin/
neuroligin [42], EphrinB/EphB, and netrinG/netrin-G ligand(NGL). New synaptic proteins continue to be reported, such as

Discussion
The successful localization of a variety of proteins by light and
EM in cultured cells as well as mitochondria in C. elegans and
SynCAM2 in intact mouse brain demonstrates the value of
miniSOG for correlated light and EM localization of specificproteins in cells and multicellular organisms. MiniSOG isadvantageous over conventional immuno-gold staining because
the protein of interest is genetically tagged before fixation and all
subsequent components (O2, DAB, and OsO4) are small moleculesthat easily permeate tissues. Tissues or cells can be fixed using
established methods for good preservation of ultrastructure
without concern for retention of antigenicity. Thus, permeabilizing
detergents such as Triton X-100 that degrade membranes tofacilitate the diffusion of bulky antibodies and secondary labels areunnecessary. This is demonstrated by the well-preserved ultrastructure in SynCAM-miniSOG labeled mice where unlabeledsynapses (arrowhead), nonsynaptic plasma membrane, andsynaptic vesicles are clearly observed (Figure 5). Such landmarks
were essential to assign the precise location of the SynCAMs.
While super-resolution fluorescence techniques [38–40] could
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Figure 5. MiniSOG produces fluorescence and EM contrast in C. elegans and reveals previously unknown localization of synaptic celladhesion molecules in mice. (A) Confocal fluorescence image of miniSOG targeted to the mitochondria in body wall muscles of C. elegans. (B–C)
Thin section EM images of a portion of C. elegans showing a subset of labeled mitochondria in the body wall muscle (arrow) and adjacent unlabeled
mitochondria in a different cell type (arrowheads). (D–E) Ultrastructural localization of miniSOG-labeled synaptic cell-adhesion molecules (SynCAMs)
in cultured cortical neurons. (D) SynCAM1 fusion reveals uniform membrane labeling at the presynaptic apposition (arrow). (E) SynCAM2 fusion shows
postsynaptic membrane labeling (pointed by arrow). Ultrastructural details including synaptic vesicles and nerve terminal substructure were wellpreserved in both (D) and (E). (F–G) Ultrastructural localization of miniSOG-labeled synaptic cell-adhesion molecule 2 (SynCAM2) in intact mousebrain. (A) A large area (,14 mm 614 mm) of one of the tissue sections imaged by serial block-face scanning electron microscopy. (B) Enlargement of
the region boxed in (A) reveals postsynaptic membrane labeling (pointed by arrow) apposing a presynaptic bouton containing vesicles.
Ultrastructural details including synaptic vesicles and membrane-bound structures of synapses were well preserved and easily recognizable (e.g.
arrowhead in the upper left). Bar A, 50 microns; B–C, 500 nm; D–E, 500 nm; F, 2 microns; G, 500 nm.
doi:10.1371/journal.pbio.1001041.g005

leucine rich repeat transmembrane proteins (LRRTMs), NGL-3,
and leukocyte common antigen-related (LAR) [43,44]. The large
variety of these molecules may be necessary to establish andsupport the great diversity of neuronal synapses; dissecting theirlocations within synapses will be a complex task.
As demonstrated here, our miniSOG-based photooxidation
technique provides a method to determine the detailed distribution
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of these and other important macromolecules. In combination
with SBFSEM, miniSOG fusion proteins should find wide
applications in the ultrastructural localization of proteins,
including 3-d reconstruction of neuronal circuits by large scale
automated SBFSEM to mark cells of interest and trace themacross large numbers of sections (Figure S13) [37]. Additionally, a
logical next step will be to further enhance the preservation of7

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cellular ultrastructure in these types of specimens by combining
chemical fixation and high pressure freezing [45] with photooxidation using miniSOG.
Spatiotemporally controlled local photogeneration of 1O2
should also be useful for rapidly inactivating proteins of interest[46], reporting protein proximities over tens of nanometers [47] by
1
O2 transfer from a SOG to a 1O2 sensitive fluorescent protein(e.g. IFP1.4) and ablating cells by photodynamic damage. Thus,
further development and application of miniSOG using 1O2
generation should greatly expand its utility in imaging andfunctional studies.

every few minutes. The samples were then illuminated using astandard FITC filter set (EX470/40, DM510, BA520) with intenselight from a 150W xenon lamp. Illumination was stopped as soon
as a very light brown reaction product began to appear in place ofthe green fluorescence as monitored by transmitted light (typically
2–10 min, depending on the initial fluorescence intensity, thebrightness of the illumination, and the optics used). Care was taken
to avoid overreacting the samples, as this can lead to overstaining
and the degradation of ultrastructure in the region of photooxidation. Multiple areas on a single dish could be reacted if thesolution was refreshed every few minutes. The cells were thenremoved from the microscope and washed in chilled buffer(562 min) and post-fixed in 1% osmium tetroxide (Electron
Microscopy Sciences) in 0.1 M sodium cacodylate buffer for
30 min on ice. Cells were washed in chilled buffer twice and rinsed
in distilled water, then en bloc stained with 2% aqueous uranyl
acetate (Ted Pella Inc.) for 1 h to overnight at 4uC. The samples
were then dehydrated in a cold graded ethanol series (20%, 50%,
70%, 90%, 100%, 100%) 2 min each, rinsed once in room
temperature anhydrous ethanol, and infiltrated in Durcupan
ACM resin (Electron Microscopy Sciences) using 1:1 anhydrous
ethanol and resin for 30 min, then 100% resin 261 h, theninto fresh resin and polymerized in a vacuum oven at 60uC for
48 h.

Materials and Methods
Gene Synthesis, Mutagenesis, and Screening
A gene encoding LOV2 domain of Phototropin 2 with codons
optimized for E. coli was synthesized by overlap extension PCR[48]. Genetic libraries were constructed by saturation and randommutagenesis and DNA shuffling [21]. Mutants were fused to
IFP1.4 by overlap extension PCR and cloned into a modified
pBAD vector containing the heme oxygenase-1 gene fromcyanobacteria [21]. Libraries were expressed in E. coli strain
TOP10 and screened by imaging the agar plates with colonies in
the IFP channel before and after blue light illumination [21].
Protein purification and spectroscopic characterization experiments were done as described [49].

Preparation of C. elegans
Transgenic worms were made by injection of cDNAs ofmitochondrially targeted miniSOG driven by myo-3 promoter at
50 ng/ml. The worms were chemically fixed with 2% glutaraldehyde, washed, and blocked as described above. The cuticle was
sharply cut to allow diffusion of DAB into the inner body forphotooxidation. After confocal imaging and fluorescence photooxidation, the worms were processed for EM imaging as describedabove.

Chimera Construction
DNA encoding miniSOG with codons optimized for mammals
was synthesized by overlap extension PCR [48]. MiniSOG fusions
were cloned into pcDNA3.1 vector. HEK293 and HeLa cells weretransfected with miniSOG or chimera cDNAs using Fugene, thenimaged 24–48 h later. Cultured cortical neurons were transfectedby Amaxa electroporation (Lonza AG, Germany) and imaged 1–
2 wk later.

Preparation, Fluorescence Imaging, and Photooxidationof Mouse Brain

Fluorescence Imaging, Photooxidation, and EM
Preparation of Transfected Cultured Cells

Endotoxin-free DNA (,3 mg) of the SynCAM2-miniSOG
fusion construct was delivered into the lateral ventricle of embryos
by in utero electroporation [50]. The offspring at p7 or p21 wereanesthetized and fixed by vascular perfusion as previously
described [51] with Ringer’s solution followed by 4% formaldehyde made fresh from paraformaldehyde (Electron Microscopy
Sciences) in 0.15 M cacodylate buffer. Brains were removed andplaced in the same fixative at 4uC for 1 h for p21 and overnight forp7. In this case we avoided glutaraldehyde in combination withparaformaldehyde due to the increased autofluorescence that
occurs with glutaraldehyde. The autofluorescence obscured
miniSOG fluorescence and made it impossible to locatetransfected neurons in the brain slices for photooxidation. Brains
were then sliced to 100 mm sections using a vibratome (Leica).
Areas of interest were identified by confocal microscopy. The
sections were then postfixed with 2% glutaraldehyde for 30 min,rinsed in cold buffer, blocked, and then photooxidized as describedabove. Subsequent procedures for EM processing were similar to
those described above except the vibratome sections were resin
embedded between two liquid release agent coated glass slides(Electron Microscopy Sciences).

Transfected cells cultured on glass bottom culture dishes (P35G0-14-C, MatTek Corp., Ashland, MA) were fixed with 2%
glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in
pH 7.4 0.1 M sodium cacodylate buffer (Ted Pella Inc., Redding,CA) for 30–60 min, rinsed several times in chilled buffer, andtreated for 30 min in blocking buffer (50 mM glycine, 10 mM
KCN, and 5 mM aminotriazole) to reduce nonspecific background reaction of diaminobenzidine (DAB). Confocal images
were taken with minimum exposure using a BioRad MRC-1024
inverted confocal microscope or similar inverted fluorescencemicroscope to identify transfected cells and for correlative lightmicroscopic imaging. Detailed protocols for performing fluorescence photooxidation of DAB have been published [2,6]. It is
important to use an inverted microscope to ensure direct open
access to the DAB solution. An objective of numerical aperture$0.7 is desirable to maximize illumination intensity. For
photooxidation, diaminobenzidine tetrahydrochloride (SigmaAldrich, St. Louis, MO) was freshly diluted to 1 mg/ml in
0.1 M sodium cacodylate buffer, pH 7.4, filtered through a 0.22
micron syringe filter (Millipore), and placed on ice and added tothe cells. The region of interest was identified by the fluorescenceand an image recorded with care not to bleach the area. A small
tube attached to an oxygen tank was placed near the top of thedish and a stream of pure oxygen was gently blown continuously
over the top of the solution. Alternately, the DAB solution on ice
was bubbled with oxygen and the solution in the dish refreshed
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Electron Microscopy
Photooxidized areas of embedded cultured cells were identified
by transmitted light and the areas of interest were sawed out usinga jeweler’s saw and mounted on dummy acrylic blocks with
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Genetically Encoded Electron Microscopy Tag

cyanoacrylic adhesive. The coverslip was carefully removed,
ultrathin sections were cut using an ultramicrotome, and electron
micrographs recorded using a 1200 TEM (JEOL) operating at
80 keV. For tissue sections, one of the glass coverslips wasremoved using a razorblade and the area of interest identified bytransmitted light microscopy. The tissue was removed from theslide, mounted, sectioned, and imaged as above. For electron
tomography, 0.5 micron thick sections of cells expressing
photooxidized H2B-miniSOG were cut and imaged using a4000 IVEM (JEOL) operated at 400 keV. Images were tilted andrecorded every 2u from 660u to 260u. The image stack wasaligned and reconstructions were obtained using R-weighed backprojection methods with the IMOD tomography package. For
serial block face scanning electron microscopy, a 3View system(Gatan Inc., Pleasanton, CA) mounted in a Quanta FEG scanningelectron microscope (FEI Company, Eindhoven, The Netherlands)
was employed. Imaging was performed as previously described[37]. Individual image planes were hand segmented to outline theplasma membrane of the target neuron and denote labeled postsynaptic densities, then thresholded and projected using Amira(Visage Imaging, Germany).

photooxidized cell expressing miniSOG-targeted mitochondria(arrows) versus a non-expressing cell (arrowheads). (C, D) Low andhigh magnification showing alpha-actinin tagged miniSOG. Bars,
500 nm.
(TIF)
Figure S10 HeLa cells transfected with miniSOG-labeled H2Bundergo mitosis. Bars, 2 microns.
(TIF)

High resolution EM reveals presynaptic labeling ofSynCAM1 tagged with miniSOG in cultured cortical neurons
from randomly selected areas. Scale bars, 500 nm.
(TIF)

Figure S11

Figure S12 Pre- and postsynaptic localization of SynCAM1 andSynCAM2 revealed by miniSOG. (A) Presynaptic localization of
SynCAM1-miniSOG in a single neuron forming synapses on itself
in micro-island culture, revealed by EM. (B) High resolution EM
reveals postsynaptic localization of SynCAM2 labeled by miniSOG in cultured cortical neurons from randomly selected areas.
Scale bars, 500 nm.
(TIF)

Supporting Information
Figure S1 Degradation of ADPA by ReAsH, KillerRed, or Rose
Bengal (RB) upon irradiation.
(TIF)

Figure S13 Stereo pair maximum intensity volume representation of a SynCam2-miniSOG serial block-face scanning electron
microscopy reconstruction. The volume consists of 224 image
planes (backscatter electron images) recorded at 60 nm intervals in z
using 6k 6 6 k pixels at 2.5 keV accelerating voltage. Once
projected, the image contrast was inverted, with the transfected
neuronal processes shown in white. The postsynaptic labeled
SynCAM2-miniSOG is shown in blue. Mitochondria in untransfected neurons are also shown. Bars, 2 microns. Movie showing theimage stack and 3-dimensional tracing of the transfected neuron
reconstruction from SBFSEM shown in Figure S13 (http://login.
ncmir.ucsd.edu/,mterada/msog/msog-syncam2b.mp4).
(TIF)

Figure S2 Destruction of IFP1.4 by Rose Bengal upon
illumination. IFP absorbance (upper left) and fluorescence (lowerleft) are decreased, proportional to irradiation (540/30 nm) time in
the presence of Rose Bengal, which absorbs maximally at 560 nm.
IFP absorbance (upper right) and fluorescence (lower right) do notchange significantly with the same irradiation time in the absenceof Rose Bengal.
(TIF)

Sequence alignment of miniSOG with its parent, theLOV2 domain of AtPhot2. Mutations are highlighted in cyan.
(TIF)

Figure S3Table S1 MiniSOG does not perturb HEK293 cell growth.

(DOC)
Figure S4 Size exclusion chromatography of miniSOG (red) andits tandem dimer td-miniSOG (blue).
(TIF)

Table S2 FMN is tightly bound in miniSOG.

(DOC)

Figure S5 Mass spectroscopy of FMN extracted from miniSOG.

Acknowledgments(TIF)
Figure S6

We thank L. Gross and S. Adams for mass spectroscopy and lightscattering, A. Royant for the measurement of singlet oxygen generation
quantum yield of ReAsH and KillerRed, T. Biederer for donation of
SynCAM1 and 2 cDNA, J. Hu for the Cx43-miniSOG construct, and G.
Sosinsky and G. Gaietta for discussions of Cx43 gap junctions.

LC/MS of untransfected HEK293 cell lysate spiked

with FMN.
(TIF)
Figure S7 LC/MS of untransfected HEK293 cell lysate.

Author Contributions(TIF)
Figure S8

The author(s) have made the following declarations about theircontributions: Conceived and designed the experiments: XS RYT.
Performed the experiments: XS VL TJD YQ EBR MWD. Analyzed thedata: XS VL TJD YQ MWD YJ MHE RYT. Contributed reagents/
materials/analysis tools: XS VL TJD YQ EBR MWD YJ MHE RYT.
Wrote the paper: XS MHE RYT. Proposal of engineering FMN-binding
phototropins as genetically encoded and efficient SOGs, with following
research design, engineering, and characterization of miniSOG: XS.
Coordination and guidance of correlated LM/EM: MHE. Coordinationand proposal of the desirability of a genetically encoded SOG: RYT.

LC/MS of miniSOG-transfected HEK293 cell lysate.

(TIF)
Figure S9 MiniSOG produces EM contrast in labeled organelles
and proteins in cells. (A) Adjacent HeLa cells showing differential
contrast between photooxidized cells expressing miniSOG taggedalpha-actinin (arrows) versus a non-expressing cell (arrowheads).
(B) Adjacent HeLa cells showing differential contrast between a

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Genetically Encoded Electron Microscopy Tag

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