Rat skeletal muscle was used as a model to develop a correlative technique for identifying NMJs by confocal and transmission electron microscopy. Muscle samples were gently fixed to maintain probe or antibody affinity and minimize autofluorescence and then cryo-protected in sucrose to prevent ice crystal formation during subsequent cryostat sectioning. Semi-thick sections (~10 microns) were suitable for good probe penetration for optical microscopy and to have sufficient material with negligible artifact at the cut interfaces or at the adhesive/slide interface used to maintain tissue through subsequent processing. Sections were screened for NMJs by confocal microscopy and then further processed for transmission electron microscopy.
The ultrastructural preservation of the motor neuron and its associated muscle fiber was maintained despite exposure to a high sucrose gradient, freezing, and post-processing. Sucrose is a commonly used cryoprotectant that is employed by the Tokuyasu method of cryoultramicrotomy for immunogold labeling of ultrathin sections (Tokuyasu, 1973
). Similar to the results with α-bungarotoxin reported here, Keller et al. (1984)
showed that sucrose-protected ultrathin cryosections could be immunogold labeled and then post-fixed in osmium, dehydrated, and embedded in resin. The morphology of such preparations was equivalent to conventionally processed samples. The current experiment demonstrates that relatively large expanses of muscle can be screened for features of interest by cryo-protection and cryostat sectioning without compromising ultrastructural integrity. However, like any study investigating the morphology of diseased NMJs, it is essential to run control muscle in parallel to rule out any possible structural changes or artifacts generated by sample processing. Due to our small probe size, our technique did not require permeabilization steps that might include Triton or saponin. However, there is no fundamental reason why antibody labeling would be precluded from performing ultrasmall gold or FluoroNanogold and silver enhancement for correlative fluorescence and electron microscopy using the procedure described here.
The most common method to identify NMJs by both light and electron microscopy uses histochemical staining techniques targeted toward acetylcholinesterase activity. Some histochemical methods applied to both light and electron microscopic observation of NMJs include those described by Koelle and Friedenwald (1949)
, Karnovsky and Roots (1964)
, and Strum and Hall-Craggs (1982)
. In Koelle and Friedenwald’s (1949)
method, tissue is incubated with the substrate acetylthiocholine and reacts with copper glycinate in the presence of acetylcholinesterase to form copper thiocholine, which further is reacted with ammonium sulfide to yield an electron dense deposit of copper sulfide. Variations of this method have also used gold and lead ions as capture reagents (Davis and Koelle, 1967
). Unlike Koelle and Friedenwald’s (1949)
method, Karnovsky and Roots (1964)
employed a single-step method in which acetylthiocholine, copper sulfate, and potassium ferricyanide reacted in the presence of acetylcholinesterase to directly form a copper ferrocyanide precipitate. The method described by Strum and Hall-Craggs (1982)
used an incubation solution of hexazotized pararosaniline and indoxyl acetate to form a more precise distribution of precipitate in the junctional folds. In all histochemical methods, an electron dense precipitate is deposited over the NMJ in the vicinity of cholinesterase activity.
Although histochemical techniques effectively delineate the location of NMJs, they have several limitations. Foremost, the histochemical reactions deposit an electron dense precipitate over the NMJs that can obscure the underlying ultrastructural details, reduce morphological preservation, and limit resolution. Incubation times in the reaction medium need to be adjusted to compromise between visibility of the neuromuscular junction by light microscopy and decreased resolution at the level of electron microscopy. Moreover, the components of the reaction medium exhibit limited penetration into tissue, and staining is restricted to superficial muscle fibers (Davis and Koelle, 1967
; Strum and Hall-Craggs, 1982
). Lastly, the reaction product has the potential to diffuse away from the source of staining (Koelle and Friedenwald, 1949
), and under certain conditions, the formation of needle-like crystals and nonspecific staining can occur (Karnovsky and Roots, 1964
More recent techniques for correlating NMJs by confocal and transmission electron microscopy have used DAB photo-oxidation. When photo-bleached with intense epi-fluorescent illumination, certain fluorescent dyes release reactive oxygen species that oxidize diaminobenzidine (DAB) to form an electron dense precipitate (Deerink et al., 1994
). DAB photo-oxidation of NMJs has been accomplished with the dye FM1-43 to demonstrate synaptic vesicle uptake at the motor neuron (Henkel et al., 1996
; Teng and Wilkinson, 2000
) and also with DiI or DiO by iontophoretic labeling (Gan et al., 1999
; Bishop et al., 2004
). As with histochemical staining of the NMJ, DAB photo-oxidation techniques also rely on the deposition of an electron dense reaction product to locate junctions by light and electron microscopy. However, the resulting reaction product is finer than those produced by histochemical methods and highly localized to the site of dye labeling. Drawbacks to DAB photo-oxidation methods include the nonspecific photo-conversion of endogenous peroxidases (Deerink et al., 1994
). Moreover, FM1-43 uptake must occur in unfixed, electrically stimulated muscle fibers (Henkel et al., 1996
), which is unsuited for fixed muscle biopsies. Lastly, iontophoretic labeling with DiO and DiI requires the injection of a dye crystal directly into individual NMJs and does not broadly label all NMJs within a sample for gross screening.
The use of sucrose-protected cryostat sections of muscle biopsies labeled with fluorescent α-bungarotoxin overcomes several limitations of histochemical acetycholinesterase staining and DAB photo-oxidation techniques for the direct correlation of NMJs by confocal and transmission electron microscopy. Cryostat sections allow large expanses of muscle tissue to be efficiently screened and mapped so that a significant fraction of junctions can be identified for transmission electron microscopy, permitting maximum utilization of often precious muscle biopsies. Moreover, the CryoJane® Tape-Transfer System used in conjunction with adhesive-coated slides allows flat, wrinkle-free sections to be collected that remain bonded to the slides throughout subsequent processing. Additionally, mapping of fluorescently labeled junctions with confocal tile scans allows junctions to be targeted for transmission electron microscopy without the introduction of an electron dense precipitate that can obscure underlying ultrastructural details. Efforts with non-sucrose protected muscle biopsies were technically possible when submerging tissue pieces in fluorescent tagged α-bungarotoxin. However, the probe only penetrated very near the cut surface, and this not only increased the likelihood that NMJs had mechanical damage but also any NMJs interior from the surface were not detectable. It must be noted that current enzymatic or photochemical electron dense reaction products will inherently have better correlative spatial resolution when compared to our described overlay method. This is due to the use of photon based detection method of light microscopy which has a diffraction limited lateral resolution of ~200 nanometers when using visible light. Although not widely available, it is certainly conceivable that refinement of this technique to overlay antibody or fluorescence protein based super-resolution techniques (Chi, 2009
) will allow lateral resolution on the order of 20nm.
Although this method was highly effective at efficiently locating NMJ in tissue blocks for subsequent high resolution imaging, some slight modifications in the technique may further streamline data acquisition and facilitate image correlation. One such improvement is the use of adhesive-coated Permanox™ slides with the CryoJane® Tape-Transfer System. Permanox™ slides exhibit no autofluorescence, are resistant to common chemicals used for TEM processing and also separate easily from polymerized resin by immersing the slides in liquid nitrogen. Using Permanox™ rather than glass slides would eliminate the danger associated with sectioning very near a glass surface with a diamond knife. Our studies tested adhesive coated Permanox™ slides to collect cryostat sections of sucrose-protected tissue, but we found that an oily residue on the slides interfered with confocal imaging of the fluorescently-labeled muscle fibers when slides were stored for prolonged periods in PBS (data not shown). An additional useful modification to this reported protocol would to include a nuclear or other counterstain with the fluorescent bungarotoxin. Labeling nuclei in addition to the NMJs would create conspicuous landmarks with the confocal images that could be easily identified in thin section TEM images and improve the ease of correlating between the two optical platforms.
The method utilized in this report holds great potential for studying difficult-to-locate NMJs in human biopsies. Indeed, data from our lab shows that we can obtain very similar results with human samples (). In some of the earliest work (De Harven and Cöers, 1959
) to study human NMJ ultrastructure by TEM, localization of NMJs was achieved by making an incision to expose the muscle fascia and electrically stimulating the muscle using a cathode electrode. The excitability threshold of the endplate was measured as a distinguishing factor in comparison with the surrounding muscle tissue, and a 2–4 mm muscle biopsy was obtained. This would provide a higher chance of finding a NMJ but would still require large areas of muscle tissue to be scanned ultrastructurally. Muscle biopsy samples obtained during surgical procedures provide little control and time for such measurements described above. Our method provides the advantage of shorter screening time and increased efficiency in accurately locating NMJs obtained from patients undergoing critical surgical procedures.
In this study, fluorescent alpha-bungarotoxin successfully labeled NMJs in 10 μm thick cryostat sections. Alpha-bungarotoxin is a small 74 amino acid peptide isolated from the venom of Bungarus multicinctus
that binds specifically to acetylcholine receptors (Lee, 1972
). One potential limitation of α-bungarotoxin is to reliably identify junctions in patients with severely reduced numbers of acetylcholine receptors. For such situations, it may be possible to use other fluorescent markers such as the ω-conopeptide derived from the venom of Conus
snails (Clark et al., 1981
), fasciculin derived from Dendroaspis angusticeps
venom (Rodriguez-Ithurralde et al., 1983
), or a fluorescent conjugate of the lectin DBA from the legume Dolichos biflorus
(Sanes and Cheney, 1982
). Fasciculin has been used in electron microscope autoradiography of NMJs (Anglister et al., 1998
) while DBA specifically labels carbohydrate moieties along the synaptic cleft, junctional folds, and basal lamina. The synthetic analog of ω-conopeptide, SNX-260, has been shown to specifically label non-N type voltage-sensitive Ca+2
channels along the presynaptic membrane of the motor neuron (Sugiura et al., 1995
). The applicability of ω-conopeptide, DBA, or fasciculin to the current method will need further investigation.
In conclusion, we describe a simple yet powerful technique to efficiently localize a structure of interest identified by light microscopy, in this case NMJs, for correlative high resolution transmission electron microscopy. The tedious nature and perhaps futility of random sectioning at the TEM level for difficult to obtain biopsy samples provided the impetus for us to develop an improved method. However, this technique certainly is neither limited to biopsies, nor NMJs and conceivably would be helpful in instances where searching for a rare structure or event is better suited for screening at the light microscopic level before attempting TEM. In this study, we took full advantage of the optical sectioning and improved resolution and contrast afforded by confocal microscopy to obtain a 3D perspective of the full targeted NMJ. We believe that this approach significantly augments any resolution detail obtained via TEM and allows the TEM data to be put into perspective due to inherent limitations (visualizing a 70nm slice from a 50 micron structure). Such a combinatorial correlative technique allows information to be collected in multiple modalities from an individual targeted structure in a way that transcends scale and is thus a valuable tool when exploring underlying pathologies. We must emphasize that while confocal microscopy provided unique capabilities/automation and facilitated certain aspects of this technique, it certainly is not an absolute requirement to locate NMJs. In fact, all screening can be easily done with conventional epifluorescence microscopes, which are far more available to many labs. Albeit with lower resolution, a single image with a low magnification objective lens can be used in place of the tile-scan montage and then higher magnification objectives used to target the structure of interest in more detail. Indeed, we also expect that any probes that are compatible with the initial paraformaldehyde fixation (i.e, antibodies, fluorescent affinity probes and green fluorescent protein-fusions) would be useful with similar efficacy.