The imaging method presented in this paper, array tomography, is based on immunostaining and imaging arrays of serial ultrathin (50-200 nm) specimen sections bonded tightly to glass slides. The individual two-dimensional section images are then aligned and collated into volumetric image stacks. Antibody stains can be stripped very efficiently, making it possible to multiplex large numbers of immunostains through repeated cycles of staining, imaging and stripping individual array slides. In addition, with array tomography GFP fluorescence is well-preserved. Fluorescence imaging can be followed by scanning electron microscopy to complement the immunofluorescence data with high resolution structural information.
Array tomography offers a number of advantages to existing imaging methods. For example, array tomography can achieve about an order of magnitude improvement in z-axis resolution over conventional immunofluorescence methods (see Fig. and ) by relying upon physical ultrathin sectioning (as low as 50 nm) rather than optical sectioning (500 nm in theory (Pawley, 1995
), but usually more than 700 nm in practice). This allows for a more precise mapping of antigen distribution, in particular when determining whether antigens are colocalized or confined to adjacent structures. Also, the detection of antigens is uniform throughout the depth of the specimen, allowing quantitative measurements of antigen distribution. For example, due to problems with antibody penetration and the difficulty of resolving small (<1 μm) and densely packed individual structures at the light microscopic level, reliable quantification of synapses has only been possible using electron microscopy, which is very time-consuming and laborious (Calhoun et al., 1996
; Geinisman et al., 1996
). Array tomography can now provide another reliable means to achieve such quantification faster and within larger tissue volumes, compared to electron microscopy. Another advantage is that tomography arrays also can be imaged at the ultrastructural level using scanning electron microscopy (SEM), as illustrated in . In the present paper, we used conventional methods for chemical fixation and tissue preparation for electron microscopy, which are not optimal for antigen preservation. However, for sensitive antigens, methods of tissue preparation which allow both good ultrastructural and antigenicity preservation are available (e.g. high-pressure freezing, freeze substitution and low temperature, partially hydrated embedding, (Newman and Hobot, 1999
) and compatible with array tomography.
While improvement in resolution was a major premise for the development of array tomography, several unexpected advantages were also discovered. For example, conventional fluorescent dyes showed remarkable photostability under the conditions of array tomography. After 6 min of constant illumination with a 100W mercury lamp, 85% of the initial fluorescence intensity remained, compared to a much faster photobleaching of the same dyes on cryostat sections, as observed by us, or in other applications, as reported in the literature (Lee et al., 2004
; Panchuk-Voloshina et al., 1999
; Sukhanova et al., 2002
). The photobleaching mechanisms of organic dyes are not completely understood, but it is known that this process depends on the environment (Song et al., 1995
; Zondervan et al., 2004
). The relative rigidity of the resin, restricting the mobility of molecules, is likely to contribute to the observed photostability. This property of array tomography allows for the collection of high quality images using longer exposure times.
In addition, in our hands antibodies were found to easily penetrate to a depth of 200 nm in LRWhite embedded material, thus increasing the amount of accessible antigens and allowing for an uninterrupted 3D reconstruction from serial sections as thick as 200 nm. There are conflicting reports about the ability of antibodies to penetrate LRWhite sections (Brorson et al., 1994
; Newman and Hobot, 1987
) and it is possible that this depends on the conditions of resin polymerization. It has been suggested that the relatively slow thermal polymerization of the resin that we employed limits cross-linking in the plastic and leads to linearity of molecular arrangement, thus facilitating antibody penetration (Newman and Hobot, 1987
Array tomography offers new opportunities for high-order multiplexing that were not easily available with previous immunoimaging methods. While antibody elution is not a new method (for example, (Kolodziejczyk and Baertschi, 1986
; Tramu et al., 1978
; Wahlby et al., 2002
), it has been limited previously by ultrastructural damage and decrease in antigenicity. Recently, a method using multiple rounds of immunolabeling, fluorescent tag bleaching and restaining of 5 μm cryostat sections, has underlined the importance and power of multiplexing approaches (Schubert et al., 2006
). LRWhite ultrathin sections on glass slides proved to be a very favorable substrate for antibody elution, and can undergo a large number of sequential cycles of staining, imaging, stripping and re-staining. We have demonstrated in this paper that quantitative staining can be carried out for nine cycles at least, and the limiting number may be much higher. Thus, where conventional indirect immunostaining methods are used to image four antigens per cycle, for example, 36 distinct antigens might be visualized by nine restaining cycles. It is now well established that most cell and tissue function results from interactions of numerous molecular species at distinct, nanometer-scale focal complexes (e.g., synaptic active zones). Array tomography is well suited for exploring the structure and function of such important focal complexes.
The numerous technical advantages of array tomography combine to offer access to critical aspects of neural circuit molecular architecture that were formerly very difficult to visualize and measure within tissue specimens. For instance, array tomography achieves easy and efficient resolution and quantification of individual synapsin immunolabeled puncta throughout large volumes of mature central nervous system neuropil. While the exact correspondence between synapsin immunolabeling and structurally identified synapses still remains to be demonstrated, all the available data strongly suggest that synapsin is a reliable marker for synapse characterization, and that it can be used as a marker in array tomography for synapse quantification. Traditionally, synapse measurements have required quite slow and circumscribed approaches such electron microscopic stereology. Given that changes in the numbers and volume densities of synapses are believed to be central to many human neurological and cognitive disorders, the potential of measuring such variables with both rigor and high throughput can be expected to have a very large impact. Furthermore, since immunofluorescence and SEM array tomography will lend themselves well to imaging human tissues (unlike methods that strictly require the expression of GFP or other transgenes), array tomography will be useful both for the direct study of human clinical specimens and for critical comparisons of human and animal disease-model tissues. The compatibility of GFP-based imaging methods with array tomography nonetheless offers additional very exciting opportunities to exploit the many special advantages of transgenic animals and to complement and extend modern in vivo imaging techniques with retrospective high-resolution analysis by array tomography. As a powerful technique for the volumetric imaging of brain tissue molecular architecture and ultrastructure, array tomography promises new avenues of attack on many issues that are now coming to the forefront of neuroscience. These include the molecular classification neural cell types, the determination of ion channel and receptor distributions within the tissue context, and circuit connectivity.