as a Guide for Electron Microscopic Study: A Simple Method ... · or hyperpolarizing (in the case of biocytin, 2-10 nA) current was passed ... Electron Miclosoopic Study. After examination

0022-1554/95/$3.30 The J o d of HistochcmiPag and CytochcmiPag Copyright 0 1995 by The Histochemical Society. Inc.

Technical Note

Vol. 43. NO. 3, pp. 329-335, 1995 Printed an U. S. A.

Using Laser Scanning Confocal Microscopy as a Guide for Electron Microscopic Study: A Simple Method for Correlation of Light and Electron Microscopy'

XUE J. SUN, LESLIE P. TOLBERT,2 and JOHN G. HILDEBRAND ARL Divirion of Neurobiology, The University of Arizona, Tucson, Arizona.

Received for publication July 18. 1994 and in revised form November 3, 1994; accepted November 21, 1994 (4T3432).

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fme branches embedded among m a n y other processes. Although electron miaoscopy provides a powerful tool for such study, the correlation of light mi-pic appearance and electron microscopic detail is very time- consuming. We report here a simple method combining laser scanning confocal miaoscopy and electron miaoscopy for study of the ~ y ~ p t i c relationships of the neurons in the antennal lobe, the fust central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and

Introduction Study of synaptic connections is fundamental to an understanding of nervous system function. Physiological recording in combination with cell staining with different dyes yields especially useful information about the functional properties and morphology of neurons in various nervous systems. Light microscopic (LM) analysis of neurons, however, often raises the question of whether synaptic C O M ~ ~ ~ ~ O Z ~ S occur at specialized regions of the neurites. Knowl- edge of whether synaptic connections between two neurons exist and where on the neuritic tree they occur provides valuable information about how signals are integrated by the neurons and thus about the role the neurons play in a given pathway. Electron microscopy (EM) provides a powerful tool for this type of study. Un- fortunately, correlation of three-dimensional LM data with EM detail is often difficult, as neurons are often large (ranging up to hundreds of micrometers in length) with complex branching patterns. Moreover, EM data usually lose most large-scale three-

' Supported by an award from the University of Arizona, Center for Insect Science (qS), by NIH grant NS-28495 (LPT, JGH), and by NIH grant

Correspondence to: Dr. L. P. Tolbert, ARL Division of Neurobiology, AI-23253 UGH).

611 Gould Simpson Bldg.. U. of Arizona, Tucson, AZ 85721.

processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in eponlaraldite. Interesting areas of the labeled neuron were imaged in the epon/araldite

sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to corre- late three-dimensional light miaoscopic information with electron miaoscopic detail, and can be very useful in studies of Synaptic connections. (J Histochem Cytochem 433329- 335, 1995) KEY WORDS: Neurobiotin; Biocytin; Confocal microscopy; Electron microscopy; Insect; hianduca sexta.

bl& with laser d g dd mi" and then thin-

dimensional information. Much work has been done to attempt to overcome these problems (3,4,8,10,14,19,21,25,26,31,34,35). To date, the most successful methods for correlating EM and three- dimensional data have been (a) three-dimensional reconstruction of thin sections, (b) semi-thin sectioning of tissue, serial reconstruction of the neuron at the light microscopic level, re-embedment, and finally thin-sectioning of selected semi-thin sections for electron microscopic study, and (c) high-voltage EM observation of relatively thick sections and reconstruction of high-voltage EM data. Although combination of these techniques or computer-assisted three-dimensional reconstruction and image processing techniques greatly facilitate the task, it is still very time-consuming to pursue this type of study.

Recently, laser scanning confocal microscopy (LSCM) has pro- vided a convenient way to obtain three-dimensional morphology of neurons, as optical sections of relatively thick tissue can be ob- tained easily and rapidly (5-7,20,24,27,28,30). Equally importantly, information is gathered as digitized optical sections and is readily displayed as three-dimensional stacks. Combination of LSCM and EM appears to be a promising way to study synaptic connections. Owing to incompatibility of staining for LSCM and EM, however, combination of these two methods is not straightforward. Deitch et al. (6) have used the reflection mode of LSCM imaging as a guide for EM study. The disadvantage of this method is that, because of weak reflecting signals, the quality of confocal images is often

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Figure 1. Multiple views of a local interneuron, labeled intracellularly with neurobiotin. in the antennal lobe of Manduca sexta; cell body indicated in A,C,D by arrowhead. (A) Conventional fluorescence micrograph of a vibratome section through the neuron before osmication and embedding in eponlaraldite plastic; x 10 objective lens. (B) Confocal micrograph of the same section before osmication and embedding in epon/araldite plastic. Projection of 18 optical sections taken at 3-pm intervals; x 20 objective lens. To minimize possible laser-induced damage, the images were acquired with minimal intensity of the laser source and a wider aperture, allowing a larger 2-step size. (C) Brightfield micrograph of same section after plastic embedding, showing immunogold labeling of the processes;

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CONFOCAL AND ELECTRON MICROSCOPY METHOD 33 1

poor or unacceptable with low-power objective lenses (7.33). We report here a method combining simultaneous immunogold and immunofluorescence labeling of neurons and an alternative way to use the LSCM to ascertain the exact site of interest for further EM study.

The material used in these experiments is the antennal lobe, the primary olfactory center, in the brain of the moth M a n d ~ c ~ sexta. put of this work has been reported in abstract form (27).

Materials and Methods Animals. Mandtuasexta(Lepidoptera, Sphingidae) were reared on U-

tificial diet under conditions described previously (L23.29). Female adults 1-4 days post emergence were used for all experiments.

Labcling of the Neurons. An isolated head preparation was used for labeling of neurons. In brief, to gain acccss to the a n t d lobe, the mouth- parts, frontal cuticle, and muscle systems of the head were removed, and the head was cut from the body and secured with insect pins in a recording chamber, where the head was superfused continuously with saline solution 1149.9 mM NaCI, 3 mM KCI. 3 mM CaClz, 25 mM sucrose, and 10 mM TES (N-Tris[hydroxvmethyl]-m~yl-2-uninocth acid), pH 6.91. These procedures and recording methods have been described in detail in previous reports (2.15). Usually, one neuron was labeled per antennal lobe and both antennal lobes of the brain were used.

Neurons were stained intracellularly with neurobiotin (Vector Labs; Burlingame, CA) or biocytin (Molecular Probes; Eugene, OR). In brief, the tip of a glass microelectrode was filled with either 4-5% neurobiotin in 1 M KCI or 4% biocytin in a solution of 0.5 M KCI in 0.05 M Tris buffer, pH 7.4. The shaft of the electrode was filled with 2 M KCI. A typical electrode filled with such a combination of solutions has a resistance of 80 megohms. After a stable impalement of a neuron or a period of intracellular recording, a depolarizing (in the case of neurobiotin, usually 2-10 nA) or hyperpolarizing (in the case of biocytin, 2-10 nA) current was passed through the electrode for a period of 5-30 min to inject dye into the cell.

Tissue Processing. Immediately after dye injection, the brain was dis- sected out of the head and fixed at 4°C overnight in freshly made fixative solution consisting of 4% paraformaldehyde, 0.32% glutaraldehyde, and 0.2% saturated picric acid in 0.1 M sodium phosphate buffer, pH 7.4. Af- ter fixation, the brain was washed briefly with 0.1 M PBS, pH 7.4, embedded in 7% agarose [low melting point agarose; gelling temperature of 2% (wlv) solution 26-30"Cl (Sigma; St Louis, MO), and sectioned at 50-80 pm with a vibratome (series 1000; Technical Products International, St Louis, MO). The sections were collected in PBS.

A standard problem in pre-embedding immunogold staining is the lack of penetration of the gold-conjugated reagent into the specimen. To enhance the penetration of the immunogold-conjugated streptavidin, we treated the vibratome sections with either 0.1% Triton X-100 (JT Baker; Phillipsburg, NJ) in PBS for a period of 10 min or a series of ethanol solutions (30, 50, 70, 50, and 30% ethanol) for 10 min at each step. The sections were incubated with a solution of 1.4-nm gold-conjugated sueptavidin (Nanoprobes; Stony Brook, NY) diluted 1:50-1:lOO in PBS for 2-4 days. Then 1:200 Cy3-conjugated streptavidin (Jackson ImmunoResearch; West Grove, PA) was added to the same solution and the sections were incubated

for an additional 8-12 hr. After this procedure, the neuron was reliably labeled by gold-conjugated streptavidin up to 40 pm into the tissue and could be detected both by LSCM (fluorescence) and by EM (gold particles). The immunogold particles then were enlarged with a silver enhancement solution (Biocell Research Labs, fkom Ted Pella; Redding, CA) to facilitate detection at lower magnification in the EM. In brief, the sections were washed several times with PBS and distilled water after the immunostain- ing and incubated in the silver enhancement solution on ice in the dark for 8 min. The sections then were warmed to room temperature (RT) for another 8 min of reaction under normal room light.

After silver enhancement, the sections were washed with sodium phosphate buffer (0.1 M, pH 7.4). As the os04 oxidizes the silver precipitates of the silver-enhanced gold particles, tissues were lightly osmicated at RT (0.1% os04 in phosphate buffer for 5-10 min) and dehydrated through a graded series of increasing concentrations of ethanol, followed by two changes of 10 min each in propylene oxide (Electron Microscopy Sciences; Fort Washington, PA). The sections were then placed in a 1:1 mixture of eponlddi te and propylene oxide for 30 min and then a 3:l mixture for 2-3 hr. The tissue was then infiltrated with pure fresh eponlaraldite overnight and finally flat-embedded between Aclar sheets (Ted Pella) (17) in eponlddite. After polymerization at 60'C overnight, selected sections were glued to blank eponlaraldite blocks with fast cyanoacrylate adhesive (Electron Microscopy Sciences) (13). The block was trimmed with a glass knife to obtain a smooth surface for LSCM observation. The bottom of the block was cut so that the bottom surface would be parallel to the section glued onto the top of the block. The block was then placed on a blank slide under the objective of the microscope and viewed with the LSCM, and thin-sectioned for EM study.

Lascr Scanning C o d d Microscopic Study. The labeled neurons were imaged with a Bio-Rad MRC 600 laser scanning confocal microscope mounted onto a Nikon Optiphot-2 microscope and equipped with a kryptonlargon laser light source (Bio-Rad; Cambridge, MA). Serial optical sections (usually at intervals of 1-2 pm) were imaged from the surface ofthe block through the depth of the vibratome sections and saved as three-dimensional stacks. Projection of these sections gave a two-dimensional reconstruction of the labeled neuron. Display of the images color-coded by depth allowed rapid determination of the depth at which features of interest lay.

Electron Miclosoopic Study. After examination with the LSCM. the epon blocks containing labeled neurons were sectioned several ways. Some blocks were thin-sectioned h o u g h the entire vibratome section and the grids were labeled according to the depth from which they came. Other blocks were alternately semi-thin- and thin-sectioned, and thin sections were taken either at regular intervals or at preselected depths of interest in the tissue. Sections were taken with a diamond knife on a MTboOO-XL microtome (RMC Tucson, AZ) or a Reichert-Jung Ultracut microtome (Leica; Deerfield, IL). The thin sections were picked up on formvar-coated slot grids or on thin- bar copper grids and post-stained with uranyl acetate and lead citrate. The thin sections were examined with a JEOL JEM-1200EX transmission electron microscope, and selected labeled processes were photographed, usually at magnifications between x 5000 and x 25,000.

Results During pre-embedding immunogold staining of biotin-labeled neu-

x 20 objective lens. (D) Conventional fluorescence micrograph of the neuron after osmication and plastic embedding; x 20 objective lens. (E) Confocal micrograph of the same neuron in epon/araldite block (dashed line indicates edge of block). Projection of 26 optical sections taken at 2-pm steps; x 20 objective lens. Image was collected at a higher level of laser illumination than in B because tissue, now fixed and embedded, was less sensitive to laser-induced damage. (F) A single optical section from the series of confocal images; arrow indicates process shown in G. (0) Electron micrograph from approximately the same optical level, showing the neurobiotin-labeled process (arrow) indicated with arrow in F. Bars: A-F 100 pm; G = 2 pm.



Figure 2. Uniglomerular projection neuron of the antennal lobe of M. sext8. (A) Confocal micrograph of vibratome section through neuron, scanned before osmication and embedding. Projection of 14 optical sections taken at 2-pm steps; x 40 objective lens. (E) Same neuron scanned after embedment in eponlaraldite block. Projection of 16 optical sections taken at 2-pm steps; x 40 objective lens. (C) Confocal micrograph of a single optical section 34 pm from the surface of the block shows a beaded process (arrowhead). @,E) Confocal micrographs of the same neuron scanned (in the plastic block) before (D) and after (E) thin- sectioning the beaded process. Arrows indicate position of beaded process, missing in E. Dashed line in D indicates edge of plastic block. Projections of 14 (D)

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CONFOCAL AND ELECTRON MICROSCOPY METHOD 333

rons, we noted that the immunogold-conjugated streptavidin did not bind to all of the injected biotin in a neurobiotin- or biocytin- labeled neuron. This phenomenon was observed even with very long incubation times (up to 5 days) and/or detergent permeabili- zation of the tissue, and therefore was not due to incomplete penetration. Taking advantage of the remaining unbound biotin, we treated sections containing labeled neurons with Cy3-conjugated streptavidin at the end of the immunogold-streptavidin staining step. Now the neuron could be visualized by LSCM, but another problem arose: the fluorescence was lost after normal processing of tissue for EM. By reexamining the fluorescence after each step in the processing of the tissue, we found that the fluorescence is retained well through most of processing steps but is lost during the osmication step, which is essential for good tissue preservation and adequate contrast for EM observation. We overcame this problem by lowering the concentration of os04 to 0.1% (one tenth of the normal concentration used) and shortening the osmication time to less than 10 min (instead of 30 min). Although the fluorescence signal was weaker than before osmication in such preparations and sometimes was barely visible by standard fluorescence microscopy, it was strong enough for LSCM imaging, More importantly, the fluorescence signal lasted for a long time: we rescanned some blocks 6 months after embedding without noticing any appreciable loss of fluorescence. The tissue preservation, although not superb, was adequate for EM observation. One of the “doubly labeled’ local interneurons of the antennal lobe of M. sexta is shown in Figure 1.

As the fluorescence is retained after embedding in eponlaraldite, the labeled neuron could be imaged even after the vibratome section containing it had been glued onto a block (Figure 1E). Using the step motor of the LSCM, we imaged the labeled neuron from the top surface of the flat-embedded tissue (which could be easily defined by a strong reflection signal, the slightly uneven surface, knife mark lines, or the edge of the plastic block) and through the entire thickness of the tissue at steps of 1-2 pm. The aperture on the LSCM was set to give optical sections estimated to be between 2 and 4 pm thick, based on overlap of images taken at 2-pm intervals.

Areas of interest could be located by examining the single optical sections and cutting semi-thin sections directly to the approximate level of the optical section before collecting thin sections (Figures 1F and 1G). However, although LSCM proved to be very useful for locating interesting areas of the labeled neurons for thin sectioning, correspondence of an optical section of LSCM with par- ticular thin sections for EM was only approximate, for several rea- sons. First, the actual thickness of a single optical section may vary, as it depends on the numerical aperture of the objective lens and the aperture of the LSCM, which depends on the strength of the fluorescence signal and the intensity of the laser light source. Sec- ond, inaccuracy of the LSCM step motor and/or microscopic stage and variation of thin-section thickness (often beyond investigator’s control) leads to a systematic error that is compounded as the num-

ber of sections increases. This will be an especial problem when the area of interest is deep in the block. Finally, the plane of section for thin-sectioning rarely corresponds exactly with the plane of optical sectioning. These problems could be overcome easily, however, by rescanning in the LSCM the same block before and after thin-sectioning and determining precisely which labeled areas had been cut away.

Figure 2 demonstrates a two-dimensional projection of a uniglomerular projection neuron scanned at 2-pm step intervals. The LSCM image (data not shown) demonstrated that the top 30 pm of the block contained tissue with only the axon of a labeled neuron; the interesting area (arrows, beaded processes; Figures 2B and 2C) spanned between 32 and 36 pm from the surface of the block. Figures 2D and 2E show images of the same block rescanned after 32 pm (Figure 2D) and 37 pm (Figure 2E) were sectioned away. The correlation between the depths of sampling with the LSCM and with the ultramicrotome was good. Figures 2F and 2G are electron micrographs of the labeled process in the interesting area taken at 35-36 pm from the surface of the block, demonstrating that the process formed output synaptic connections onto unlabeled neurites.

Discussion Several different techniques have been used to bridge the gap between LM and EM (3,4,8,10,14,19,21,25,26,31,34,35). One common way is to section the block into semi-thin sections, make a three- dimensional reconstruction of the neuron at the LM level, and then thin-section areas of interest (4,8,10,19,21). However, three-dimensional reconstruction from sections is tedious, and valuable tissue may be lost during reembedding and resectioning. High-voltage EM is a reasonable alternative, as relatively thicker sections can be observed, thus reducing the number of sections needed to span a neuron. This method, however, requires access to a high-voltage EM. We have developed a technique using LSCM as a guide for EM study. A biotin-labeled neuron is rendered detectable by both fluorescence (for LSCM) and immunogold (for EM). After such dou- ble labeling, interesting areas of physiologically identified and intracellularly labeled neurons can be investigated at the EM level to see where synaptic connections are formed. Although with our method three-dimensional reconstruction might also be needed, the number of vibratome sections needed to span a neuron is small (two to four in our case with 80-pm sections). This greatly reduces the task.

The major challenge in correlation of LSCM and EM is the com- patibility of the labeling methods. Visualization methods using diaminobenzidine (either in a peroxidase reaction or in a reaction catalyzed by photo-oxidation of fluorescent dyes) and Golgi staining have been used widely for both LM and EM observations (see, e.g., 8,26,35). The light- and electron-dense label, however, is not suitable for LSCM imaging, which is best performed on fluores-

and 10 (E) optical sections taken at 2-pm steps; both scanned with x 40 objective lens; E magnified electronically. (F,G) Electron micrographs of the indicated beaded process (arrowheads in C and D) demonstrate that the process forms output synapses (arrows) onto unlabeled processes. Bars: A-D = 25 pm; E = 12.5 pm; F,G = 0.5 pm.



334 SUN, TOLBERT, HILDEBRAND

cent labels. One possibility is to use fluorescent labels, perform LSCM, and, after LSCM imaging, convert the fluorescent dyes into electron-dense materials by using either photo-oxidation of fluorescent dyes [such as Lucifer Yellow (16) or DiI as suggested by Vischer and Durrenberger (32)] in the presence of diaminobenzidine or an antibody against the dye injected into the cell. It is possible to determine the position of an interesting area of a neuron by LSCM with this method, but relocating the same area after plastic embedding is much more difficult than the presently reported method because of shrinkage of the tissue during tissue processing for EM and the inability to monitor the exact position by LSCM during sectioning. The reflection mode of LSCM has been applied successfully to detect electron-dense label in several systems (6,7,22,33). With this mode of imaging, Deitch et al. (6) have proposed another way of combining LSCM and EM. Reflection signals in the LSCM are often weak, however, and do not take full advantage of the excellent resolving capabilities of the confocal microscope. Moreover, to obtain a useful image with the reflection mode of LSCM, high-power objective lenses (usually x 40 or higher) are needed (7,33). This requires scanning the same neuron several times and making montages of the neuron. The technique presented here provides an alternative way to combine LSCM and EM. This technique takes full advantage of LSCM and minimizes the loss of tissue inherent in resectioninglreembedment procedures, as only few vibratome sections are needed to cover an entire neuron.

An additional advantage of this method is that, because the neuron is labeled with immunogold particles, the internal struc- ture of the labeled processes is not significantly masked by the labeling as it is with the peroxidase-based labeling techniques. This is particularly advantageous when a small process is presynaptic to another process. In the limit, however, the label can be barely detectable above background in very fine processes. We overcome this problem by examining more than one thin section to verify that a given process contains gold particles in adjacent sections.

We found that the major restriction of the technique is that the osmicated tissue is barely translucent, and the depth that can be imaged by LSCM is restricted to about 80 pm (Figure 2). Al- though it is possible to increase the transparency of the tissue by reducing the time and concentration of Os04 treatment even further, this reduces the quality of the ultrastructural preservation of the tissue to unacceptable levels.

Electrophysiological and morphological characteristics of neurons are important for understanding the function of the nervous system. Intracellular recording and subsequent intracellular staining yield a great deal of information, both morphologically and physiologically. Combination of these techniques with EM study of the physiologically characterized neuron, however, is usually difficult. The method we have described for simultaneous fluorescent- and gold-tagging of injected dyes helps to bridge the gap between light and EM. Although we have applied this technique only to neurons of the antennal lobe of the moth M. sextu, we believe that the technique could be successfully applied to other neural tissue, since the dyes we have used have been applied broadly and successfully in other preparations (9,11,12,18,20).

Acknowledgments We wish to thank Patricia Jansma for excellent technical assistance with kaser scanning confocdmicmscopy and Charles Hedgcock, R. B.P, forprepar-

ing thefina/photographicp&tes. We dso thank A. Osmun for rearhag Man- duca sexta and Drs James Buckner andJames Svoboda (USDA) forpmvid- ing M. sexta eggs.

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as a Guide for Electron Microscopic Study: A Simple Method ... · or hyperpolarizing (in the case of biocytin, 2-10 nA) current was passed ... Electron Miclosoopic Study. After examination