- Email: [email protected]
[9] Combining laser scanning confocal microscopy and electron microscopy
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art technique in the continuing investigation of cell structure and function in normal and pathological conditions.
Acknowledgments This work was supported by an M R C C grant to Dr. G h a s s a n Bkaily. The authors thank Ms. Susann Topping for secretarial assistance.
[9] C o m b i n i n g
Laser Scanning Confocal Microscopy and Electron Microscopy
By
XUEJUN SUN
Introduction Laser scanning confocal microscopy (confocal microscopy) provides a exciting new tool for biological research. For the first time, it is possible to obtain clear optical sections of relatively thick specimens without physically sectioning the tissue. Additionally, because of the high Z-axis resolution of preregistered images it provides, confocal microscopy enables rapid three-dimensional (3D) reconstruction of specimens. The combination of confocal microscopy with powerful computer image-processing techniques gives unprecedented possibilities for visualizing and measuring stained cells in three dimension. 1 The technology has gained wide acceptance in biological research in just one decade of its commercialization. Although it is true that confocal microscopy provides higher resolution than conventional light microscopy, it is still fundamentally light microscopy, limited in resolution by the wavelength of the light source and the numerical aperture of the lens. This is at best about 150 nm with current optics. In many fields of biological research, one needs subcellular resolution better than 150 nm. For example, the establishment of an association of a gene product with a type of organelle leads to important insights into the role of the product in the cell. For subcellular study, electron microscopy (EM) is still an indispensable tool for the unambiguous identification of organelles. EM provides a resolution that cannot be matched by any other imaging tool currently available. EM, however, suffers from a lack of 3D l H. Chen, J. R. Swedlow, M. Grote, J. W. Sedat, and D. A. Agard, in " H a n d b o o k of Biological Confocal Microscopy" (J. B. Pawley, ed.), 2nd ed., p. 197. Plenum Press, New York, 1995.
METHODS IN ENZYMOLOGY,VOL. 307
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information. Because of the poor penetrating power of electrons, observation with E M is often limited to a fraction (nanometer range) of the cellular structure. High-voltage E M provides an alternative that yields both high resolution and some degree of 3D information. It is, however, not readily accessible to most biologists and even with high-voltage EM, specimens no thicker than 1 /zm can be observed. Three-dimensional reconstruction is still required to have a 3D view of cells. N e u r o a n a t o m y provides an extreme situation in which 3D information is particularly important. Neurons often are large and have complicated 3D structure. The intrinsic 3D morphology reflects the connections of neurons within a neuronal network. Yet, ultimately, the discrete synaptic connections, and the neurotransmitter(s) used by them, rule the exact roles a particular neuron plays within the network. Study of stained neurons at the level of light microscopy often pinpoints particular loci of interest. Whether there are synaptic connections in the pinpointed areas provides necessary information for understanding the neuron and the circuit. Assessment of the three-dimensional morphology of neurons requires light microscopic study whereas synaptic information is available only through ultrastructural study. Therefore, many investigators have attempted to combine E M with LM. 2-5 All currently available methods require a certain degree of 3D reconstruction, which, even with advanced computer imaging technology, is still labor-intensive and sometimes impossible for large neurons. The combination of confocal microscopy and E M is a promising avenue for biologists attempting to solve this problem of 2D versus 3D. Confocal microscopy can provide a valuable overview and 3D information. E M provides ultrastructural detail. A combination of these two technologies is, however, not straightforward because the two technologies require very different tissue processing procedures and staining. First, for confocal microscopy, a specimen must be stained with a fluorescent marker, which is not necessarily electron dense and therefore not suitable for E M study. Second, for confocal observations, the tissue must be translucent, whereas for electron microscopy, heavy metals are used that are opaque to light. Third, for E M observation, tissue is usually embedded in plastic. This often introduces a large amount of tissue shrinkage and distortion, which render the correlation of light microscopic information with ultrastructural data difficult. A combination of confocal microscopy and E M has been attempted
z K. Hama, T. Arii, and T. Kosaka, Microsc. Res. Tech. 29, 357 (1994). 3j. DeFelipe and A. Fair6n, J. Histochem. Cytochem. 41, 769 (1993). 4j. K. Stevens, T. L. Davis, N. Friedman, and P. Sterling, Brain Res. 2, 265 (1980). 5R. W. Ware and V. LoPresti, Intl. Rev. Cytol. 40, 325 (1975).
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using the reflection mode of imaging with the confocal microscope. 6 With this method, a cell labeled with an electron-dense marker can be imaged in the confocal microscope by bouncing light off of it. This method, however, is not widely applied due to the generally poor quality of reflection in confocal images. We have been studying the neuronal connections within insect central nervous systems by combining standard confocal microscopy with EM. 7-1° The two methods described in this article rely on a very specific interaction between biotin and avidin. Although the methods were developed primarily for neuroanatomy with biotin-stained neurons, it can be adapted to other tissues to localize various substances within cell using biotinylated antibodies or D N A / R N A probes, as shown in this article with an antibody against serotonin, a neuromodulator in insects. I1'I2
Experimental Procedures Overview of Methods
The methods require the introduction of biotin or biotin derivatives into cells. The biotin can subsequently be detected using a fluorescent tag for confocal microscopy and an electron-dense tag for E M observation. Confocal microscopy can provide a guiding tool for thin sectioning in order to facilitate the correlation of confocal information with E M results. Both methods involve these basic steps of introduction of biotin, introduction of appropriate tags for the biotin, confocal observation, and E M observation. Both methods have been applied on insect central nervous tissue. Adult central nervous systems of two insect species were used: the moth M a n d u c a sexta (Lepidoptera, Sphingidae) and the white-eyed mutant housefly M u s c a d o m e s t i c a (Diptera, Muscidae). The detailed rearing procedures have been described previously (for moth, see Bell and Joachim13; for fly, see Fr6hlich and Meinertzhagena4).
6j. S. Deitch, K. L. Smith, J. W. Swann, and J. N. Turner, J. Microsc. 160, 265 (1990). 7X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Histochem. Cytochem. 43, 329 (1995). 8X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Comp. Neurol. 379, 2 (1997). 9X. J. Sun, L. P. Tolbert, J. G. Hildebrand, and I. A. Meinertzhagen, J. Histochem. Cytochem. 46, 263 (1998). 10L. P. Tolbert, X. J. Sun, and J. G. Hildebrand, J. Neurosci. Methods 69, 25 (1996). 11A. R. Mercer, J. H. Hayashi, and J. G. Hildebrand, J. Exp. Biol. 198, 613 (1995). 12A. R. Mercer, P. Kloppenburg, and J. G. Hildebrand, J. Comp. Physiol. A 178, 21 (1996). 13R. A. Bell and F. A. Joachim, Ann. Entomol. Soc. Am. 69, 365 (1976). 14A. Fr6hlich and I. A. Meinertzhagen, J. Neurocytol. U, 159 (1982).
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Method 1: I m m u n o g o l d Fluorescence Method This method takes advantage of the finding that immunogold reagents do not bind to all the available biotin in biotin-stained cells. 7 Therefore, the biotin-stained neurons can be incubated with immunogold-conjugated avidin first and then the remaining biotin molecules can be tagged with fluorescent avidin. Cell Staining with Biotin
Biotin was introduced into cells by one of three methods. Intracellular Labeling of Neurons. Biotin derivatives, Neurobiotin (Vector Labs., Inc., Burlingame, CA) or biocytin (Molecular Probes, Inc., Eugene, OR), are injected intracellularly into neurons by electrophoresis through a micropipette. For this, an isolated head preparation is used. To gain access to the brain, the mouth parts, frontal cuticle, and muscle systems of the head are removed surgically, and the head is cut off the body and secured with insect pins in a recording chamber where it is superfused continuously with saline solution [149.9 m M NaC1, 3 m M KC1, 3 m M CaC12, 25 m M sucrose, and 10 m M TES (N-tris[hydroxymethyl]-methyl-2aminoethanesulfonic acid), pH 6.9]. A standard electrophysiological recording setup is used to inject the dye as described previously. 15 The tip of a sharp glass microelectrode is filled with either 4% Neurobiotin in 1 M KC1 or 4% biocytin in a solution of 0.5 M KC1 in 0.05 M Tris buffer (pH 7.4). The shaft of the electrode is filled with 2 M KC1 solution. 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 is passed through the electrode for a period of 5-30 min to inject label into the cell. Back-Filling of Neurons. Biotin derivatives can also be introduced by back-filling,7'9 a staining technique widely applied in other animals (reviewed by McDonalda6). Back-filling is accomplished by cutting surgically accessible axons of the neurons to be labeled. The cut end of the nerve is then exposed to distilled water for approximately 1 min in order to open the damaged neurites. A drop of biotin solution (a few crystals of Neurobiotin in distilled water, estimated concentration <0.2%) is placed over the cut area. The whole animal is then placed in a humidified chamber for 5-20 min for the label to be picked up by the damaged cells. The incubation time depends on the distance that the biotin has to travel. Ten minutes is sufficient for about 2 mm of diffusion. Because biotin travels rather quickly within 15S. G. Matsumoto and J. G. Hildebrand, Proc. R. Soc. Lond. B 213, 249 (1981). 16A. J. McDonald,Neuroreport 3, 821 (1992).
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dendrites, there is no need for additional diffusion time. The area of the cut is flushed with saline after staining to wash out the excess biotin. The brain is then dissected out and processed as described later. Immunocytochemical Staining by Biotinylated Antibody. Biotin can also be introduced as common biotin-conjugated probes, such as biotinylated secondary antibodies. We tested this method with a biotinylated secondary antibody. The detail procedure for this type of application is described later in a separate section.
Tissue Processing Fixation. The fixation requirements for confocal microscopy and EM are very different. Confocal applications do not tolerate glutaraldehyde fixation because the high autofluorescence introduced by glutaraldehyde will potentially interfere with the fluorescent label. For EM study, a combination of paraformaldehyde and glutaraldehyde is the most common ehemical fixation procedure. We find that lowering the concentration of glutaraldehyde to 0.15-0.40% is compatible with most confocal applications while maintaining adequate tissue preservation for EM. Additionally, using longemission wavelength fluorophores such as Cy3 reduces the problem associated with autofluorescence. For EM, the fixative solution must be fresh. Immediately after injection of biotin, the brain is dissected out of the head and fixed at 4 ° overnight in freshly made fixative solution comprising 4% paraformaldehyde, 0.32% glutaraldehyde, and 0.2% saturated picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Picric acid is optional. It has been suggested that picric acid stabilizes membranes and precipitates proteins for EM observation. 17 It introduces, however, some additional degree of autofluorescence. We do not find a noticeable difference in preservation of ultrastructure in preparations processed with or without picric acid. Fixation time can vary, depending on tissue size. We routinely fix moth brains overnight at 4° for convenience (approximate size of the brain is 2 × 3 mm). However, fixation times as short as 30 min can be applied at room temperature. Tissue Sectioning. It is necessary to cut the tissue into thin slabs because (1) the immunogold reagent has a limited penetration power and (2) the lenses used for confocal microscopy have a short working distance. After fixation, the brain is washed briefly with sodium phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and embedded in 7% agarose [low melting point agarose; gelling temperature of 2% (w/v) solution: 26-30°; Sigma, St. Louis, 17M. G. Zomboniand C. de Martino,J. CellBiol. 35, 148A (1967).
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MO]. The brains are then sectioned at 50-80/xm with a Vibratome (series 1000; Technical Products International, Inc., St. Louis, MO). The floating sections are collected in PBS. Permeabilization and Incubation with Immunogold-Conjugated Streptavidin. A common problem in immunogold staining before embedment is the lack of penetration of the gold-conjugated reagent into the specimen because of the relatively large size of the gold particles. To enhance the penetration of the immunogold-conjugated streptavidin, we treat the Vibratome sections with either 0.1% Triton X-100 (J. T. Baker, Inc., Phillipsburg, NJ) in PBS for a period of 10 min or a series of ethanol solutions (30, 50, 70, 50, and 30% ethanol) for 5-10 min at each step. Optimal penetration is achieved with such permeabilization and using streptavidin conjugated to small (1.4 nm) gold particles. We have attempted to use smaller gold particles (0.8 nm, Nanoprobes Inc., Stony Brook, NY) to enhance the penetration of the reagent, but the 0.8-nm gold particle-conjugated streptavidin penetrated even less than the 1.4-nm gold. After permeabilization, the sections are incubated with a solution of 1.4-nm gold-conjugated streptavidin (Nanoprobes Inc.) diluted 1:50-1 : 100 in PBS for 2-4 days on a shaker in an 8° cold room. Then 1:200 Cy3conjugated streptavidin (Jackson ImmunoResearch Labs., Inc., West Grove, PA) is added to the same solution, and the sections are incubated for an additional 8-12 hr in the cold room on the shaker. Following this procedure, the biotin-injected neuron is labeled reliably by gold-conjugated and fluorescently tagged streptavidin up to 40/zm into the tissue and could be detected both by confocal microscopy (fluorescence) and by EM (gold particles) (Fig. 1).
Confocal Observation Stained slices of brain are mounted on slides in saline or glycerol-based mounting medium (Vectashield, Vector Labs., Inc.). Care must be taken to avoid drying the tissue for potential ultrastructural damage. We normally use nail polish to seal the slide. We use a Bio-Rad MRC 600 confocal microscopy (Bio-Rad, Cambridge, MA) mounted on a Nikon Optiphot-2 microscope and a Zeiss LSM 410 mounted on a Zeiss Axiovert M100 microscope, both equipped with a krypton/argon laser light source for confocal observations. The tissue is usually imaged with a 20x (NA 0.75) Nikon objective lens (Bio-Rad) or with a 25x (NA 0.8) multi-immersion Plan Neofluar objective lens (Zeiss). A multi-immersion lens is highly suitable for such a study in order to minimize the effects of refractive index mismatch. Serial optical sections, usually at intervals of 1-2/zm, are imaged through the depth of the labeled
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A.
|}
FIG. 1. Example of examination of a Neurobiotin-injected neuron using the immunogold fluorescent method to combine confocal and EM. (A) Intracellular staining of a local interneuron in the antennal lobe of the moth Manduca sexta. The neuron was imaged in saline, before plastic embedding, at 3-/zm intervals for a total of 18 optical sections. The neuron ramifies extensively throughout the neuropile (antennal lobe). The soma is out of the field of view. (B) Bright-field image of the same neuron after silver enhancement and plastic embedding shows enhanced immunogold staining. The cell body is indicated with an arrowhead. (C) Confocal image of the Epon block demonstrates that the fluorescence is retained. Dashed line indicates the edge of the block. Comparison with confocal image obtained before plastic embedding (A) shows that most of the small processes remain detectable. (D) Single optical section from the 3D image stack at the level of the ultrathin section taken in E. Arrow indicates the corresponding position of the process. (E) Thin section of the same neuron at EM level showing the ultrastructure of the labeled process, which contains numerous gold particles. Bars: ( A - D ) 100/zm, (E) 2 k~m. Adapted, with permission, from X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Histochem. Cytochem. 43, 329 (1995).
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neurons and saved as three-dimensional image stacks. Projecting this image stack onto a single two-dimensional plane generates a 2D image of the reconstructed labeled neuron. The image stack is also studied with appropriate software to visualize the neuron in three dimensions. Areas of interest are defined within the stack. There is no evidence of ultrastructural damage by laser radiation on fixed tissue. However, to avoid potential ultrastructural damage by strong laser radiation with the confocal microscope, we try to minimize both the scanning time and the laser intensity by choosing an acceptable fast scan speed and a low laser power setting. This usually involves a tradeoff of image quality, imaging duration, and laser intensity.
Silver Enhancement of Gold Particles The 1.4-nm gold particles, too small to allow easy observation under the EM, are enlarged with a silver enhancement solution (Biocell Res. Labs., from Ted Pella, Inc., Redding, CA) in order to facilitate detection in EM. The silver enhancement procedure is modified from the protocol suggested by the manufacture of the kit. In brief, after confocal observation, the selected sections are washed three times (10 minutes each) with PBS to remove the mounting medium. They are then washed in deionized water (two times at 3 min each) and incubated in the silver enhancement solution on ice in the dark for 8 min to allow diffusion of the solution into the sections. The container containing the sections is subsequently transferred to a water bath at room temperature for another 8 min of reaction in the dark. Timing of the reaction is important and care must be taken to avoid a self-nucleation reaction. The silver enhancement reaction is stopped by several washes with deionized water (three times at 3 min each). The sections are then transferred to sodium phosphate buffer (0.1 M, pH 7.4) for further processing for EM study.
Pos~xation for EM Study For EM, it is necessary to stabilize cell membranes by postfixation and then to embed in plastic. A common postfixative for EM is osmium tetroxide. As it was suggested that osmium tetroxide oxidizes silver precipitates of the silver-enhanced gold particles, TMtissues are lightly osmicated at room temperature (0.1% osmium tetroxide in phosphate buffer for 5-10 min). The postfixation process is stopped by replacing the solution with phosphate buffer. This procedure stabilizes the cell membranes enough for plastic embedding while retaining enough silver precipitate on the gold particles 18A. I. Basbaum,J. Histochem. Cytochem. 37, 1811 (1989).
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for EM observation. Osmium tetroxide and its vapor are highly toxic. This postfixation step must be carried out under a well-ventilated fume hood and the waste solution disposed of properly.
Plastic Embedding The postfixed brain slices are dehydrated through a graded series of increasing concentrations of ethanol (50, 70, 95, and twice 100% for 10 min at each step), followed by two changes of 10 min each in propylene oxide (Electron Microscopy Sciences, Fort Washington, PA). The sections are then placed in a 1 : 1 mixture of Epon/Araldite and propylene oxide for 30 min. The tissue is infiltrated with pure fresh Epon/Araldite overnight on a rotatory shaker. To flat embed the sections, sections are sandwiched between two Aclar sheets (Ted Pella, Inc., Redding, CA) in Epon/Araldite, and this assembly is further sandwiched between two microscope slides with two paper clips to hold the whole assembly together. The whole assembly is then put into a 60° oven overnight for polymerization. One Aclar sheet can be peeled off easily and the sections can be examined with bright-field microscopy (Fig. 1B) or with the confocal microscope. Selected sections are cut out with a razor blade and glued to blank Epon/Araldite blocks with fast cyanoacrylate adhesive (Electron Microscopy Sci., Inc., Ft. Washington, PA) for microtomy.
Confocal Microscopy as Guide for Microtomy We found that a certain degree of Cy3 fluorescence is retained after processing tissue as described earlier. Therefore, it is possible to use a confocal microscope as a guide for microtomy. For viewing the block on the confocal microscope, the block is trimmed with a glass knife in order to obtain a smooth surface for confocal observation. It is then held in a specially fabricated brass slide with a hole in the middle. In this way, the block can be imaged in either an inverted or an upright position depending on the configuration of the microscope. These arrangements allow, moreover, a slight tilt of the surface of the block, if the surface of the block is not totally parallel to the image plane, which is a critical prerequisite for determination of the depth of the area of interest. The confocal microscope stage and the z-axis motor are calibrated and the reproducibility of both checked carefully so as to provide accurate depth information. The surface of the block is defined easily by its strong reflecting signal, giving the starting depth of 0/xm. The area of interest can be relocalized with confocal microscopy by reference to the initial set of confocal images. Through the readout of the z-axis motor, the actual depth of the area can be obtained
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for guiding microtomy. To ease the effort of relocalizing the fiber of interest under EM, the block is trimmed as small as possible.
Electron Microscopic Study After examination with the confocal microscope, the Epon blocks containing labeled neurons are thin sectioned either at regular intervals by alternating thin sectioning and semithin sectioning or at preselected depths of interest in the tissue defined by the confocal study. Sections are cut with a diamond knife on a MT6000-XL microtome (RMC Inc., Tucson, AZ) or on a Reichert-Jung Ultracut microtome (Leica, Deerfield, IL). To facilitate localization of the chosen depth, thin sections are collected so that the ribbon on each grid contains a defined thickness of tissue. Because of the refractive index mismatch and inaccuracies of the microscopic stage, the depth information obtained using the confocal microscope may not be precisely accurate. Additionally, estimates of section thickness on the microtomes will incorporate some error. Therefore, additional thin sections are collected before and after the supposed depth of interest in order to ensure that the area of interest is contained in the thin sections collected. Thin sections are picked up on Formvar-coated slot grids or on thin-bar copper grids and poststained with saturated aqueous uranyl acetate solution and with lead citrate for 10 min each. The thin sections are examined with a J E O L JEM-1200EX transmission EM or with a Philips 201C EM. The labeled processes are selectively photographed, usually at magnifications between x5000 and x15,000 for the identification of synapses.
Correlation of Confocal Microscopy and EM In the majority of cases, depth information and the shape of the labeled process in the sections enable easy correlation of the confocal overview with EM detail. However, this correlation must be made with care, as a confocal optical section at the resolution of the lenses used in these experiments may contain the same amount of tissue as is included in as many as 40 thin sections. Moreover, localizing small, labeled profiles in a relative large area under EM is not an easy task. This can be eased by trimming the book as small as possible to limit the area of view for EM. One example using the immunogold fluorescence method to combine confocal microscopy and EM is presented in Fig. 1. A neuron in the antennal lobe of the moth brain is stained intracellularly with Neurobiotin and viewed with confocal microscopy before plastic embedding (Fig. 1A) and again in the plastic block (Fig. 1C). The transmitted bright-field microscopy (Fig. 1B) shows the immunogold labeling at the light microscopic level. An
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electron micrograph (Fig. 1D) of the neuron is shown. Use of this method to investigate the properties of insect neurons has been published in separate papers, s Method 2: Avidin-Biotin Complex Method This method uses a commercially available kit [avidin-biotinperoxidase complex (ABC) from Vector Labs. Inc.] to convert some of the fluorescence signal in a fluorescently stained cell into an electron-dense form using 3,3'-diaminobenzadine (DAB) as substrate. It is based on the finding that avidin conjugated to a fluorescent tag can be bridged again with the ABC complex. 9 Then the peroxidase in the ABC complex can be used to convert D A B to an electron-dense reaction product for EM viewing.
Introduction of Biotin Introduction of biotin to cell is accomplished exactly as the immunogold fluorescence method described earlier.
Fixation and Tissue Sectioning Fixation is carried out as described earlier for the immunogold fluorescence method. Because the ABC complex can penetrate much more deeply into the tissue than the immunogold reagent, it is possible to investigate much thicker tissue (up to 150 txm) with this method than with the immunogold fluorescence method. Nevertheless, the maximum thickness of the slices is also limited by the working distance of the objective used for confocal imaging. The 25 X multi-immersion lenses used in these experiments has a working distance of 130/xm. Therefore, tissue is usually sectioned on the Vibratome at 100 ~m as described earlier and the sections are collected in PBS.
Detection of Labeled Neurons with Fluorescent Marker Labeled cells are rendered fluorescent by incubating the floating sections in a fluorescently conjugated avidin (or streptavidin). A variety of different fluorophores conjugated to avidin or streptavidin are available commercially (e.g., Molecular Probes, Inc.). We mostly use Cy3 for its photostability and brightness and because it is well suited for imaging with a krypton/ argon laser. Because streptavidin or avidin penetrates into tissue much more readily than immunogold particles, the incubation time is much shorter (1-3 hr) than in the immunogold fluorescence method. Moreover, permeabiliza-
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tion is not necessary in this case. We mostly incubate the sections in a Cy3-conjugated streptavidin (Jackson ImmunoResearch Labs., Inc., West Grove, PA) solution (0.5/xg/ml of PBS) for 3 hr to overnight (for convenience). The sections are then washed with PBS and mounted without dehydration in Vectashield under a cover glass sealed with nail polish. Again, for good tissue preservation, care must be taken not to let the tissue dry in this process.
Confocal Microscopy Confocal microscopy is carried out as described in the immunogold method described previously. However, because the fluorescence signal is no longer detectable after ABC conversion, all confocal examinations must be carried out before the ABC conversion, unlike the immunogold method where some fluorescence is retained in the plastic block.
A vidin-Biotin-Peroxidase Complex Conversion After confocal microscopic observation, the sections are transferred to PBS and incubated in an avidin-biotin-peroxidase complex (ABC Elite, Vector Labs., Inc.) solution for 2 hr to overnight in a cold room (8°) on a shaker. The concentration used is suggested by the kit supplier. The preparations are then washed with PBS, incubated with DAB (0.25% in PBS) for 15 min, and finally reacted with DAB in the presence of H202 (0.01% in DAB solution) for 5-10 min. Because the reaction is somewhat light sensitive, it is preferable to carry it out under darkness. It is, however, necessary to check the reaction periodically under a dissecting microscope and to stop the reaction before the background stain rises. The reaction is stopped with several washes of PBS. After such a reaction, the labeled neurons become dark and the fluorescent label is no longer visible.
Postfixation and Plastic Embedding Postfixation with osmium tetroxide would enhance DAB staining. However, one of the common problems with DAB staining for EM is masking of the internal structure of the labeled cell. We found that a good balance between overall tissue preservation and maintenance of visibility of internal structure is achieved by osmicating tissue slices in 0.5% osmium tetroxide in sodium phosphate buffer (0.1 M, pH 7.4) for 15 min. The sections are then dehydrated and embedded in plastic as described earlier for the immunogold method. The plastic sheet containing the labeled neurons is initially examined at the light microscopic level. Even though the image quality is somewhat impaired by the thickness of the slices and the density of osmi-
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cation, the filled cells can be identified clearly under bright-field microscopy. Selected plastic-embedded Vibratome sections containing the labeled neuron are cut out with a razor blade and glued to a blank block as described earlier for thin sectioning.
Defining Depth of Areas of Interest with Confocal Microscopy Because the fluorescence signal is no longer visible after plastic embedding with this method, locating the area of interest in plastic poses a particular challenge. This is overcome by using either the reflection mode of the confocal microscope or the transmitted light detector of the confocal microscope if it is so equipped. Although neither of the methods gives images of high enough quality to allow another 3D reconstruction of the stained cell, both methods allow easy comparison with the original confocal images for identification of the area of interest. Then the depth of that area can be defined from the readout of the z-axis motor of confocal and microtomy can be conducted as described in the immunogold fluorescence method.
EM Study Once the depth of a particular region of interest is defined, the plastic block is thin sectioned and examined by E M as described earlier for the immunogold fluorescence method. An example of a Neurobiotin-stained neuron visualized using this method is presented in Fig. 2. Preservation of ultrastructure is good judging by the appearance of cell membranes and well-preserved synaptic vesicles (Figs. 2D and 2E).
Application of the M e t h o d to I m m u n o c y t o c h e m i s t r y The biotin-avidin interaction is well exploited in many fields of biological research because of the specific and strong interaction between biotin and avidin. The methods described earlier, in theory, should be applicable with any biotinylated probe. As an example, we use an antibody against serotonin, a neurotransmitter/modulator, to demonstrate the use of the method for immunocytochemistry. A requirement of the method is that the antigen must survive use of the small amount of glutaraldehyde that is required for adequate tissue preservation for E M study. This is the case for this specific antibody against serotonin. 19 19X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Comp.NeuroL338, 1 (1993).
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L\
FIG. 2. Example of the use of the ABC conversion method on a Neurobiotin-labeled neuron. (A) Reconstruction of a Neurobiotin-labeled neuron from the fly Musca domestica. Neuron was imaged in a 100-/xm Vibratome slice. Optical sections were obtained every 2/zm over a depth of 80/xm. Selected varicosities (arrow and arrowhead) indicate areas of interest (varicosities) for further EM investigation. (B) Using the reflecting mode of the confocal microscope, it is possible to locate areas of interest (arrow) in the plastic block to determine the depth of the region (24.5/zm). (Inset) The strong reflecting image of the surface of the block (0/zm). (C) Partial reconstruction from transmitted detector images of confocal indicates that the stain is restricted to the labeled neuron only. Arrow and arrowhead indicate the same regions of the neurons in (A). (D and E) Electron micrographs of the same neurons in the arrowhead (D) and arrow (E) regions in (A-C). Asterisks indicate locations of possible synaptic connections. The quality of tissue preservation is good as indicated by well-preserved cell membrane and synaptic vesicles. Bars: (A) 50/zm, (B) 20/~m, (C) 50 tzm, (D, E) 0.5 /xm. Adapted, with permission, from X. J. Sun, L. P. Tolbert, J. G. Hildebrand, and I. A. Meinertzhagen, J. Histochem. Cytochem. 46, 263 (1998).
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Serotonin immunocytochemical staining is carried out as described previouslyI9 except that a biotinylated secondary antibody is used to introduce biotin to the labeled neurons. In brief, tissue is fixed and sectioned on a Vibratome as described previously. Then, the tissue is incubated in primary antibody (rabbit antiserotonin, Incstar Corp., Stillwater, MN) in PBS overnight. It is subsequently washed in PBS and incubated with biotinylated goat antirabbit antibody [used at the concentration suggested by the supplier (1:200), Vector Labs, Inc.] for 3 hr. The staining is detected with Cy3conjugated streptavidin for confocal microscopy. Then, the DAB reaction is carried out as described earlier for the ABC complex. An example of such staining is shown in Fig. 3. Discussion and Conclusion The two methods described in this article are complementary. The immunogold method provides better detection than the ABC method because the internal structure is not masked by the stain. Additionally, immunogold particles do not diffuse like the DAB reaction product does. Moreover, the immunogold method provides the possibility to observe the tissue with confocal microscopy after embedding in plastic. Therefore, it avoids the problem of discrepancies caused by tissue shrinkage after plastic embedding when correlating 3D information with ultrastructural detail. The immunogold fluorescence method, however, is slow due to the long incubation time required for the immunogold reagent. Additionally, the immunogold method limits section thickness to about 80/~m, which creates additional problems for large cells. The ABC complex method, however, is rapid. It can be carried out in a single day if necessary. Moreover, the ABC method allows much thicker sections to be studied. In summary, the ABC method is suitable for routine applications, whereas the immunogold fluorescence method should be used when better tissue preservation and detection are required.
Limitations of Methods The combination of confocal microscopy and EM allows the investigator to correlate 3D information with ultrastructural characteristics with relative ease. However, identifying the same structure under EM and confocal still requires a high degree of skill. This problem is compounded by the fact that confocal optical sections are much thicker than a single thin section. The problem may be minimized by performing a small 3D reconstruction from serial thin sections. Both confocal-EM methods require the introduction of biotin. This may not be applicable in tissues with a high level of endogenous biotin.
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J~
/
FIG. 3. Extension of the methods to immunocytochemical labeling using labeling with antiserotonin antibody as an example. (A) Single optical section of a serotonin-immunoreactive fiber (which belongs to neuron LBO5HT 21) in the optical path (lamina, LN) of the fly brain. Arrow indicates a varicosity of interest. (B) Transmitted light image of the same fiber after DAB reaction and plastic embedding. (C) Reconstruction of the entire optical stack (40 optical sections at 1.2-/xm intervals) shows the overall view of the neurons in the Vibratome slice. (D) Electron micrograph of the varicosity in B shows that it contains numerous darkly labeled vesicles and is surrounded by glia (gl). A neighboring photoreceptor terminal (R), characterized by capitate projections (arrowheads), shows good tissue preservation. Bars: (A-C) 25/zm, (D) 0.5 /zm. Adapted, with permission, from X. J. Sun, L. P. Tolbert, J. G. Hildebrand, and I. A. Meinertzhagen, J. Histochem. Cytochem. 46, 263 (1998).
W e u s e d Cy3 a l m o s t exclusively as t h e f l u o r o p h o r e o f c h o i c e f o r c o n f o c a l o b s e r v a t i o n b e c a u s e o f its b r i g h t n e s s a n d p h o t o s t a b i l i t y . A d d i t i o n a l l y , Cy3 r e t a i n s a fair d e g r e e o f f l u o r e s c e n c e w i t h t h e i m m u n o g o l d f l u o r e s c e n c e m e t h o d , a l l o w i n g c o n f o c a l m i c r o s c o p i c s t u d y o f t h e l a b e l e d cell in plastic. H o w e v e r , s t u d y i n g p l a s t i c s e c t i o n s with c o n f o c a l m i c r o s c o p y p o s e s o t h e r challenges. O p a c i t y o f t h e tissue a f t e r o s m i c a t i o n m a d e it p a r t i c u l a r l y diffi-
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cult to image deep into the sections. Moreover, mismatches in refractive indexes between plastic and tissue introduce additional inaccuracies for depth determination. Adaptation to Other Systems Major obstacles of combining confocal microscopy and E M are the incompatibilities of the markers used for confocal microscopy and for EM. This article describes methods of simultaneously or sequentially labeling insect neurons with fluorescence for confocal microscopy and with electrondense labels for EM. The methods should be adaptable to other systems if certain facts are kept in mind. The requirements of tissue fixation for confocal microscopy are much less demanding than those for EM. For EM, however, there is no universal, ideal fixative for every tissue type. Often, a trial-and-error test is needed to find the best fixation procedure for a specific tissue. Finding the best fixative procedure for E M is a good starting point for developing a combined c o n f o c a l - E M method for the tissue. By systematically modifying the concentration and/or the time of fixation as we did, one should be able to achieve optimum balance between ultrastructural preservation and good signal-to-noise ratios for fluorescent and electrondense labels. Similarly, different types of tissues and different antigens (for immunocytochemical labeling) will require different methods and amounts of permeabilization for the access of gold particles. For example, some antigens will not survive dehydration, so alcohol cannot be used as the permeabilization agent. O t h e r methods such as freeze-thawing 2° to permeabilize the tissue m a y be better. In summary, we have reviewed two methods we developed for combining the advantages of confocal and electron microscopy. Although other c o n f o c a l - E M methods have b e e n described, 6 the methods described here are simpler and less tedious. We have proven t h e m to be practical and useful for the combination of confocal microscopy and E M for neurobiological research. Acknowledgments The work was carded out in the Laboratories of Drs. Leslie P. Tolbert and John G. Hildebrand at ARL Division of Neurobiology, the University of Arizona, and in the laboratory of Dr. Ian A. Meinertzhagen at the Department of Psychology,Dalhousie University, Canada. The author thanks them for their support. I also thank Dr. L. P. Tolbert for critical reading 2oA. N. van de Pol, J. Neurosci. 6, 877 (1986). 21D. R. N~ssel, Progr. Neurobiol. 30, 1 (1987).
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of the manuscript and suggestions. Additionally, the author thanks Patty Jasma, A. A. Osman, Lesley Varney, and Maggie Klonowska for technical assistance. The work was supported by an award from the University of Arizona, Center for Insect Science to X.J.S., grants to I.A.M. (NIH EY-03592, NSERC OPG 0000065), to L.P.T. and J.G.H. (NIH NS-28495), and to J.G.H. (NIH AI-23253).
[10] C o n s t r u c t i o n
of Line-Scan Confocal Microscope for Physiological Recording
By NICK
CALLAMARAS a n d IAN PARKER
Introduction The use of confocal laser scanning microscopes (CLSM) together with fluorescent Ca 2÷ indicator dyes allows detailed imaging studies of the spatiotemporal aspects of intracellular Ca 2+ signaling. For example, the high spatial and temporal resolution of this technique has revealed that release of Ca 2÷ from intracellular organeUes into the cytosol occurs as transient, elementary signals, arising at specific subcellular release sites. These elementary events include Ca 2+ "puffs" mediated through inositol 1,4,5-trisphosphate (InsP3) receptors in Xenopus oocytes and HeLa cells I-4 and Ca 2÷ "sparks" mediated through ryanodine receptors in cardiac muscle. 5 This article describes the construction and performance of a CLSM, combined with a UV illumination system for the photolysis of caged compounds, presently in use in the authors' laboratory for studies of InsP3-mediated intracellular Ca 2÷ release in Xenopus oocytes. This system can be adapted readily to virtually any inverted microscope and offers greater versatility and equal or better performance than comparable commercial instruments for a fraction of the cost. Commercial confocal microscopes fall broadly into two classes: those designed for morphological studies of fixed or slowly changing specimens and video-rate (30 Hz) full-field imaging systems for monitoring dynamic cellular signals. In either case, the application of these instruments for physiological studies often presents several difficulties. First, design constraints in commercial instruments tend to complicate the optical paths, I I. Parker and Y. Yao, Proc. R. Soc. (Lond.) B. Biol. Sci. 246, 269 (1991). 2 y . Yao, J. Choi, and I. Parker, J. Physiol. (Lond.) 482, 533 (1995). a M. Bootman, E. Niggli, M. Berridge, and P. Lipp, J. Physiol. (Lond.) 499, 307 (1997). 4 X.-P. Sun, N. Callamaras, and I. Parker, J. Physiol. (Lond.) 509, 67 (1998). H. Cheng, W. J. Lederer, and M. B. Cannell, Science 262, 740 (1993).
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art technique in the continuing investigation of cell structure and function in normal and pathological conditions.
Acknowledgments This work was supported by an M R C C grant to Dr. G h a s s a n Bkaily. The authors thank Ms. Susann Topping for secretarial assistance.
[9] C o m b i n i n g
Laser Scanning Confocal Microscopy and Electron Microscopy
By
XUEJUN SUN
Introduction Laser scanning confocal microscopy (confocal microscopy) provides a exciting new tool for biological research. For the first time, it is possible to obtain clear optical sections of relatively thick specimens without physically sectioning the tissue. Additionally, because of the high Z-axis resolution of preregistered images it provides, confocal microscopy enables rapid three-dimensional (3D) reconstruction of specimens. The combination of confocal microscopy with powerful computer image-processing techniques gives unprecedented possibilities for visualizing and measuring stained cells in three dimension. 1 The technology has gained wide acceptance in biological research in just one decade of its commercialization. Although it is true that confocal microscopy provides higher resolution than conventional light microscopy, it is still fundamentally light microscopy, limited in resolution by the wavelength of the light source and the numerical aperture of the lens. This is at best about 150 nm with current optics. In many fields of biological research, one needs subcellular resolution better than 150 nm. For example, the establishment of an association of a gene product with a type of organelle leads to important insights into the role of the product in the cell. For subcellular study, electron microscopy (EM) is still an indispensable tool for the unambiguous identification of organelles. EM provides a resolution that cannot be matched by any other imaging tool currently available. EM, however, suffers from a lack of 3D l H. Chen, J. R. Swedlow, M. Grote, J. W. Sedat, and D. A. Agard, in " H a n d b o o k of Biological Confocal Microscopy" (J. B. Pawley, ed.), 2nd ed., p. 197. Plenum Press, New York, 1995.
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information. Because of the poor penetrating power of electrons, observation with E M is often limited to a fraction (nanometer range) of the cellular structure. High-voltage E M provides an alternative that yields both high resolution and some degree of 3D information. It is, however, not readily accessible to most biologists and even with high-voltage EM, specimens no thicker than 1 /zm can be observed. Three-dimensional reconstruction is still required to have a 3D view of cells. N e u r o a n a t o m y provides an extreme situation in which 3D information is particularly important. Neurons often are large and have complicated 3D structure. The intrinsic 3D morphology reflects the connections of neurons within a neuronal network. Yet, ultimately, the discrete synaptic connections, and the neurotransmitter(s) used by them, rule the exact roles a particular neuron plays within the network. Study of stained neurons at the level of light microscopy often pinpoints particular loci of interest. Whether there are synaptic connections in the pinpointed areas provides necessary information for understanding the neuron and the circuit. Assessment of the three-dimensional morphology of neurons requires light microscopic study whereas synaptic information is available only through ultrastructural study. Therefore, many investigators have attempted to combine E M with LM. 2-5 All currently available methods require a certain degree of 3D reconstruction, which, even with advanced computer imaging technology, is still labor-intensive and sometimes impossible for large neurons. The combination of confocal microscopy and E M is a promising avenue for biologists attempting to solve this problem of 2D versus 3D. Confocal microscopy can provide a valuable overview and 3D information. E M provides ultrastructural detail. A combination of these two technologies is, however, not straightforward because the two technologies require very different tissue processing procedures and staining. First, for confocal microscopy, a specimen must be stained with a fluorescent marker, which is not necessarily electron dense and therefore not suitable for E M study. Second, for confocal observations, the tissue must be translucent, whereas for electron microscopy, heavy metals are used that are opaque to light. Third, for E M observation, tissue is usually embedded in plastic. This often introduces a large amount of tissue shrinkage and distortion, which render the correlation of light microscopic information with ultrastructural data difficult. A combination of confocal microscopy and E M has been attempted
z K. Hama, T. Arii, and T. Kosaka, Microsc. Res. Tech. 29, 357 (1994). 3j. DeFelipe and A. Fair6n, J. Histochem. Cytochem. 41, 769 (1993). 4j. K. Stevens, T. L. Davis, N. Friedman, and P. Sterling, Brain Res. 2, 265 (1980). 5R. W. Ware and V. LoPresti, Intl. Rev. Cytol. 40, 325 (1975).
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using the reflection mode of imaging with the confocal microscope. 6 With this method, a cell labeled with an electron-dense marker can be imaged in the confocal microscope by bouncing light off of it. This method, however, is not widely applied due to the generally poor quality of reflection in confocal images. We have been studying the neuronal connections within insect central nervous systems by combining standard confocal microscopy with EM. 7-1° The two methods described in this article rely on a very specific interaction between biotin and avidin. Although the methods were developed primarily for neuroanatomy with biotin-stained neurons, it can be adapted to other tissues to localize various substances within cell using biotinylated antibodies or D N A / R N A probes, as shown in this article with an antibody against serotonin, a neuromodulator in insects. I1'I2
Experimental Procedures Overview of Methods
The methods require the introduction of biotin or biotin derivatives into cells. The biotin can subsequently be detected using a fluorescent tag for confocal microscopy and an electron-dense tag for E M observation. Confocal microscopy can provide a guiding tool for thin sectioning in order to facilitate the correlation of confocal information with E M results. Both methods involve these basic steps of introduction of biotin, introduction of appropriate tags for the biotin, confocal observation, and E M observation. Both methods have been applied on insect central nervous tissue. Adult central nervous systems of two insect species were used: the moth M a n d u c a sexta (Lepidoptera, Sphingidae) and the white-eyed mutant housefly M u s c a d o m e s t i c a (Diptera, Muscidae). The detailed rearing procedures have been described previously (for moth, see Bell and Joachim13; for fly, see Fr6hlich and Meinertzhagena4).
6j. S. Deitch, K. L. Smith, J. W. Swann, and J. N. Turner, J. Microsc. 160, 265 (1990). 7X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Histochem. Cytochem. 43, 329 (1995). 8X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Comp. Neurol. 379, 2 (1997). 9X. J. Sun, L. P. Tolbert, J. G. Hildebrand, and I. A. Meinertzhagen, J. Histochem. Cytochem. 46, 263 (1998). 10L. P. Tolbert, X. J. Sun, and J. G. Hildebrand, J. Neurosci. Methods 69, 25 (1996). 11A. R. Mercer, J. H. Hayashi, and J. G. Hildebrand, J. Exp. Biol. 198, 613 (1995). 12A. R. Mercer, P. Kloppenburg, and J. G. Hildebrand, J. Comp. Physiol. A 178, 21 (1996). 13R. A. Bell and F. A. Joachim, Ann. Entomol. Soc. Am. 69, 365 (1976). 14A. Fr6hlich and I. A. Meinertzhagen, J. Neurocytol. U, 159 (1982).
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Method 1: I m m u n o g o l d Fluorescence Method This method takes advantage of the finding that immunogold reagents do not bind to all the available biotin in biotin-stained cells. 7 Therefore, the biotin-stained neurons can be incubated with immunogold-conjugated avidin first and then the remaining biotin molecules can be tagged with fluorescent avidin. Cell Staining with Biotin
Biotin was introduced into cells by one of three methods. Intracellular Labeling of Neurons. Biotin derivatives, Neurobiotin (Vector Labs., Inc., Burlingame, CA) or biocytin (Molecular Probes, Inc., Eugene, OR), are injected intracellularly into neurons by electrophoresis through a micropipette. For this, an isolated head preparation is used. To gain access to the brain, the mouth parts, frontal cuticle, and muscle systems of the head are removed surgically, and the head is cut off the body and secured with insect pins in a recording chamber where it is superfused continuously with saline solution [149.9 m M NaC1, 3 m M KC1, 3 m M CaC12, 25 m M sucrose, and 10 m M TES (N-tris[hydroxymethyl]-methyl-2aminoethanesulfonic acid), pH 6.9]. A standard electrophysiological recording setup is used to inject the dye as described previously. 15 The tip of a sharp glass microelectrode is filled with either 4% Neurobiotin in 1 M KC1 or 4% biocytin in a solution of 0.5 M KC1 in 0.05 M Tris buffer (pH 7.4). The shaft of the electrode is filled with 2 M KC1 solution. 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 is passed through the electrode for a period of 5-30 min to inject label into the cell. Back-Filling of Neurons. Biotin derivatives can also be introduced by back-filling,7'9 a staining technique widely applied in other animals (reviewed by McDonalda6). Back-filling is accomplished by cutting surgically accessible axons of the neurons to be labeled. The cut end of the nerve is then exposed to distilled water for approximately 1 min in order to open the damaged neurites. A drop of biotin solution (a few crystals of Neurobiotin in distilled water, estimated concentration <0.2%) is placed over the cut area. The whole animal is then placed in a humidified chamber for 5-20 min for the label to be picked up by the damaged cells. The incubation time depends on the distance that the biotin has to travel. Ten minutes is sufficient for about 2 mm of diffusion. Because biotin travels rather quickly within 15S. G. Matsumoto and J. G. Hildebrand, Proc. R. Soc. Lond. B 213, 249 (1981). 16A. J. McDonald,Neuroreport 3, 821 (1992).
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dendrites, there is no need for additional diffusion time. The area of the cut is flushed with saline after staining to wash out the excess biotin. The brain is then dissected out and processed as described later. Immunocytochemical Staining by Biotinylated Antibody. Biotin can also be introduced as common biotin-conjugated probes, such as biotinylated secondary antibodies. We tested this method with a biotinylated secondary antibody. The detail procedure for this type of application is described later in a separate section.
Tissue Processing Fixation. The fixation requirements for confocal microscopy and EM are very different. Confocal applications do not tolerate glutaraldehyde fixation because the high autofluorescence introduced by glutaraldehyde will potentially interfere with the fluorescent label. For EM study, a combination of paraformaldehyde and glutaraldehyde is the most common ehemical fixation procedure. We find that lowering the concentration of glutaraldehyde to 0.15-0.40% is compatible with most confocal applications while maintaining adequate tissue preservation for EM. Additionally, using longemission wavelength fluorophores such as Cy3 reduces the problem associated with autofluorescence. For EM, the fixative solution must be fresh. Immediately after injection of biotin, the brain is dissected out of the head and fixed at 4 ° overnight in freshly made fixative solution comprising 4% paraformaldehyde, 0.32% glutaraldehyde, and 0.2% saturated picric acid in 0.1 M sodium phosphate buffer (pH 7.4). Picric acid is optional. It has been suggested that picric acid stabilizes membranes and precipitates proteins for EM observation. 17 It introduces, however, some additional degree of autofluorescence. We do not find a noticeable difference in preservation of ultrastructure in preparations processed with or without picric acid. Fixation time can vary, depending on tissue size. We routinely fix moth brains overnight at 4° for convenience (approximate size of the brain is 2 × 3 mm). However, fixation times as short as 30 min can be applied at room temperature. Tissue Sectioning. It is necessary to cut the tissue into thin slabs because (1) the immunogold reagent has a limited penetration power and (2) the lenses used for confocal microscopy have a short working distance. After fixation, the brain is washed briefly with sodium phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and embedded in 7% agarose [low melting point agarose; gelling temperature of 2% (w/v) solution: 26-30°; Sigma, St. Louis, 17M. G. Zomboniand C. de Martino,J. CellBiol. 35, 148A (1967).
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MO]. The brains are then sectioned at 50-80/xm with a Vibratome (series 1000; Technical Products International, Inc., St. Louis, MO). The floating sections are collected in PBS. Permeabilization and Incubation with Immunogold-Conjugated Streptavidin. A common problem in immunogold staining before embedment is the lack of penetration of the gold-conjugated reagent into the specimen because of the relatively large size of the gold particles. To enhance the penetration of the immunogold-conjugated streptavidin, we treat the Vibratome sections with either 0.1% Triton X-100 (J. T. Baker, Inc., Phillipsburg, NJ) in PBS for a period of 10 min or a series of ethanol solutions (30, 50, 70, 50, and 30% ethanol) for 5-10 min at each step. Optimal penetration is achieved with such permeabilization and using streptavidin conjugated to small (1.4 nm) gold particles. We have attempted to use smaller gold particles (0.8 nm, Nanoprobes Inc., Stony Brook, NY) to enhance the penetration of the reagent, but the 0.8-nm gold particle-conjugated streptavidin penetrated even less than the 1.4-nm gold. After permeabilization, the sections are incubated with a solution of 1.4-nm gold-conjugated streptavidin (Nanoprobes Inc.) diluted 1:50-1 : 100 in PBS for 2-4 days on a shaker in an 8° cold room. Then 1:200 Cy3conjugated streptavidin (Jackson ImmunoResearch Labs., Inc., West Grove, PA) is added to the same solution, and the sections are incubated for an additional 8-12 hr in the cold room on the shaker. Following this procedure, the biotin-injected neuron is labeled reliably by gold-conjugated and fluorescently tagged streptavidin up to 40/zm into the tissue and could be detected both by confocal microscopy (fluorescence) and by EM (gold particles) (Fig. 1).
Confocal Observation Stained slices of brain are mounted on slides in saline or glycerol-based mounting medium (Vectashield, Vector Labs., Inc.). Care must be taken to avoid drying the tissue for potential ultrastructural damage. We normally use nail polish to seal the slide. We use a Bio-Rad MRC 600 confocal microscopy (Bio-Rad, Cambridge, MA) mounted on a Nikon Optiphot-2 microscope and a Zeiss LSM 410 mounted on a Zeiss Axiovert M100 microscope, both equipped with a krypton/argon laser light source for confocal observations. The tissue is usually imaged with a 20x (NA 0.75) Nikon objective lens (Bio-Rad) or with a 25x (NA 0.8) multi-immersion Plan Neofluar objective lens (Zeiss). A multi-immersion lens is highly suitable for such a study in order to minimize the effects of refractive index mismatch. Serial optical sections, usually at intervals of 1-2/zm, are imaged through the depth of the labeled
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A.
|}
FIG. 1. Example of examination of a Neurobiotin-injected neuron using the immunogold fluorescent method to combine confocal and EM. (A) Intracellular staining of a local interneuron in the antennal lobe of the moth Manduca sexta. The neuron was imaged in saline, before plastic embedding, at 3-/zm intervals for a total of 18 optical sections. The neuron ramifies extensively throughout the neuropile (antennal lobe). The soma is out of the field of view. (B) Bright-field image of the same neuron after silver enhancement and plastic embedding shows enhanced immunogold staining. The cell body is indicated with an arrowhead. (C) Confocal image of the Epon block demonstrates that the fluorescence is retained. Dashed line indicates the edge of the block. Comparison with confocal image obtained before plastic embedding (A) shows that most of the small processes remain detectable. (D) Single optical section from the 3D image stack at the level of the ultrathin section taken in E. Arrow indicates the corresponding position of the process. (E) Thin section of the same neuron at EM level showing the ultrastructure of the labeled process, which contains numerous gold particles. Bars: ( A - D ) 100/zm, (E) 2 k~m. Adapted, with permission, from X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Histochem. Cytochem. 43, 329 (1995).
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neurons and saved as three-dimensional image stacks. Projecting this image stack onto a single two-dimensional plane generates a 2D image of the reconstructed labeled neuron. The image stack is also studied with appropriate software to visualize the neuron in three dimensions. Areas of interest are defined within the stack. There is no evidence of ultrastructural damage by laser radiation on fixed tissue. However, to avoid potential ultrastructural damage by strong laser radiation with the confocal microscope, we try to minimize both the scanning time and the laser intensity by choosing an acceptable fast scan speed and a low laser power setting. This usually involves a tradeoff of image quality, imaging duration, and laser intensity.
Silver Enhancement of Gold Particles The 1.4-nm gold particles, too small to allow easy observation under the EM, are enlarged with a silver enhancement solution (Biocell Res. Labs., from Ted Pella, Inc., Redding, CA) in order to facilitate detection in EM. The silver enhancement procedure is modified from the protocol suggested by the manufacture of the kit. In brief, after confocal observation, the selected sections are washed three times (10 minutes each) with PBS to remove the mounting medium. They are then washed in deionized water (two times at 3 min each) and incubated in the silver enhancement solution on ice in the dark for 8 min to allow diffusion of the solution into the sections. The container containing the sections is subsequently transferred to a water bath at room temperature for another 8 min of reaction in the dark. Timing of the reaction is important and care must be taken to avoid a self-nucleation reaction. The silver enhancement reaction is stopped by several washes with deionized water (three times at 3 min each). The sections are then transferred to sodium phosphate buffer (0.1 M, pH 7.4) for further processing for EM study.
Pos~xation for EM Study For EM, it is necessary to stabilize cell membranes by postfixation and then to embed in plastic. A common postfixative for EM is osmium tetroxide. As it was suggested that osmium tetroxide oxidizes silver precipitates of the silver-enhanced gold particles, TMtissues are lightly osmicated at room temperature (0.1% osmium tetroxide in phosphate buffer for 5-10 min). The postfixation process is stopped by replacing the solution with phosphate buffer. This procedure stabilizes the cell membranes enough for plastic embedding while retaining enough silver precipitate on the gold particles 18A. I. Basbaum,J. Histochem. Cytochem. 37, 1811 (1989).
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for EM observation. Osmium tetroxide and its vapor are highly toxic. This postfixation step must be carried out under a well-ventilated fume hood and the waste solution disposed of properly.
Plastic Embedding The postfixed brain slices are dehydrated through a graded series of increasing concentrations of ethanol (50, 70, 95, and twice 100% for 10 min at each step), followed by two changes of 10 min each in propylene oxide (Electron Microscopy Sciences, Fort Washington, PA). The sections are then placed in a 1 : 1 mixture of Epon/Araldite and propylene oxide for 30 min. The tissue is infiltrated with pure fresh Epon/Araldite overnight on a rotatory shaker. To flat embed the sections, sections are sandwiched between two Aclar sheets (Ted Pella, Inc., Redding, CA) in Epon/Araldite, and this assembly is further sandwiched between two microscope slides with two paper clips to hold the whole assembly together. The whole assembly is then put into a 60° oven overnight for polymerization. One Aclar sheet can be peeled off easily and the sections can be examined with bright-field microscopy (Fig. 1B) or with the confocal microscope. Selected sections are cut out with a razor blade and glued to blank Epon/Araldite blocks with fast cyanoacrylate adhesive (Electron Microscopy Sci., Inc., Ft. Washington, PA) for microtomy.
Confocal Microscopy as Guide for Microtomy We found that a certain degree of Cy3 fluorescence is retained after processing tissue as described earlier. Therefore, it is possible to use a confocal microscope as a guide for microtomy. For viewing the block on the confocal microscope, the block is trimmed with a glass knife in order to obtain a smooth surface for confocal observation. It is then held in a specially fabricated brass slide with a hole in the middle. In this way, the block can be imaged in either an inverted or an upright position depending on the configuration of the microscope. These arrangements allow, moreover, a slight tilt of the surface of the block, if the surface of the block is not totally parallel to the image plane, which is a critical prerequisite for determination of the depth of the area of interest. The confocal microscope stage and the z-axis motor are calibrated and the reproducibility of both checked carefully so as to provide accurate depth information. The surface of the block is defined easily by its strong reflecting signal, giving the starting depth of 0/xm. The area of interest can be relocalized with confocal microscopy by reference to the initial set of confocal images. Through the readout of the z-axis motor, the actual depth of the area can be obtained
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for guiding microtomy. To ease the effort of relocalizing the fiber of interest under EM, the block is trimmed as small as possible.
Electron Microscopic Study After examination with the confocal microscope, the Epon blocks containing labeled neurons are thin sectioned either at regular intervals by alternating thin sectioning and semithin sectioning or at preselected depths of interest in the tissue defined by the confocal study. Sections are cut with a diamond knife on a MT6000-XL microtome (RMC Inc., Tucson, AZ) or on a Reichert-Jung Ultracut microtome (Leica, Deerfield, IL). To facilitate localization of the chosen depth, thin sections are collected so that the ribbon on each grid contains a defined thickness of tissue. Because of the refractive index mismatch and inaccuracies of the microscopic stage, the depth information obtained using the confocal microscope may not be precisely accurate. Additionally, estimates of section thickness on the microtomes will incorporate some error. Therefore, additional thin sections are collected before and after the supposed depth of interest in order to ensure that the area of interest is contained in the thin sections collected. Thin sections are picked up on Formvar-coated slot grids or on thin-bar copper grids and poststained with saturated aqueous uranyl acetate solution and with lead citrate for 10 min each. The thin sections are examined with a J E O L JEM-1200EX transmission EM or with a Philips 201C EM. The labeled processes are selectively photographed, usually at magnifications between x5000 and x15,000 for the identification of synapses.
Correlation of Confocal Microscopy and EM In the majority of cases, depth information and the shape of the labeled process in the sections enable easy correlation of the confocal overview with EM detail. However, this correlation must be made with care, as a confocal optical section at the resolution of the lenses used in these experiments may contain the same amount of tissue as is included in as many as 40 thin sections. Moreover, localizing small, labeled profiles in a relative large area under EM is not an easy task. This can be eased by trimming the book as small as possible to limit the area of view for EM. One example using the immunogold fluorescence method to combine confocal microscopy and EM is presented in Fig. 1. A neuron in the antennal lobe of the moth brain is stained intracellularly with Neurobiotin and viewed with confocal microscopy before plastic embedding (Fig. 1A) and again in the plastic block (Fig. 1C). The transmitted bright-field microscopy (Fig. 1B) shows the immunogold labeling at the light microscopic level. An
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electron micrograph (Fig. 1D) of the neuron is shown. Use of this method to investigate the properties of insect neurons has been published in separate papers, s Method 2: Avidin-Biotin Complex Method This method uses a commercially available kit [avidin-biotinperoxidase complex (ABC) from Vector Labs. Inc.] to convert some of the fluorescence signal in a fluorescently stained cell into an electron-dense form using 3,3'-diaminobenzadine (DAB) as substrate. It is based on the finding that avidin conjugated to a fluorescent tag can be bridged again with the ABC complex. 9 Then the peroxidase in the ABC complex can be used to convert D A B to an electron-dense reaction product for EM viewing.
Introduction of Biotin Introduction of biotin to cell is accomplished exactly as the immunogold fluorescence method described earlier.
Fixation and Tissue Sectioning Fixation is carried out as described earlier for the immunogold fluorescence method. Because the ABC complex can penetrate much more deeply into the tissue than the immunogold reagent, it is possible to investigate much thicker tissue (up to 150 txm) with this method than with the immunogold fluorescence method. Nevertheless, the maximum thickness of the slices is also limited by the working distance of the objective used for confocal imaging. The 25 X multi-immersion lenses used in these experiments has a working distance of 130/xm. Therefore, tissue is usually sectioned on the Vibratome at 100 ~m as described earlier and the sections are collected in PBS.
Detection of Labeled Neurons with Fluorescent Marker Labeled cells are rendered fluorescent by incubating the floating sections in a fluorescently conjugated avidin (or streptavidin). A variety of different fluorophores conjugated to avidin or streptavidin are available commercially (e.g., Molecular Probes, Inc.). We mostly use Cy3 for its photostability and brightness and because it is well suited for imaging with a krypton/ argon laser. Because streptavidin or avidin penetrates into tissue much more readily than immunogold particles, the incubation time is much shorter (1-3 hr) than in the immunogold fluorescence method. Moreover, permeabiliza-
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tion is not necessary in this case. We mostly incubate the sections in a Cy3-conjugated streptavidin (Jackson ImmunoResearch Labs., Inc., West Grove, PA) solution (0.5/xg/ml of PBS) for 3 hr to overnight (for convenience). The sections are then washed with PBS and mounted without dehydration in Vectashield under a cover glass sealed with nail polish. Again, for good tissue preservation, care must be taken not to let the tissue dry in this process.
Confocal Microscopy Confocal microscopy is carried out as described in the immunogold method described previously. However, because the fluorescence signal is no longer detectable after ABC conversion, all confocal examinations must be carried out before the ABC conversion, unlike the immunogold method where some fluorescence is retained in the plastic block.
A vidin-Biotin-Peroxidase Complex Conversion After confocal microscopic observation, the sections are transferred to PBS and incubated in an avidin-biotin-peroxidase complex (ABC Elite, Vector Labs., Inc.) solution for 2 hr to overnight in a cold room (8°) on a shaker. The concentration used is suggested by the kit supplier. The preparations are then washed with PBS, incubated with DAB (0.25% in PBS) for 15 min, and finally reacted with DAB in the presence of H202 (0.01% in DAB solution) for 5-10 min. Because the reaction is somewhat light sensitive, it is preferable to carry it out under darkness. It is, however, necessary to check the reaction periodically under a dissecting microscope and to stop the reaction before the background stain rises. The reaction is stopped with several washes of PBS. After such a reaction, the labeled neurons become dark and the fluorescent label is no longer visible.
Postfixation and Plastic Embedding Postfixation with osmium tetroxide would enhance DAB staining. However, one of the common problems with DAB staining for EM is masking of the internal structure of the labeled cell. We found that a good balance between overall tissue preservation and maintenance of visibility of internal structure is achieved by osmicating tissue slices in 0.5% osmium tetroxide in sodium phosphate buffer (0.1 M, pH 7.4) for 15 min. The sections are then dehydrated and embedded in plastic as described earlier for the immunogold method. The plastic sheet containing the labeled neurons is initially examined at the light microscopic level. Even though the image quality is somewhat impaired by the thickness of the slices and the density of osmi-
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cation, the filled cells can be identified clearly under bright-field microscopy. Selected plastic-embedded Vibratome sections containing the labeled neuron are cut out with a razor blade and glued to a blank block as described earlier for thin sectioning.
Defining Depth of Areas of Interest with Confocal Microscopy Because the fluorescence signal is no longer visible after plastic embedding with this method, locating the area of interest in plastic poses a particular challenge. This is overcome by using either the reflection mode of the confocal microscope or the transmitted light detector of the confocal microscope if it is so equipped. Although neither of the methods gives images of high enough quality to allow another 3D reconstruction of the stained cell, both methods allow easy comparison with the original confocal images for identification of the area of interest. Then the depth of that area can be defined from the readout of the z-axis motor of confocal and microtomy can be conducted as described in the immunogold fluorescence method.
EM Study Once the depth of a particular region of interest is defined, the plastic block is thin sectioned and examined by E M as described earlier for the immunogold fluorescence method. An example of a Neurobiotin-stained neuron visualized using this method is presented in Fig. 2. Preservation of ultrastructure is good judging by the appearance of cell membranes and well-preserved synaptic vesicles (Figs. 2D and 2E).
Application of the M e t h o d to I m m u n o c y t o c h e m i s t r y The biotin-avidin interaction is well exploited in many fields of biological research because of the specific and strong interaction between biotin and avidin. The methods described earlier, in theory, should be applicable with any biotinylated probe. As an example, we use an antibody against serotonin, a neurotransmitter/modulator, to demonstrate the use of the method for immunocytochemistry. A requirement of the method is that the antigen must survive use of the small amount of glutaraldehyde that is required for adequate tissue preservation for E M study. This is the case for this specific antibody against serotonin. 19 19X. J. Sun, L. P. Tolbert, and J. G. Hildebrand, J. Comp.NeuroL338, 1 (1993).
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L\
FIG. 2. Example of the use of the ABC conversion method on a Neurobiotin-labeled neuron. (A) Reconstruction of a Neurobiotin-labeled neuron from the fly Musca domestica. Neuron was imaged in a 100-/xm Vibratome slice. Optical sections were obtained every 2/zm over a depth of 80/xm. Selected varicosities (arrow and arrowhead) indicate areas of interest (varicosities) for further EM investigation. (B) Using the reflecting mode of the confocal microscope, it is possible to locate areas of interest (arrow) in the plastic block to determine the depth of the region (24.5/zm). (Inset) The strong reflecting image of the surface of the block (0/zm). (C) Partial reconstruction from transmitted detector images of confocal indicates that the stain is restricted to the labeled neuron only. Arrow and arrowhead indicate the same regions of the neurons in (A). (D and E) Electron micrographs of the same neurons in the arrowhead (D) and arrow (E) regions in (A-C). Asterisks indicate locations of possible synaptic connections. The quality of tissue preservation is good as indicated by well-preserved cell membrane and synaptic vesicles. Bars: (A) 50/zm, (B) 20/~m, (C) 50 tzm, (D, E) 0.5 /xm. Adapted, with permission, from X. J. Sun, L. P. Tolbert, J. G. Hildebrand, and I. A. Meinertzhagen, J. Histochem. Cytochem. 46, 263 (1998).
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Serotonin immunocytochemical staining is carried out as described previouslyI9 except that a biotinylated secondary antibody is used to introduce biotin to the labeled neurons. In brief, tissue is fixed and sectioned on a Vibratome as described previously. Then, the tissue is incubated in primary antibody (rabbit antiserotonin, Incstar Corp., Stillwater, MN) in PBS overnight. It is subsequently washed in PBS and incubated with biotinylated goat antirabbit antibody [used at the concentration suggested by the supplier (1:200), Vector Labs, Inc.] for 3 hr. The staining is detected with Cy3conjugated streptavidin for confocal microscopy. Then, the DAB reaction is carried out as described earlier for the ABC complex. An example of such staining is shown in Fig. 3. Discussion and Conclusion The two methods described in this article are complementary. The immunogold method provides better detection than the ABC method because the internal structure is not masked by the stain. Additionally, immunogold particles do not diffuse like the DAB reaction product does. Moreover, the immunogold method provides the possibility to observe the tissue with confocal microscopy after embedding in plastic. Therefore, it avoids the problem of discrepancies caused by tissue shrinkage after plastic embedding when correlating 3D information with ultrastructural detail. The immunogold fluorescence method, however, is slow due to the long incubation time required for the immunogold reagent. Additionally, the immunogold method limits section thickness to about 80/~m, which creates additional problems for large cells. The ABC complex method, however, is rapid. It can be carried out in a single day if necessary. Moreover, the ABC method allows much thicker sections to be studied. In summary, the ABC method is suitable for routine applications, whereas the immunogold fluorescence method should be used when better tissue preservation and detection are required.
Limitations of Methods The combination of confocal microscopy and EM allows the investigator to correlate 3D information with ultrastructural characteristics with relative ease. However, identifying the same structure under EM and confocal still requires a high degree of skill. This problem is compounded by the fact that confocal optical sections are much thicker than a single thin section. The problem may be minimized by performing a small 3D reconstruction from serial thin sections. Both confocal-EM methods require the introduction of biotin. This may not be applicable in tissues with a high level of endogenous biotin.
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J~
/
FIG. 3. Extension of the methods to immunocytochemical labeling using labeling with antiserotonin antibody as an example. (A) Single optical section of a serotonin-immunoreactive fiber (which belongs to neuron LBO5HT 21) in the optical path (lamina, LN) of the fly brain. Arrow indicates a varicosity of interest. (B) Transmitted light image of the same fiber after DAB reaction and plastic embedding. (C) Reconstruction of the entire optical stack (40 optical sections at 1.2-/xm intervals) shows the overall view of the neurons in the Vibratome slice. (D) Electron micrograph of the varicosity in B shows that it contains numerous darkly labeled vesicles and is surrounded by glia (gl). A neighboring photoreceptor terminal (R), characterized by capitate projections (arrowheads), shows good tissue preservation. Bars: (A-C) 25/zm, (D) 0.5 /zm. Adapted, with permission, from X. J. Sun, L. P. Tolbert, J. G. Hildebrand, and I. A. Meinertzhagen, J. Histochem. Cytochem. 46, 263 (1998).
W e u s e d Cy3 a l m o s t exclusively as t h e f l u o r o p h o r e o f c h o i c e f o r c o n f o c a l o b s e r v a t i o n b e c a u s e o f its b r i g h t n e s s a n d p h o t o s t a b i l i t y . A d d i t i o n a l l y , Cy3 r e t a i n s a fair d e g r e e o f f l u o r e s c e n c e w i t h t h e i m m u n o g o l d f l u o r e s c e n c e m e t h o d , a l l o w i n g c o n f o c a l m i c r o s c o p i c s t u d y o f t h e l a b e l e d cell in plastic. H o w e v e r , s t u d y i n g p l a s t i c s e c t i o n s with c o n f o c a l m i c r o s c o p y p o s e s o t h e r challenges. O p a c i t y o f t h e tissue a f t e r o s m i c a t i o n m a d e it p a r t i c u l a r l y diffi-
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cult to image deep into the sections. Moreover, mismatches in refractive indexes between plastic and tissue introduce additional inaccuracies for depth determination. Adaptation to Other Systems Major obstacles of combining confocal microscopy and E M are the incompatibilities of the markers used for confocal microscopy and for EM. This article describes methods of simultaneously or sequentially labeling insect neurons with fluorescence for confocal microscopy and with electrondense labels for EM. The methods should be adaptable to other systems if certain facts are kept in mind. The requirements of tissue fixation for confocal microscopy are much less demanding than those for EM. For EM, however, there is no universal, ideal fixative for every tissue type. Often, a trial-and-error test is needed to find the best fixation procedure for a specific tissue. Finding the best fixative procedure for E M is a good starting point for developing a combined c o n f o c a l - E M method for the tissue. By systematically modifying the concentration and/or the time of fixation as we did, one should be able to achieve optimum balance between ultrastructural preservation and good signal-to-noise ratios for fluorescent and electrondense labels. Similarly, different types of tissues and different antigens (for immunocytochemical labeling) will require different methods and amounts of permeabilization for the access of gold particles. For example, some antigens will not survive dehydration, so alcohol cannot be used as the permeabilization agent. O t h e r methods such as freeze-thawing 2° to permeabilize the tissue m a y be better. In summary, we have reviewed two methods we developed for combining the advantages of confocal and electron microscopy. Although other c o n f o c a l - E M methods have b e e n described, 6 the methods described here are simpler and less tedious. We have proven t h e m to be practical and useful for the combination of confocal microscopy and E M for neurobiological research. Acknowledgments The work was carded out in the Laboratories of Drs. Leslie P. Tolbert and John G. Hildebrand at ARL Division of Neurobiology, the University of Arizona, and in the laboratory of Dr. Ian A. Meinertzhagen at the Department of Psychology,Dalhousie University, Canada. The author thanks them for their support. I also thank Dr. L. P. Tolbert for critical reading 2oA. N. van de Pol, J. Neurosci. 6, 877 (1986). 21D. R. N~ssel, Progr. Neurobiol. 30, 1 (1987).
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of the manuscript and suggestions. Additionally, the author thanks Patty Jasma, A. A. Osman, Lesley Varney, and Maggie Klonowska for technical assistance. The work was supported by an award from the University of Arizona, Center for Insect Science to X.J.S., grants to I.A.M. (NIH EY-03592, NSERC OPG 0000065), to L.P.T. and J.G.H. (NIH NS-28495), and to J.G.H. (NIH AI-23253).
[10] C o n s t r u c t i o n
of Line-Scan Confocal Microscope for Physiological Recording
By NICK
CALLAMARAS a n d IAN PARKER
Introduction The use of confocal laser scanning microscopes (CLSM) together with fluorescent Ca 2÷ indicator dyes allows detailed imaging studies of the spatiotemporal aspects of intracellular Ca 2+ signaling. For example, the high spatial and temporal resolution of this technique has revealed that release of Ca 2÷ from intracellular organeUes into the cytosol occurs as transient, elementary signals, arising at specific subcellular release sites. These elementary events include Ca 2+ "puffs" mediated through inositol 1,4,5-trisphosphate (InsP3) receptors in Xenopus oocytes and HeLa cells I-4 and Ca 2÷ "sparks" mediated through ryanodine receptors in cardiac muscle. 5 This article describes the construction and performance of a CLSM, combined with a UV illumination system for the photolysis of caged compounds, presently in use in the authors' laboratory for studies of InsP3-mediated intracellular Ca 2÷ release in Xenopus oocytes. This system can be adapted readily to virtually any inverted microscope and offers greater versatility and equal or better performance than comparable commercial instruments for a fraction of the cost. Commercial confocal microscopes fall broadly into two classes: those designed for morphological studies of fixed or slowly changing specimens and video-rate (30 Hz) full-field imaging systems for monitoring dynamic cellular signals. In either case, the application of these instruments for physiological studies often presents several difficulties. First, design constraints in commercial instruments tend to complicate the optical paths, I I. Parker and Y. Yao, Proc. R. Soc. (Lond.) B. Biol. Sci. 246, 269 (1991). 2 y . Yao, J. Choi, and I. Parker, J. Physiol. (Lond.) 482, 533 (1995). a M. Bootman, E. Niggli, M. Berridge, and P. Lipp, J. Physiol. (Lond.) 499, 307 (1997). 4 X.-P. Sun, N. Callamaras, and I. Parker, J. Physiol. (Lond.) 509, 67 (1998). H. Cheng, W. J. Lederer, and M. B. Cannell, Science 262, 740 (1993).
METHODSIN ENZYMOLOGY,VOL.307
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