Journal of Chemical Neuroanatomy, Vol. 4:387-395 (1991)

Confocal Microscopy in Chemical Neuroanatomy P e t e r Wall$n

The Nobel Institute for Neurophysiology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden ABSTRACT The application of computer-assisted confocal laser scanning microscopy in chemical neuroanatomy is briefly reviewed and illustrated with examples taken mainly from morphological studies of neurons in the spinal cord of the lamprey, a lower vertebrate. The principle of operation of the confocal microscopy system used, with emphasis on the computer-controlled image data acquisition and further processing of the large image volumes, is described. The imaging characteristics of confocal laser scanning microscopy in combination with fluorescence labelling, in particular the capacity to perform three-dimensional reconstructions of high optical resolution, are also described and illustrated with projection images from different viewing angles and with stereo pair reconstructions. Different possibilities for image processing, e.g. surface shading and surface extraction, are also described and illustrated. KEYWORDS: Scanninglaser microscopy Three-dimensionalreconstruction Optional sectioning Computer-controlled image processing Fluorescence Neuronal morphology

THE PRINCIPLE OF CONFOCAL MICROSCOPY In a confocal microscope the object under study is illuminated point-by-point by a thin beam of light, and only the light from the illuminated spot is detected (Fig. 1; Wilson and Sheppard, 1984). This point illumination-point detection principle is implemented in a confocal microscope by letting the optics of a conventional microscope focus the illumination light into a small spot in the focal plane, and by focussing the light emanating from this spot onto a small 'pinhole' aperture in front of the detector (photomultiplier tube); see Fig. 1A. In comparison with conventional microscopy this principle gives several advantages, the two main ones being: (1) a dramatic reduction of stray light since only one small spot, rather than the whole object, is being illuminated at one time, and (2) a considerable improvement of resolution, particularly in the depth dimension, since only light from structures in the focal plane will pass through the aperture and reach the detector (Fig. 1A). Thus, it is possible to record thin 'optical sections' through, for example, a neuron in a thick piece of tissue, as illustrated in Fig. 1C. For comparison, the corresponding non-confocal image, obtained after removal of the pinhole aperture, is shown in Fig. 1B. To perform such optical sectioning, however, the illuminating light spot has to be scanned across the whole field of view in the focal plane. This can be done in different ways: object stage scanning, where the object is being moved and the light beam remains fixed (e.g. 0891-0618/91/050387-09 $05.00 © 1991 by John Wiley and Sons Ltd

Brakenhoff et al., 1979), and beam scanning, where the light spot is moved across a stationary object (e.g. Davidovits and Egger, 1971; Aslund et al., 1983). A variant of the latter principle is the tandemscanning confocal microscope, where a rotating Nipkow disc with many small holes produces spot illumination and detection over the whole field of view simultaneously, thus akllowing for direct viewing of a confocal optical section in real time (Petr/m et al., 1968). The most common type of confocal microscopes is of the beam scanning kind. The optical sectioning capacity of the confocal microscope opens the possibility of producing detailed and accurate three-dimensional reconstructions of the object, by combining several optical sections sampled with a slight shift of focus level in between each. Serial optical sectioning thus results in a stack of sections forming a three-dimensional image volume (Fig. 2) containing, for example, a stained neuron. Fluorescence labelling is most suitable for such three-dimensional reconstructions, whereas reflected light scanning can be used for two-dimensional reconstructions of, for example, Golgi-stained material. In confocal fluorescence microscopy, laser light is the light source of choice due to its intensity and parallelism. The degree of optical resolution in both the lateral dimension and in depth is mainly determined by the quality and numerical aperture of the microscope objective (Carlsson, 1988). Thus, the larger the numerical aperture, the better the depth resolution and the thinner the optical sections will be (typically below 1 ~tmwith an objective with 1.3 in numerical aperture).

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Fig. 1. (A) Ray-path in a confocal microscope. Light from structures in the focal plane will pass through the pinhole aperture in front of the detector, while light from out-of-focus structures (dashed lines) will not reach the detector, allowing thin optical sections to be made through the specimen. (B) Non-confocal image of Lucifer Yellow-filled lamprey neuron, obtained after removal of the pinhole aperture. (C) Corresponding confocal image (single optical section) obtained with the pinhole aperture in place. Only the structures in the focal plane are recorded. Objective: 100 x/1.4 NA.

COMPUTER CONTROL AND TREATMENT OF IMAGE DATA IN CONFOCAL MICROSCOPY The principle of confocal microscopy was first described 30 years ago (Minsky, 1961), but only recently affordable computer systems with adequate storage and data processing capabilities have become available. Similarly, in the last few years, smaller, reasonably priced lasers suitable for fluorescence excitation have appeared. Following these technological steps, several computer-controlled laser scanning confocal microscope systems are now commercially available. In principle, these different systems operate in a similar fashion, but the follow-

ing description relates primarily to the Sarastro 1000 system (Molecular Dynamics Inc.), which is a beam scanning system built around a conventional fluorescence microscope, and which was originally developed at the Royal Institute of Technology in Stockholm (Carlsson and ~slund, 1987; Carlsson and Liljeborg, 1988). Clearly the control of the scanning unit and the acquisition and treatment of the image data require a fast computer with adequate storage capacity (Fig. 2). Data volumes can become quite large; a volume of 200 optical sections may take up more than 50 Mbytes of computer memory. The Sarastro system is operated by a UNIX-based, so-called RISC computer (Silicon Graphics Personal Iris).

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Fig. 2. Schematic diagram of the confocal laser scanning microscope system used in this study. A UNIX-based computer controls the scanner and the focus motor of the microscope via an electronics unit. The scanner unit contains the laser, the scanning mirrors, beam splitters, filters and the detectors. A conventional fluorescence microscope is connected to the scanner via the photo tube. Recorded optical sections result in a three-dimensional image volume which is stored on the computer hard disk and on tape cassette and/or optical disk. Calculated projection images are displayed on the computer screen and also sent to a colour video printer (Kodak SV6560) for illustrations.

The scanning movements of the laser spot across the focal plane are accomplished by two mirrors within the scanning unit, controlled from an electronics unit upon command from the computer (Fig. 2). The scanned area is divided into a grid of up to 1024 by 1024 picture elements (pixels), and the detected light intensity of each pixel is stored as an 8-bit digital number, giving a grey scale resolution of 256 levels. To perform serial optical sectioning in three dimensions, the object stage of the microscope is moved in small steps (down to 0.1 ~tm) between each section by means of a stepping motor controlled from the computer. The resulting image data volume is stored on the hard disk of the computer for subsequent processing. The scanning time for a single 512 by 512 pixel optical section is approximately 10s, giving a total time for the (automatic) scanning of a volume with 100 sections of about 17 min. After scanning and storage of the image data volume, computer-generated reconstructions of the object from user-defined viewing angles can be produced, as well as stereo reconstructions, allowing visualization of the three-dimensional structure of, for example, a neuron (Wall6n et al., 1988; Figs 3-5). With the entire three-dimensional structural information available in digital form in the computer, processing of the data can also be performed. For instance, borderlines and edges of the cell can be emphasized using a surface extraction routine (Fig. 4C). Another technique is surface shading, simulating a light source illuminating the cell from an oblique angle, revealing the surface structure (Fig.

4B; Forsgren, 1990). The computation time for producing a reconstructed image of the object depends on the size of the data volume. The data volume is usually reduced in size prior to further processing, by means of a vectorization algorithm where pixels with a light level below a certain user-defined threshold, are discarded (Forsgren, 1990). For a volume containing a neuron with its thin dendrites, most pixels may be 'empty', often resulting in a reduction of more than 90% of the original volume. After this data reduction step, the computation of an image typically takes less than 1 min. Storage of the original data volume and the computed images requires a large amount of hard disk space. It is obvious that even a hard disk of several hundred Mbytes may soon be filled. Therefore longterm storage must be made on other media, like tape stations or optical disks. The latter alternative has proven extremely useful, since the optical disk is incorporated into the file system of the computer, and is fast enough to allow storage of the data directly during scanning. VISUALIZATION REQUIREMENTS IN CHEMICAL NEUROANATOMY A common goal in chemical neuroanatomy is to acquire as detailed knowledge as possible about the structure and location of histochemicaUy identified neurons or neuronal ensembles. Most often cells groups are identified on the basis of their content of,

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Fig. 3. Confocal projection images of a motoneuron intracellularty filled with Lucifer Yellow in the lamprey spinal cord (whole-mount preparation). (A,B) Zero-degree (A) and 90° (B) projection images of the soma and proximal dendrites. Note the initial part of the axon pointing left in (B). Maximum intensity images, 512 by 512 pixels, pixel size 0.25 lam.Objective: 40 x / 1.3 NA. (C) Stereo pair images of the same cell, with a 6° difference in projection angles. A surface shading routine was used here.

for example, transmitter substances, by means of immunohistochemical methods. In addition, retrograde or anterograde staining techniques, as well as intracellular labelling, are often used. In order to achieve accurate information on the detailed structure, location, dendritic branching and axonal projection paths o f the different neurons and cell groups, an extensive histological analysis with sectioning of large volumes o f tissue is often required. In this analysis it is clearly o f great value not only to be

able to visualize the labelled neurons in two dimensions, but also to achieve a true three-dimensional picture. This is o f particular importance when investigating structural interrelations between different groups o f neuronal elements, for instance the pattern of afferent terminations onto a central neuron. F o r an accurate localization o f the cell groups of interest, it is obvious that the general structure of the surrounding nervous tissue also needs to be adequately visualized.

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Fig. 4. Differentimage processingroutines used on the same projectionimage of the motoneuron of Fig. 3. (A) Standard look-through image with intensityscaling, and with no further image processing. (B) Surfaceshading highlights the structure of the cell surface. (C) Surfaceextractionimage,emphasizingcontours and borders of the cell. (D) Depth-codedimage,withclosebystructuresreproducedwith high intensityand deep structuresappearing darker (of. Fig. 3B).Sameimagevolumeas in Fig. 3.

W H A T C O N F O C A L M I C R O S C O P Y CAN DO In the light o f the above-mentioned visualization requirements, confocal microscopy appears as a very useful analysis tool. The improved resolution in depth and efficient reduction of out-of-focus stray light allow optical sections to be made through thick pieces of tissue, without a need for physical sectioning, thereby leaving the tissue intact. The optical sections can be made thinner (less than 1 ~tm) than

what is easily obtainable with standard histological sectioning. A general problem when reconstructing the threedimensional morphology of neurons and fibres using conventional techniques, is the necessity for accurate realignment of consecutive sections--an often tedious and time-consuming procedure even if modern computer-assisted routines are employed. With confocal microscopy these problems are avoided; the scanning of a whole tissue volume is

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Fig. 5. (A,B) Area of possible synaptic contact between an intraspinal stretch receptor neuron and an interneuron in the lamprey spinal cord (of. Viana Di Priseo et al., 1990). (A) Stereo pair reconstruction, showing putative contact points between the upper interneuron dendrite branch and the (less bright) receptor cell axon. The lower dendrite branch runs above the receptor cell axon. (B) After a 90 ° rotation around the vertical axis, the lower dendrite branch is seen not to be in contact with the receptor cell axon. Image height: 100 ~tm. Objective: 100 x/1.4 NA. (C) 5-Hydroxytryptamine (5-HT) immunofluorescent neurons in the lamprey spinal cord. The ventral half of the spinal cord was scanned (40 x objective). Image height: 250 lain. Adapted from Wall6n et al. (I 989). (D) Stereo pair reconstruction of a 5-HT immunoreactive neuron in the rat nucleus raphe obscurus. Surface-shaded image. Scale bar: 50 I~m.Objective: 100 x. Adapted from Brodin et al. (1988).

done automatically and relatively fast, and the individual optical sections are perfectly aligned. In addition, the capability of performing thin optical sections at short intervals in depth gives possibilities for detailed reconstructions of neuronal elements as viewed from any desired projection angle. As mentioned above, for three-dimensional visualization, confocal microscopy is best combined with fluorescence, and the potential analysing power together with immunofluorescence techniques is obvious. Of particular interest is the possibility to detect simultaneously more than one

fluorophore during the same scanning session, by means of multiple detectors, adequate beam splitting mirrors and filters. Dual detection scanning is now possible with several of the commerical instruments. The simultaneous detection of more than one fluorophore has the obvious advantage that the risk for tissue displacement between separate scanning sessions for the different fluorophores is avoided. Thus, the confocal images of two different fluorophores demonstrating, for example, transmitter coexistence in a neuron will be in perfect register to each other.

Confocal Microscopy While the advantages of confocal laser scanning microscopy (CLSM) are manyfold, there are certain factors that should be kept in mind to minimize problems. First, thick tissue sections (or whole-mounts) may absorb rather much of the excitation light, so that at deeper levels the light intensity and thereby the fluorescence becomes weaker than at the top. This could be compensated for by successively increasing the laser power or the detector sensitivity when scanning at deeper levels, or by using a tissueclearing agent to increase transparency (Wallrn et al., 1988). A related problem is that a reduction in resolution may occur due to light' refraction and scattering in the tissue. It therefore seems of importance to utilize a mounting procedure that gives the tissue a homogeneous refraction index similar to that of the mounting medium. This refractive index should, furthermore, match the refractive index of the immersion liquid used for the objective. Another factor of importance when scanning through thicker pieces of tissue is clearly the working distance of the objective. Microscope objectives with long working distances also have lower numerical apertures, leading to poorer performance in optical sectioning and depth resolution (cf. above). Thus, there is always a compromise to be made between working distance and resolution. However, this is a general fact applicable also to conventional microscopes, and in confocal microscopy the resolution is always improved. An additional factor relating to thick preparations is the limited penetration of antisera when utilizing immunofluorescence. In such cases this may be the limiting factor when scanning thick tissue, rather than the working distance of the objective (Brodin et al., 1988; Carlsson et al., 1989). ANALYSIS OF N E U R O N A L STRUCTURE IN THREE DIMENSIONS USING CONFOCAL MICROSCOPY To illustrate how the often complex morphology of neurons can be visualized with CLSM and computerized image processing, examples of reconstructions of fluorescence-labelled neurons in the lamprey central nervous system will be described. During intracellular recording in an in vitro preparation (e.g. Grillner et al., 1991), neurons were filled with Lucifer Yellow (Stewart, 1978) from the microelectrode, to allow a morphological characterization of the same, functionally identified, cell (cf. Viana Di Prisco et al., 1990). As mentioned above, after scanning through the preparation (in these examples typically a wholemount of the flattened, 250 lain thick, lamprey spinal cord) in the CLSM, the computer software can produce images of the neuron as viewed from any desired angle. Fig. 3A is a zero-degree projection image of a motoneuron in the lamprey spinal cord, as viewed from above. Rotation by 90 ° produces a side view of the cell, as shown in Fig. 3B, which is thus a reconstruction of the whole cell, and not

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analogous to a conventional cross-section. Note the axon exiting from the soma towards the left. The faithful reconstruction also of the thinner dendritic branches is evident in the lateral dimension (A) as well as in depth (B). A useful way to investigate the three-dimensional morphology of a cell is to examine stereo pair reconstructions. These can be produced by making two projection images with a small angular shift in between them (e.g. 6°). Fig. 3C shows such a stereo pair reconstruction of the same neuron. When viewed in stereo, preferably with the aid of stereo glasses, the three-dimensional structure of the cell becomes vividly apparent. The axon points towards the viewer, and the dendritic branches to the left are in the background (cf. B). In this stereo pair reconstruction a surface shading routine was used. Fig. 4 illustrates the effects of employing various image processing routines on the same projection image of the neuron as in Fig. 3A. Fig. 4A is a socalled look-through projection, i.e. with no further processing of the image. Similarly, the images of Fig. 3A,B are look-through projections, but with a different scaling of light intensity. Surface shading results in the image in Fig. 4B, and Fig. 4C shows the same reconstruction after surface extraction (see above). As an alternative to stereo pair reconstructions, another image processing routine, depth coding, can be used (Fig. 4D). Here the pixels located close to the viewer are given high intensity values, whereas pixels far from the viewer become darker. The bright axon in the front is apparent, as are some closeby dendritic branches in the lower left corner (cf. Fig. 3B). When studying the interation between neurons subserving a specific function, it is of significant value to be able to visualize the areas where the cells make synaptic contact. Since, however, the dendritic arborizations of the neurons are spread in all three dimensions to receive synaptic input from various sources, conventional two-dimensional reconstructions may not allow conclusive determinations of the points of contact. This is illustrated in Fig. 5A,B, where an area of possible synaptic contact between an intraspinal stretch receptor neuron and an interneuron in the lamprey spinal cord was scanned at high resolution (100 x ) after intracellular injection of Lucifer Yellow into both cells in a doublemicroelectrode experiment (Viana Di Prisco et al., 1990). In each of the two stereo images in Fig. 5A the brightly fluorescing stem dendrite of the interneuron is seen to send a few branches towards the stretch receptor neuron axon to the left. If viewed only in two dimensions, these images would suggest that there might be two or three points of possible synaptic contact between the two cells in this area. However, when viewed in three dimensions, it can be seen that the upper branch appears to make contact with the stretch receptor axon; indeed, the branch seems to terminate on a thin axon collateral. The lower interneuron dendrite branch clearly runs

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above the stretch receptor neuron axon. If the whole image volume is rotated around the vertical axis (Fig. 5B), it becomes obvious that the lower branch in A does not point in the direction of the receptor cell axon (now partly hidden underneath the interneuron dendrite). It is thus obvious that performing three-dimensional reconstructions of areas of possible synaptic contact allows a more accurate structural analysis, and determinations based on conventional two-dimensional images may in fact be incorrect. VISUALIZING TRANSMITTERIDENTIFIED NEURONS USING IMMUNOFLUORESCENCE AND CLSM As indicated above, the combination of immunofluorescence techniques and confocal microscopy appears as a powerful tool when investigating the morphology, location and distribution of nerve cells and fibres identified with respect to, for example, their transmitter content. As an example, Fig. 5C shows a confocalimage ofserotonergic cells and fibre plexa on either side of the midline in the lamprey spinal cord (Wall6n et al., 1989). The preparation had been incubated with 5-hydroxytryptamine (5-HT) antiserum and secondary fluorescein isothiocyanate (FITC)-coupled antibodies, before scanning in the confocal microscope. Also with immunofluorescence labelling, a high degree of resolution of fine details can be achieved. Fig. 5D is a stereo pair reconstruction from a high magnification (100 x objective) scanning ofa 5-HT neuron (FITC immunofluorescence) in the nucleus raphe obscurus of the rat (Brodin et al., 1988). The thin dendritic branches of this neuron are clearly discernible.

For the simultaneous detection of several fluorophores in multiply-labelled specimens, one might foresee a further development of new fluorescent markers as well as of new filters with improved separation capabilities. New lasers producing alternative wavelengths of excitation light are now becoming available. For example, promising results have recently been obtained with a confocal system combined with an ultraviolet-laser (Ulfhake et al., t991). A complementary approach to improve further the separation of multiple fluorophores is the use of computer software image processing (Mossberg, 1991). The continuous development of faster and even more powerful computers is naturally most beneficial for the CLSM technique and its application in, for example, chemical neuroanatomy, presumably limitations with regard to data storage capacity will continue to be of some concern, but will probably present less of a problem with faster storage media of high size capacity, like optical disk systems. Finally, the development of new image processing software techniques, and the application of existing ones to confocal microscopy, holds promise to become of great value, for example for photometric measurements in three dimensions. ACKNOWLEDGEMENTS The valuable comments on the manuscript from Dr Kjell Carlsson, the Royal Institute of Technology,Stockholm,are gratefully acknowledged. This work was supported by the SwedishMedicalResearchCouncil(projectno. 3026)and by the Swedish Council for Planning and Coordinationof Research (project no. 9302). REFERENCES

CONCLUDING REMARKS In the structural analysis ofimmunohistochemically or physiologically characterized neuronal elements, confocal laser scanning microscopy is becoming an important new technique in several research laboratories. The capacity for optical sectioning and the possibility of obtaining detailed three-dimensional reconstructions are the most prominent features of the CLSM technique. In particular, the threedimensional distribution of a neuron in a large volume of tissue can be faithfully reconstructed without the need to individually analyse and realign hundreds of histological sections. The technical obstacles inherent in an optical investigation of a thick piece of intact nervous tissue should, however, be taken into consideration. Hopefully the increased use of confocal microscopy systems will lead to the development of better microscope objectives with sufficient working distance, yet with high numerical apertures. New histological procedures giving even better tissue transparency etc., would also be most helpful.

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Accepted 2 July 1991