Extended confocal microscopy of myocardial laminae and collagen network

Extended confocal microscopy of myocardial laminaeand collagen network

A. A. YOUNG, I. J. LEGRICE, M. A. YOUNG & B. H. SMAILLDepartment of Physiology, School of Medicine, University of Auckland, Private Bag 92019,Auckland, New Zealand

Key words. Confocal fluorescence microscopy, extended volume image, myocardialblood vessels, myocardial collagen, myocyte organization, picrosirius red, three-dimensional cardiac microstructure.

Summary

Ventricular myocardium has a complex three-dimensionalstructure which has previously been inferred from two-dimensional images. We describe a technique for imagingthe 3D organization of myocytes in conjunction with thecollagen network in extended blocks of myocardium. Rathearts were fixed with Bouin’s solution and perfusion-stained with picrosirius red. Transmural blocks from the leftventricular free wall were embedded in Agar 100 resin andmounted securely in an ultramicrotome chuck. Confocalfluorescence laser scanning microscopy was used to obtain3D images to a depth of 60 mm in a contiguous mosaicacross the surface. Approximately 50 mm was then cut offthe surface of the block with an ultramicrotome. Thissequence was repeated 20 times. Images were assembledand registered in 3D to form an extended volume3800 × 800 × 800 mm3 spanning the heart wall fromepicardium to endocardium. Examples are given of howdigital reslicing and volume rendering methods can beapplied to the resulting dataset to provide quantitativestructural information about the 3D organization ofmyocytes, extracellular collagen matrix and blood vesselnetwork of the heart.

Introduction

Quantitative information about the 3D architecture ofventricular myocardium is required for the analysis ofmyocardial mechanics and activation in normal andpathological hearts (Spotnitz et al., 1974; Dolber & Spach,1987; Robinson et al., 1988; LeGrice et al., 1995). Inparticular, we require detailed information about theorganization of and spatial relationships between indivi-dual myocytes, muscle layers, cardiac connective tissueand blood vessels. To date, myocardial structure has been

studied using predominantly two-dimensional (2D) ima-ging techniques such as light and polarization microscopy,with transmission or scanning electron microscopy (TEM,SEM) used for higher resolution. These techniques yieldimportant morphological information and have providedinsight into the three-dimensional (3D) arrangement ofuniformly varying cardiac structures. However, theycannot be used to reconstruct complex spatial relation-ships as required to fully characterize myocardial archi-tecture. An imaging modality which is ideal for this task isconfocal microscopy (Pawley, 1995). This techniqueenables volume reconstruction from serial optical sectionsthrough thick tissue specimens and so provides a powerfulmethod for elucidating complex 3D structure in biologicaltissues.

Picrosirius red (PSR) has previously been used as aspecific stain for collagen types I, II and III. This dye isknown to enhance the birefringence of collagen fibres and isalso fluorescent in the Texas Red/Rhodamine range (Sweatet al., 1964; Junqueira et al., 1979; Dolber & Spach, 1987;MacKenna et al., 1996). The proposed mechanism ofcollagen staining is by the interaction between thesulphonic acid groups of sirius red and the basic aminoacids of the collagen molecule in the low-pH environmentprovided by picric acid (Junqueira et al., 1979). PSR doesnot bind to collagen in a stoichiometric manner (Puchtleret al., 1988) and therefore collagen content cannot bedetermined by the intensity of PSR fluorescence, althoughthe relative size and morphology of the collagen networkcan be clearly identified.

Dolber & Spach (1993) have used confocal fluorescencemicroscopy together with PSR staining to study the 3Dmorphology of cardiac collagenous structures. Cytoplas-mic background fluorescence was reduced after immersionin phosphomolybdic acid (PMA) prior to PSR staining.Although fine endomysial collagen could not be resolvedwith these methods, perimysial collagenous weaves and

Journal of Microscopy, Vol. 192, Pt 2, November 1998, pp. 139–150.Received 19 December 1997; accepted 22 June 1998

139q 1998 The Royal Microscopical Society

Correspondence to: A. A. Young. Tel: (649) 3737599; fax: (649) 3737499; e-

mail: [email protected]

cords were clearly identified. Their immersion stainingtechnique allows optical sections to be acquired up to10 mm deep into the tissue with acceptable signal-to-noiseratio (SNR). However, this is inadequate for a quantitative3D analysis of the laminar architecture of myocardiumand its relationship to the collagen network, since thewidth of a typical muscle layer is 50 mm and large-diametercollagen fibres can extend over significant distances(several myocyte lengths) before branching (Robinsonet al., 1988).

This paper presents a technique for tissue processing andextended 3D visualization of blocks of ventricular myocar-dium. The technique employs Langendorff perfusion fortissue fixation and staining, TEM tissue preparationmethods, optical and physical serial sectioning and compu-ter image processing. A block of rat left ventricular (LV)tissue spanning from epicardium to endocardium wasreconstructed in 3D from registered serial optical sections,resulting in a transmural image 3800 × 800 × 800 mm3 involume at a resolution of 1·56 mm per (cubic) voxel side.The distribution and morphology of myofibres, collagen,extracellular spaces and major vessels within this blockwere examined and compared with previous results fromstudies which used 2D techniques (light and polarizationmicroscopy, TEM and SEM). Finally, estimates of collagendimensions made using extended confocal microscopy werecompared with measurements of the same structures inTEM images at identical sites.

Methods

Staining

Experimental protocols were approved by the University ofAuckland Animal Ethical Committee. Adult Wistar ratswere anaesthetized with 3% halothane in oxygen. Heartswere rapidly excised and arrested by immersion in chilledcardioplegic solution (St Thomas’ Hospital Solution 1). Theaorta was mounted on a cannula for Langendorff perfusion.Warm (37 8C) oxygenated Krebs–Henseleit buffer was usedto re-establish spontaneous heart rhythm and when amaximal rate (typically 200 beats min¹1) was achieveddiastolic arrest was induced by switching the perfusate toSt Thomas’ Hospital Solution 1 (Robinson et al., 1984).Hearts were then perfused with Bouin’s fixative for 15 min,followed by PMA (2·5% aqueous solution) for a further15 min and finally picrosirius red (PSR: 0·1% solution ofsirius red F3–BA in saturated aqueous picric acid, pH < 2)at 1 mL min¹1 for approximately 2 h. In some experiments,the PMA step was omitted. Hearts were stored in Bouin’sfixative and further processed after 1 week. The aboveprotocol was determined after experimentation with differ-ent fixatives and staining procedures. In particular, resultswere compared with hearts fixed in formaldehyde (3%) anda formaldehyde–glutaraldehyde mixture (1·5% each).

Embedding

An equatorial ring < 2 mm thick was removed from theventricles (Fig. 1a). From this, a transmural segment of LVfree wall < 2 mm wide was cut between the papillarymuscles (Fig. 1b). The tissue segment was dehydrated ingraded ethanols (70, 80, 95, 100, 100%) and then inpropylene oxide, each step being 45 min in duration. Thetissue was infiltrated with a 50:50 mix of propylene oxide

Fig. 1. Schematic showing the location and orientation of LV tissueblock. (a) Equatorial ring (dashed lines) excised from the heart. (b)Segment cut from lateral LV free wall between the papillary mus-cles. (c) Tissue block set in resin mould. Cardiac coordinate system:C – circumferential direction, L – longitudinal direction, R – radialdirection. Note that the cross-hatched L–R (longitudinal–radial)surface which is vertical in (b) is uppermost in (c).

Fig. 2. Mounting assembly showing the resin block fixed in anultramicrotome chuck and glued into a plastic holder shaped likea microscope slide. (a) Plan view. (b) Section through PP. Hatchedregion indicates glue.

140 A. A. YOUNG ET AL.

q 1998 The Royal Microscopical Society, Journal of Microscopy, 192, 139–150

and Agar 100 resin (45 min) and then immersed overnightin 100% resin. The infiltrated segment was flat embeddedfor imaging in the longitudinal–radial (L–R) plane (Fig. 1c)and polymerized at 60 8C for 48 h. Some tissue sections werealso mounted in glycerol or CleariumTM for comparison.

Mounting

The tissue block was tightly secured in a Reichert–Jungultramicrotome chuck with the L–R surface orientatedparallel to the base. The chuck was fitted into a custom-made Perspex holder as shown in Fig. 2. This was designed(i) to fit into the slide recess of the microscope stage, (ii) toposition the upper surface of the block approximately in theplane of the stage and (iii) to fit interchangably into themicrotome and the microscope stage. The chuck was gluedto the holder to guarantee reproducible positioning of thespecimen in the microscope.

Confocal imaging and tissue sectioning

The block was trimmed to expose the tissue surface, whichwas imaged using a Leica TCS 4D Kr–Ar confocal laserscanning microscope (CLSM). A local coordinate system (seeFig. 3) was defined in which the x and y directions wereparallel to the microscope imaging plane aligned with thelongitudinal and radial directions, respectively, in the heart,and the z direction was vertical (perpendicular to theimaging plane in the circumferential direction). A stepper-motor-driven stage (nominally accurate to 1 mm) allowedtranslation in the x and y directions of up to 30 mm, usingeither a manual joystick or software command. Initially, thefour upper corners of the block were imaged in reflectancemode to assess the orientation of the surface with respect tothe imaging plane. A 10-mm vertical (z) displacementbetween diagonal corners was deemed acceptable. Other-wise, the block surface was trimmed using a glass knife withthe angle settings on the microtome altered to ensuresectioning parallel to the imaging plane. Once set, theultramicrotome settings were fixed for all further sectioning.

The Leica CLSM uses a mirror scanning system toacquire the image in the x and y directions, and a stage tiltmechanism to acquire the optical section planes. As thestage tilts the specimen may move in the y direction. Thismovement is zero if the image plane is co-planar with thestage; however, it increases the further the image plane isabove or below the stage. In order to avoid the distortionsinherent in this tilt mechanism the specimen was held sothat the top surface of the resin block was approximately atthe level of the microscope stage (see Fig. 2). We estimatedthat the tilt mechanism contributed up to 5% error over theextent of the reconstructed volume, with variations withinthis range depending on the position of the image planerelative to the stage.

Confocal images of the embedded specimen wereacquired without a coverslip using a 25× oil-immersionlens (NA 0·75). A Rhodamine/Texas Red filter set (568 nmexcitation wavelength, >590 nm detection filter) wasemployed, with the pinhole set at 11 optical units. Eightline averages were performed. With these settings opticalsections with acceptable SNR could be obtained up to adepth of 60–80 mm.

We adopt the following nomenclature to describedifferent components of the imaging and registration process(see Fig. 3): ‘slice’ denotes a single optical section, ‘stack’ refersto a single 3D acquisition of 40 optical sections. A ‘slicemosaic’ refers to a set of slices at the same z location (or lateralplane) and a ‘stack mosaic’ refers to a set of stacks obtained atthe same z location (i.e. covering the same range of lateralplanes). Finally a ‘stack volume’ refers to an extended 3Dimage data set built up from a series of stack mosaics.

Stacks comprising 40 optical sections, each with a400 × 400-mm field of view and 256 × 256-pixel imagematrix, were obtained through a depth of 62·5 mm. Thisimaging geometry resulted in cubic voxels (1·56 mm perside), which are preferred for digital reslicing and volumerendering. Although undersampled in the lateral plane,the axial resolution was comparable to the FWHMresponse of the microscope in this direction. The gain ofthe photomultiplier was manually adjusted with depth


Fig. 3. Schematic indicating the three-dimen-sional organization of the image volume in rela-tion to the specimen. The volume is assembledfrom an array of contiguous image stacks eachconsisting of 40 slices (optical sections). Theaxes LCR represent the cardiac coordinate sys-tem, while the xyz axes represent the imagingcoordinate system.

EXTENDED CONFOCA L MICROSCOP Y 141

during the acquisition to compensate for the progressiveloss of signal in deeper slices. Contiguous 3D stacks (with asmall overlap) were acquired by sequentially translatingthe stage through 392 mm in x and y directions (see Fig. 3).Immediately after acquisition of a stack, a printout of themiddle slice was obtained and incorporated into a hard-copy slice mosaic to ensure that there were no errors in thetranslation sequence. The bottom slice of each corner ofthe stack mosaic was also printed and used to repositionthe stage after each trimming step.

Once imaging of a stack mosaic was complete, the top40–50 mm of the block was trimmed off with a glass knife inthe Reichert–Jung ultramicrotome. The block was thenreturned to the microscope and repositioned by matchingnewly acquired images with the printouts of the bottomcorner images from the previous stack mosaic. The overlapof at least 12 mm in the z direction provided a safety marginagainst overtrimming. Imaging and trimming cycles wererepeated until a total of 20 overlapping stack mosaics wereobtained to a total depth of < 800 mm.

3D digital registration

The 3D stacks were registered by calculating the x, y, z offsetof each stack with respect to the origin. This was done usingcustom software using the OpenGL library on a SiliconGraphics workstation, making use of accelerated renderinghardware. Within a stack mosaic, individual stacks wereregistered relative to one corner. Since each stack mosaicwas imaged in a single session, there was no movement inthe z direction and neighbouring stacks could be aligned bytranslational offsets in the x and y directions only. Softwarewas written to adjust adjacent stacks in x and y untilstructural elements (collagen cords and cell boundaries)were matched across slice boundaries. Registration betweenstack mosaics was carried out as follows. The x, y and zoffsets of the four corners of adjacent stack mosaics weredetermined by matching 3D structure in overlappingregions of the corner stacks. Linear interpolation was thenused to calculate offsets for the intermediate stacks.

The offsets determined above were used to assemble thestack volume as a fully registered 3D image. In regions ofoverlap, only the upper slices from the lower stack mosaicwere retained since these had superior SNR. The stackvolume was partitioned and archived on magneto-opticaldisc. In order to visualize the entire imaged volume at once,a reduced resolution stack volume was generated in whicheach voxel was the average of a 3 × 3 × 3-voxel neighbour-hood in the original image.

Image segmentation

In order to aid visualization of the 3D arrangementof myocytes and collagen, the registered images were

processed using the public domain NIH Image program(developed at the US National Institutes of Health andavailable on the Internet at http://rsb.info.nih.gov/nih-image/). Signal due to collagen was separated from thatdue to both myocytes and background on the basis of signalintensity and grey-scale morphology. Slices were initiallysegmented using high- and low-intensity thresholds chosento label collagen and background unambiguously. However,there was considerable overlap of intensity for myocytes andcollagen between these thresholds. The ‘rolling ball’ back-ground subtraction algorithm of NIH Image (a spatial high-pass filter) was employed to select fine structure above acertain threshold in intensity, which was also labelled ascollagen. All collagen pixels were linearly mapped into thegrey-scale range 128–255, while pixels labelled back-ground were mapped into the range 0–29. Pixelswhich were neither collagen nor background werelabelled as myocytes and mapped into the grey-scale range30–127.

Accuracy of depth measurement

In confocal microscopy, resolution and scaling in the zdirection usually differs from that in the x–y plane.Spherical aberration, due to mismatch between therefractive indices of the embedding media and the immer-sion oil, degrades image resolution and introduces a scalingin the z direction (Hell et al., 1993). These issues wereinvestigated in a number of ways. Firstly, the refractiveindex of the resin was measured using a refractometer.Secondly, the effect of the embedding medium on depthmeasurement was determined by imaging two thin metalstrips offset and glued to each other and fixed to a glassslide. The difference in heights between the top of the twostrips was measured using the same oil-immersion lensbefore and after embedding in resin. Finally, the anisotropyof image acquisition was directly measured in resin-embedded tissue by comparing images obtained in differentorientations. A block was positioned for imaging in thecircumferential–longitidunal (C–L) aspect and a400 × 400 × 40-mm stack was acquired. The tissue blockwas then rotated 908 in the chuck and imaged in thecircumferential–radial (C–R) plane. An extended series ofC–R stack mosaics was acquired and registered as describedabove. This stack volume was then digitally resliced in theC–L plane, allowing comparison of the dimensions ofstructures imaged in the two orthogonal planes.

Accuracy of collagen dimension measurement

In order to determine the accuracy of estimates of collagenbundle diameter from confocal micrographs, comparisonswere made between images of the same bundles as seen inthe confocal microscope and the TEM. A region on the



surface of the block < 1500 × 1500 mm in size was selectedfor investigation and imaged with the confocal microscopeusing the 25× oil-immersion lens. Areas containing largecollagen bundles were then selected for further imaging to adepth of 20 mm with the 63× oil-immersion lens. Ultrathinsections (< 90 nm) were cut from the surface of the blockand prepared for transmission electron microscopy. Sectionswere picked up on Formvar-coated slot grids, treated withuranyl acetate and lead citrate, and collagen enhanced withtannic acid. One of the ultrathin sections was then viewedin the TEM (Philips 410) and the collagen bundles selectedpreviously were identified and then recorded at highmagnification (2000–9000×). The confocal stack contain-ing each collagen bundle was then re-examined in order toidentify the slice which most closely matched the plane ofthe TEM image containing that bundle. Correspondingmaximum and minimum diameters were then determinedfor each collagen bundle using NIH Image software and therelative differences calculated.

Results

Results describing the development of the technique arepresented from a number of experiments, together with anextended volume image of LV myocardium obtained from asingle heart.

Staining

Figure 4(a) shows a typical slice taken from a midwallregion of a transmural LV free wall block at 25×magnification. A higher power (63× lens) image of a similar

region is shown in Fig. 4(b). Staining was uniformthroughout the heart wall in all cases where the perfusionprocedure was successful. Collagen staining with PSR wasbest following fixation with Bouin’s solution. Formaldehyde-fixed hearts did not stain well for collagen while glutar-aldehyde fixation led to excessive autofluorescence. ForBouin’s-fixed hearts examined immediately after perfusion,PSR was found to be concentrated in the capillaryendothelial cell nuclei. Maximal collagen staining wasonly achieved 3–5 days after perfusion. Stained tissue wastherefore stored in Bouin’s solution for this period prior toembedding in resin.

Resin-embedded tissue had a similar appearance tosections mounted in glycerol or CleariumTM; however, thesignal intensity from myocytes was higher in resin-embedded specimens. For sections subsequently mountedin glycerol or Clearium, pre-PSR treatment with PMAreduced the myocyte signal. This was not observed in resin-embedded tissue, in which the only apparent effect of PMAwas to reduce the signal from the endomysial collagen.Endomysial collagen was most evident in higher magnifica-tion images and was clearly seen in non-PMA-treated tissue(Fig. 4b). HCl treatment after staining with PSR haspreviously been used to improve the collagen signal inmounted sections (Dolber & Spach, 1993) but we did notobserve any difference with HCl treatment in resin-embedded tissue.

Registration

The overlap in the x and y directions between adjacentstacks in the same stack mosaic was consistently 2–5


Fig. 4. Image slices from LV midwall. (a) Typical slice from PMA-treated specimen: 25× objective, 256 × 256-pixel image of 400 × 400-mmfield. (b) Higher magnification image from non-PMA-treated specimen: 63× objective, 512 × 512-pixel image of 150 × 150-mm field. Endo-mysial collagen surrounding individual myocytes is evident. Myocytes (M), collagen cords (C), vascular structures (V) and cleavage planes(P) are indicated.


pixels. However, the overlap in the z direction betweenadjacent stacks was more variable (up to 15 voxels). Allstacks could be registered to the nearest voxel bymatching collagen structures in the overlapping regions.Figure 5 is a fully registered extended 2D image (slicemosaic) spanning the LV wall from epicardium toendocardium. Figure 6 is a rendered 3D projectionshowing the entire stack volume spanning3600 × 800 × 800 mm. It is evident that this provides acoherent view of the 3D organization of ventricularmyocardium. The slightly striated texture of the frontsurface is due to variation in intensity with depth in eachstack. This was minimized by progressive adjustment ofphotomultiplier gain during each 3D acquisition.

Three-dimensional organization of ventricular myocardium

Figure 6 provides a macroscopic view of the 3D organizationof ventricular myocardium. The top and front faces of thereconstructed volume are (in cardiac coordinates) the L–Rand C–R aspects of the block. The most striking features ofthis 3D image are the laminar organization of myocytes andthe extensive cleavage planes between layers. When viewedfrom the L–R aspect, the layers have an approximatelyradial orientation, becoming more longitudinal in the

subendocardial and subepicardial regions, with a chevronor pinnation pattern in the C–R aspect. These orientationsare consistent with those seen previously in rats and dogsusing 2D techniques (Hort, 1960; Spotnitz et al., 1974;LeGrice et al., 1995). Connective tissue (white) is organizedin close association with the muscle layers and apparentlyincreases in density near the epicardial and endocardialsurfaces (left and right sides of the figure, respectively). Theextensive collagen network covering the epicardial surface isobvious in this figure, as is the convoluted nature of theendocardial surface.

With an extended volume image, such as that presentedin Fig. 6, it is possible to investigate local microstructuresystematically in its 3D context. Structures observed in 2Dextended sections can be interpreted with greater con-fidence because they may be followed in any directionthroughout the adjacent volume using a wide variety ofviewing modes. Preliminary results obtained in this way areoutlined below.

Myocyte orientation and laminar structure

In Fig. 7, the stack volume has been digitally sectionedapproximately parallel to the epicardial surface and 2Dviews are presented at five representative sites through the

Fig. 5. Slice mosaic through the registered stack volume spanning the heart wall from epicardium to endocardium.

Fig. 6. Three-dimensional representation of the registered stack volume. The longitudinal radial (L–R) surface of the block is uppermost andthe front face is the most basal C–R aspect of the block. Epicardial and endocardial surfaces are indicated.



LV wall. Myocytes in longitudinal section can be identifiedat this magnification and the laminar arrangement ofmyocytes is evident. Layers are typically three to four cellsthick and muscle bridges between adjacent laminae arerelatively sparse. It is evident that there is some reductionof image quality when reslicing an extended confocalvolume of this type, due to the reduced resolution in the zdirection.

The apparent orientation of myocytes (and layers)varied progressively through 1208 from subepicardium tosubendocardium, in agreement with previous histologicalresults (Streeter, 1979). In the past, the transmuraldistribution of muscle ‘fibre’ orientation has been quanti-fied using equivalent histological sections cut parallel tothe epicardial tangent plane, assuming that the imbrica-tion angle (the angle between the myocyte axis and theepicardial tangent plane) is negligible (Streeter, 1979).This assumption is tested in Fig. 8, which shows thecomplete block sectioned in five different orientations (allperpendicular to the epicardial tangent plane) chosen tocoincide with muscle layer orientations seen at the fivesites shown in Fig. 7. Imbrication angles can therefore beviewed directly at these sites and are negligible in thisspecimen.

Organization of connective tissue network

Extended confocal microscopy techniques also make itpossible to study the 3D arrangement of major elementsof the cardiac connective tissue matrix. Networks ofcollagen cords which span cleavage planes to connectadjacent muscle layers are evident in both Fig. 5 and Fig. 6.Large collagen cords which are aligned approximately withthe myocyte axis and extend over many cell lengths areobserved in the cleavage planes (Figs. 4–7). There alsoappears to be a relatively high density of these cords withinmuscle layers. These longitudinal cords have a convolutedrather than a coiled appearance.

Analysis of the extended volume image also demon-strates the problems inherent in attempts to quantifyconnective tissue organization from 2D transmural ventri-cular sections. In Fig. 8(b), the section plane is perpendi-cular to the myocyte axis at the subendocardial siteidentified by the broken line, while in Fig. 8(e), the sectionplane contains the myocyte axis at this site. The appearanceof collagen density in these two views is markedly different.In order to make valid comparisons of connective tissueorganization at different transmural sites it is thereforenecessary to account for local myocyte orientation.


Fig. 7. Transmural variation of muscle layer orientation. The stack volume is digitally sectioned approximately parallel to the epicardial sur-face at five sites distributed through ventricular wall from subepicardium (a) to subendocardium (e). The transmural location of these sites isindicated by the solid white lines in the corresponding panels a to e in Fig. 8. Note: the right-hand side of each panel in Fig. 7 corresponds tothe upper surface of the block.


The 3D organization of the perimysial collagen networkand its spatial relationship with the muscle layers is shownin Fig. 9(a,b). Collagen, segmented as outlined in theMethods section, is displayed in the presence and absence ofbackground signal due to myocytes. These rendered 3Dprojections show the density of the convoluted collagencords aligned with the myocyte axis and demonstrate thatthese cords are present within and between muscle layers.

Blood vessel network

Various components of the coronary circulation can be alsoidentified in cleavage planes and muscle layers. Typically,blood vessels are closely associated with the connectivetissue matrix and are aligned with the large collagen cords.However, the vascular spaces seen intersecting musclelayers do not follow this pattern. These structures aresurprisingly extensive and may traverse several layers. Theycan be identified by a thin wall which stains lightly withPSR and an interior with no discernible structure (unlikethe gaps between laminae, which contain collagen cords).

These vascular sinuses, which can be clearly seen in Figs. 4and 5, were manually segmented and volume rendered toobtain the 3D projection in Fig. 9(c,d).

Depth measurement validation

The refractive index of the polymerized resin was found tobe < 1·52 using a standard refractometer. This matchedthe refractive index of the immersion oil (1·515). Themeasured thickness of a metal strip imaged in oil and thenin resin differed by an average 5% (with the resinproducing slightly greater distances than oil). Similarly,there was good agreement between observed dimensions ofstructures imaged in two orthogonal orientations. Dis-tances measured between structural features in C–Limages from resin-embedded tissue and in equivalentimages reconstructed from C–R stacks showed less than6% difference. These results indicate that the acquired 3Dimages were approximately isotropic, with no significantdistortion in the z direction due to refractive indexmismatch.

Fig. 8. Transmural variation of imbrication angleand connective tissue organization. The volume isdigitally sectioned in five different orientations.Section orientation in (a)–(e) is parallel to the mus-cle layer orientation in Fig. 7(a–e), respectively.The solid white lines in each panel indicate thepositions of corresponding sections in Fig. 7.Thus the solid line in each panel indicates thetransmural site at which the myocyte axis is inplane with the section, while broken white linesindicate sites at which the myocyte axis is perpen-dicular to the section plane.



Comparison with TEM

Comparison of TEM images with confocal images of thesame tissue confirmed that brightly fluorescent structures inthe confocal micrographs were collagen. This is illustratedin Fig. 10, where two collagen bundles labelled A and B areseen in cross-section in both the TEM and the confocalimages. The resolution of the digitized TEM image was0·024 mm, whereas in the confocal image it was 0·31 mm.Major and minor diameters of bundles A and B are 6·76,4·05, 5·04 and 3·57 mm in the TEM image and correspond-ing measurements in the confocal micrographs are 8·0,5·4, 6·7 and 4·4 mm. The measurements correspond tooverestimates of collagen bundle diameters of 18%, 33%,33% and 23%. Measurements from confocal images ofseven other bundles overestimated collagen bundle diameterby an average of 0·9 mm (23 6 16%) as compared with TEMimages; in no case did the confocal image underestimatecollagen bundle dimension.

Discussion

The techniques outlined in this paper provide a frameworkfor quantitative analysis of 3D structure in relatively large

biological specimens. The method employs confocal lasermicroscopy and was developed to study the arrangement ofcardiac myocytes, connective tissue and blood vessels inventricular myocardium. However, it could be used in avariety of tissues. A repeated sequence of optical andphysical sectioning was employed under conditions whichenable accurate spatial registration to be preserved. This hasenabled us to assemble an extended transmural image of ratventricular myocardium 3800 × 800 × 800 mm3 in volumewith a resolution of 1·5 mm per voxel side. As far as we areaware this is the most extensive data set of this type atpresent available.

Staining embedding and imaging protocol

We found that it was possible to stain uniformly for collagenthroughout thick myocardial specimens by perfusing PSRthrough the coronary arteries and storing the tissue inBouin’s fixative for at least 3 days prior to resin embedding.Hearts fixed or stored in formalin will not maintain stainingas well as hearts fixed and stored in Bouin’s fixative. Resinembedding was employed because it provides long-termspecimen stability, with reduced distortion under sectioning


Fig. 9. Three-dimensional representation of segmented subvolumes showing the arrangement of collagen (a,b) and vascular sinuses (c,d). Thevolume is 800 × 800 × 100 mm. Collagen segmented and rendered (a) in the presence of background signal due to myocytes and (b) withbackground removed. Manually segmented vascular sinuses rendered (c) in the presence of background signal due to myocytes and (d)with background removed.


and increased imaging depth compared with other mount-ing methods. Tissue could be imaged with acceptable SNRto a depth of 60–80 mm. The degradation of image qualitywas probably due to the accumulating effect with depth ofabsorbance and scattering of light by the tissue.

Phosphomolybdic acid (PMA) has previously been usedto minimize the cytoplasmic fluorescence (Dolber & Spach,1987, 1993). However, in resin-embedded specimens, PMAhad little effect on background fluorescence and finestructures such as endomysial collagen could be moreclearly identified without PMA treatment. The refractiveindex of the resin-embedded tissue did not changeappreciably when PMA was used, whereas PMA increasesthe refractive index of mounted tissue sections (Dolber &Spach, 1987). We conclude that the effects of PMA arelargely nullified during the dehydration and embeddingprocess. The perfusion and embedding protocol resulted in agreater myocyte signal than in sections mounted in glycerolor Clearium; however, this myocyte signal facilitatedanalysis of relationships between muscle and connectivetissue architecture.

At present, the dimensions of the specimens which canbe studied using these techniques are limited by theprocessing and sectioning methods employed. The cores oflarge tissue blocks are difficult to dehydrate and embed withresin, and the glass knives used in the ultramicrotomechipped specimens when removing sections more than2 mm transverse to the knife.

Accuracy of imaging and reconstruction

Cubic voxels were aquired in this study to simplify theprocesses of 3D image reconstruction, volume rendering

and digital reslicing of the image volume. The samplingdensity was set to reduce the volume of data acquired whileretaining structures of interest such as cardiac myocytesand the perimysial collagen network. Spherical aberrationdue to mismatch between the refractive indices wasminimized and errors in measured dimensions in the zdirection were thus relatively small.

We found that the motion of the specimen was notexactly specified by the stage scanning software, althoughwith sufficient bonding of the chuck to the holder the errorwas minimal. An overlap of 5 mm was sufficient to matchadjacent 400-mm-wide images. The overlap in the zdirection between adjacent stacks was more variable (upto 15 voxels) due to the inability to specify accurately theamount of material trimmed by the glass knife. Relativerotations of the image plane due to positioning inaccuraciesbetween imaging sessions were ignored in the reconstruc-tion process; however, structures could be registered to thevoxel resolution in all cases, implying that the rotationswere very small.

The precision with which collagen dimensions can bemeasured in PSR-stained myocardial specimens usingextended confocal microscopy has been determined bycomparing the cross-sectional dimensions of correspondingcollagen cords identified in confocal and TEM images.Collagen dimensions are consistently overestimated usingfluorescence confocal microscopy. The error is probably dueto the greater optical section thickness in the confocalmicroscope relative to the physical section thickness withTEM and the relatively large pixel size used in the confocalimages. This comparison provides a basis for assessing thereliability of collagen dimension measured using fluores-cence confocal microscope. However, it should be noted that

Fig. 10. Comparison of confocal image slice with TEM image of the same structure. (a) TEM image of myocyte (at left-hand margin) andcollagen cords (A,B) in cross-section. (b) Confocal image slice at same site. PSR-stained collagen cords are identified by the intensity of fluor-escence signal and are labelled appropriately.



the resolution of the confocal images precludes analysis offine collagen structures.

Measurements of collagen cord diameter may be affectedby fixation with Bouin’s solution, which can cause collagenswelling. Measurements of myocyte orientation and col-lagen density may also be affected by tissue shrinkage anddistortion in the fixation, dehydration and embeddingprocess. These artefacts are considerably less with resinthan with paraffin embedding and can be further minimizedby careful dehydration in suitably graded ethanol solutions.Previous studies (Hayat, 1989) report measurement arte-facts of less than 10% using the techniques employed here.

In the present application the collagen fibres provided anintrinsic network of fiducial markers which were used toregister adjacent overlapping data sets. Since the collagenfibres were not strictly parallel, their relative positionschanged appreciably from slice to slice, allowing registrationto within a pixel. Registration errors should therefore berelatively random from layer to layer and should notaccumulate over large distances. In other applications theuse of implanted fiducial markers is required in order toavoid the accumulation of errors. This is a difficult problemsince the markers must be small, well defined andstructured so as to allow accurate registration in threedimensions.

Collagen and myocyte structure

Rather than provide a detailed description of cardiacmicrostructure, the purpose of this paper was to describetechniques developed for the 3D analysis of that structure.The results presented demonstrate the advantages of usingan appropriate volume data set to analyse the 3Dorganization of complex biological structures such asventricular myocardium. They also highlight the need torefer morphological measurements to local coordinatesystems which reflect the characteristic microstructure ofthe tissue studied. Two specific examples are consideredbelow: imbrication angle and collagen density.

The extent of imbrication (orientation of myocyte axiswith respect to ventricular surfaces) at different sites withinthe left ventricle has long been the subject of controversy.Evidence has been presented by Streeter (1979) and co-workers that imbrication angles are present in the LV freewall but are typically less than 108. Using a transmuralvolume image, the 3D orientation of myocytes can bereliably determined and imbrication angles may therefore bequantified throughout the ventricular wall. In this paper,the image volume has been sequentially resliced so that thetransmural variation of imbrication angle can be visualized.We conclude that imbrication angle was negligible for theone myocardial specimen considered.

We have also shown that the appearance of perimysialcomponents of the cardiac extracellular connective tissue

matrix is very different in planes parallel with andtransverse to the local myocyte axis. Clearly, it is necessaryto account for this when comparing the organization ofperimysial collagen at different sites in the ventricular wall.Within this context, there was a clear transmural variationin the extent and the arrangement of perimysial connectivetissue in the one myocardial specimen considered here.There was also a high density of convoluted collagen cordswhich are aligned with the myocyte axis distributedthroughout muscle layers.

Disadvantages of the extended confocal microscopytechnique are that it is time-consuming and produces alarge amount of data. The reconstruction presented herespans the lateral LV wall of a rat heart and represents avolume of 2·43 mm3. About 2 h was needed to image onestack mosaic. Additional time is required to register theimages; however, this step may be automated (Becker et al.,1996). The extended volume consists of 20 stack mosaicsand requires 0·5 Gbytes of computer memory. It would beimpractical to use this approach to visualize the structuralorganization of an entire heart in three dimensions.However, the method is well suited to providing precisequantitative information about the 3D arrangement ofcomplex structures in relatively large tissue samples. Thetechniques described here are currently being used in ourlaboratory to characterize microstructure at selected sites inthe right and left ventricles of rats and to quantifymorphological changes associated with the development ofhypertrophy in spontaneously hypertensive rats.

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Extended confocal microscopy of myocardial laminae and collagen network

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