PROCEEDINGS OF THE PHYSIOLOGICAL SOCIETY MEETING AT IMPERIAL COLLEGE LONDON
13-14 December 1991 These abstracts were accepted by Members of the Society present at the Meeting
2P
Journal of Physiology (1992). 452, 2P
DEMONSTRATIONS Three-dimensional reconstruction of lesions in the rat brain M.J. Watson, V.A. Moss and 0. Holmes Department of Physiology, University of Glasgow, Glasgow G12 8QQ In studying the critical mass of neocortex which will support synchronous electrical discharges in the anaesthetized rat in vivo, it became necessary to reconstruct partial isolations of cuboids of cortex. After the physiological experiment, the rat's brain was removed and laid with its pial aspect on a flat surface. Subcortical structures were dissected away and the cortex was flattened between two slides before fixing in thionine formalin. Subsequently, sections of 10 or 20 gm parallel with the cortical surface were cut on a cryostat. The sections were viewed through a camera lucida microscope; cuts through the cortex and bridges of connecting neural tissue which had escaped section-
ing were traced with a digitizing tablet and a manual cursor. The data were captured and displayed (Moss, 1990) as shown in Fig. 1. The calibrations are based on measurements of the stereotaxic frame of the spacing of lesions. A
B
IV
Fig. 1. A, side view of 35 sections spanning the cerebral cortex. The shading shows uninterrupted bridges of laterally connecting neural tissue. Gaps correspond to sections which contained artefact and could not be read. B, section through the cortex perpendicular to the cortical surface to show lamination corresponding to the reconstruction. C, an angled view to show bridges of remaining lateral connections of the partially-isolated block. Calibration bars: 400 ,um.
M.J.W. is a Scottish Home and Health Department scholar. We thank Dr I. Montgomery for assistance and the Wellcome Trust and the Epilepsy Association of Scotland for support. REFERENCE
Moss, V.A. (1990). Trans. Royal Microsc. Soc. 1, 389-392.
3P
Journal of Physiology (1992), 452, 3P
Wide-field volume visualization of thick microscope sections by computed nearest neighbour deconvolutions N.F. Clinch*, C.J. Daly, J.F. Gordon, V.A. Moss and N.C. Spurway Department of Physiology, University of Glasgow, Glasgow G12 8QQ and *Fairfield Imaging, Forest Row, Sussex RH18 5EZ
It is now possible to reconstruct 3-D images by making optical sections of a relatively thick specimen using wide-field microscopy. Such images are built from a sequence of images, captured at regular intervals through the thickness of the specimen. Reduction of the out-of-focus contributions to these images is essential to avoid blurring of the final image. Attention has recently been directed to the possibility of achieving satisfactory optical sectioning through the use of the point-spread function (PSF) of the microscope to compute the 'out-of-focus' component of each 2-D image (Agard, 1984; Carrington et al. 1990). Agard's nearest neighbour algorithm was used to deconvolve sequences of serial images, including those obtained from vascular preparations stained using the nuclear dye Hoechst 33342 (bisbenzimide) which gives excellent fluorescent visualization (A = 0.49 pm) of the long nuclei in smooth muscle cells (Daly et al. 1992). A sequence of 20 images (each 512 x 512 pixels) was captured using a Cohu monochrome video camera mounted on a Leitz Orthoplan microscope (x 40 water-immersion objective NA 0.75) with an inter-slice spacing 1 pm. Using an IBM 386AT computer equipped with a DSP board (188 Labs), each of these images was processed with the image above and below, after first computing the PSF for the microscope objective. From the resultant 18 deconvolved images, 3-D volumetric images were reconstructed using alpha-blending (Drebin, 1988) to render the images transparent to all but the higher intensity values. To approximate true spatial relationships, an interpolated slice is placed between successive data slices. These images can be rotated to show side or oblique views and used to generate animation sequences. The process of interactive image deconvolution and computer-generated displays of 3-D volumes obtained before and after deconvolution will be demonstrated. This technique has also been applied to images obtained using transmitted-light microscopy, including colour images which were captured as sequences of separate red, green and blue images either by using a colour video camera or using a monochrome camera and a wedge interference filter to change the colour of the light source. We have found that this method gave excellent results as long as the inter-slice sampling interval was kept within the range of longitudinal resolution (approximately equal to 2 A/NA2, 2 pm for a NA 0.7 lens or 0.5 pm with NA 1.4); beyond this point, image quality was reduced due to excessive image contrast in the deconvolved images. REFERENCES
Agard, D.A. (1984). Ann. Rev. Biophys. Bioeng. 13, 191-219. Carrington, W.A., Fogarty, K.E., Lifschitz, L. & Fay, F.S. (1989). Handbook of Biological Confocal Microscopy, ed. Pawley, J., pp. 137-146. Madison, WI:IMR Press. Drebin, A. (1988). Computer Graphics (SIGGRAPH '88 Proceedings) 22, 65-74. Daly, C.J, Gordon, J. & McGrath, J.C. (1992). J. Vasc. Res. 1 (in the Press).
13-14 December 1991 These abstracts were accepted by Members of the Society present at the Meeting
2P
Journal of Physiology (1992). 452, 2P
DEMONSTRATIONS Three-dimensional reconstruction of lesions in the rat brain M.J. Watson, V.A. Moss and 0. Holmes Department of Physiology, University of Glasgow, Glasgow G12 8QQ In studying the critical mass of neocortex which will support synchronous electrical discharges in the anaesthetized rat in vivo, it became necessary to reconstruct partial isolations of cuboids of cortex. After the physiological experiment, the rat's brain was removed and laid with its pial aspect on a flat surface. Subcortical structures were dissected away and the cortex was flattened between two slides before fixing in thionine formalin. Subsequently, sections of 10 or 20 gm parallel with the cortical surface were cut on a cryostat. The sections were viewed through a camera lucida microscope; cuts through the cortex and bridges of connecting neural tissue which had escaped section-
ing were traced with a digitizing tablet and a manual cursor. The data were captured and displayed (Moss, 1990) as shown in Fig. 1. The calibrations are based on measurements of the stereotaxic frame of the spacing of lesions. A
B
IV
Fig. 1. A, side view of 35 sections spanning the cerebral cortex. The shading shows uninterrupted bridges of laterally connecting neural tissue. Gaps correspond to sections which contained artefact and could not be read. B, section through the cortex perpendicular to the cortical surface to show lamination corresponding to the reconstruction. C, an angled view to show bridges of remaining lateral connections of the partially-isolated block. Calibration bars: 400 ,um.
M.J.W. is a Scottish Home and Health Department scholar. We thank Dr I. Montgomery for assistance and the Wellcome Trust and the Epilepsy Association of Scotland for support. REFERENCE
Moss, V.A. (1990). Trans. Royal Microsc. Soc. 1, 389-392.
3P
Journal of Physiology (1992), 452, 3P
Wide-field volume visualization of thick microscope sections by computed nearest neighbour deconvolutions N.F. Clinch*, C.J. Daly, J.F. Gordon, V.A. Moss and N.C. Spurway Department of Physiology, University of Glasgow, Glasgow G12 8QQ and *Fairfield Imaging, Forest Row, Sussex RH18 5EZ
It is now possible to reconstruct 3-D images by making optical sections of a relatively thick specimen using wide-field microscopy. Such images are built from a sequence of images, captured at regular intervals through the thickness of the specimen. Reduction of the out-of-focus contributions to these images is essential to avoid blurring of the final image. Attention has recently been directed to the possibility of achieving satisfactory optical sectioning through the use of the point-spread function (PSF) of the microscope to compute the 'out-of-focus' component of each 2-D image (Agard, 1984; Carrington et al. 1990). Agard's nearest neighbour algorithm was used to deconvolve sequences of serial images, including those obtained from vascular preparations stained using the nuclear dye Hoechst 33342 (bisbenzimide) which gives excellent fluorescent visualization (A = 0.49 pm) of the long nuclei in smooth muscle cells (Daly et al. 1992). A sequence of 20 images (each 512 x 512 pixels) was captured using a Cohu monochrome video camera mounted on a Leitz Orthoplan microscope (x 40 water-immersion objective NA 0.75) with an inter-slice spacing 1 pm. Using an IBM 386AT computer equipped with a DSP board (188 Labs), each of these images was processed with the image above and below, after first computing the PSF for the microscope objective. From the resultant 18 deconvolved images, 3-D volumetric images were reconstructed using alpha-blending (Drebin, 1988) to render the images transparent to all but the higher intensity values. To approximate true spatial relationships, an interpolated slice is placed between successive data slices. These images can be rotated to show side or oblique views and used to generate animation sequences. The process of interactive image deconvolution and computer-generated displays of 3-D volumes obtained before and after deconvolution will be demonstrated. This technique has also been applied to images obtained using transmitted-light microscopy, including colour images which were captured as sequences of separate red, green and blue images either by using a colour video camera or using a monochrome camera and a wedge interference filter to change the colour of the light source. We have found that this method gave excellent results as long as the inter-slice sampling interval was kept within the range of longitudinal resolution (approximately equal to 2 A/NA2, 2 pm for a NA 0.7 lens or 0.5 pm with NA 1.4); beyond this point, image quality was reduced due to excessive image contrast in the deconvolved images. REFERENCES
Agard, D.A. (1984). Ann. Rev. Biophys. Bioeng. 13, 191-219. Carrington, W.A., Fogarty, K.E., Lifschitz, L. & Fay, F.S. (1989). Handbook of Biological Confocal Microscopy, ed. Pawley, J., pp. 137-146. Madison, WI:IMR Press. Drebin, A. (1988). Computer Graphics (SIGGRAPH '88 Proceedings) 22, 65-74. Daly, C.J, Gordon, J. & McGrath, J.C. (1992). J. Vasc. Res. 1 (in the Press).
