Usukura E et al. (2017), A Cryosectioning Technique for the Observation ...

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Sci Rep 2017 Jul 25;71:6462. doi: 10.1038/s41598-017-06942-1.

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A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM).

Usukura E , Narita A , Yagi A , Sakai N , Uekusa Y , Imaoka Y , Ito S , Usukura J .

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The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.

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	An illustration showing the difference in imaging between resin sections (A) and cryosections (B) with AFM. A: Polymerized resin is hard to remove; therefore, the cantilever just traces the flat surface of the resin section. B: For the cryosection, the embedding medium (sucrose) was removed by immersing the sections in PBS. The fine structures in cells and tissues were then exposed, which enabled the cantilever to trace along any undulations that appeared
	AFM images of the cryosections of photoreceptor rod outer segments after removing the sucrose embedding medium (A,B,C). Double-headed arrows indicate the scanning direction of the cantilever. (A) Highly ordered piling disks were observed, but the luminal spaces of the disks were almost collapsed, except at the incisures and the peripheral region (compared to the thin section EM image, D). (B) When enlarging the images of part of the disks, the luminal spaces look like slits. The unit membrane structure of the disks that appeared in D was not observed, but the structure appears to contain globular substances. (C) The marginal surfaces of the disk membranes are exposed but partially covered with cell membrane (asterisk) when the sections were near the peripheral region. (D) Thin-section EM image of the ROS inserted for a comparative analysis and better understanding of the ROS structure. The insets in A and D are Fourier transforms of A and B, respectively. The inset in A shows periodicity of disks piling up, but a high-resolution zone was obscured by another random structure, and there seems to be subtle information in the lateral direction of the disk membranes (arrows). Although the inset in D shows highly ordered periodicity of disks due to the contrast enhanced by the staining effect as though it were a line drawing, there is no information in the lateral direction of the disks.
	The AFM images of the connecting region between the outer and inner segment showing the outside and inside of the connecting cilium (CC). (A) The ciliary necklaces (an arrow) are exposed to the external surface of the connecting cilium because of the removal of sucrose. The upper section area contained microtubules (MT) that should be doubled in size. The finer structure of the microtubules is not observable. (B) The same image from A at a higher magnification. The external surfaces of the connecting cilium and microtubules are coloured in green and yellow, respectively. (C) A conventional thin-section EM image included as a reference.
	Atomic force micrographs of the synaptic active zone of photoreceptor cells (A,B,C). (A) The synaptic ribbons (SR) are tethered onto the cytoplasmic surface (green) of the pre-synaptic cell membrane with short filaments (arrow). The arrowheads indicate the characteristic circular structure on the cytoplasmic surface. (B) The post-synaptic membrane facing the pre-synaptic active zone contains several trans-membrane particles (arrow). An asterisk shows the cytoplasmic surface of the post-synaptic membrane. SR: synaptic ribbon. (C) The external surface of the post-synaptic membrane facing the active zone contains highly ordered particles (arrow). (D) A conventional thin section EM image in the synaptic active zone is provided in the inset for comparison. SR: synaptic ribbon.
	AFM immunocytochemistry identifying anti-opsin antibody binding (A,B). The anti-opsin antibody was used to evaluate whether AFM visualized the antibody binding because opsin is the major constituent protein that localizes to ROS. A: Immunofluorescence images of ROS labelled with the anti-opsin antibody and the secondary antibody conjugated to Alexa 568. The inset shows an enlargement of the white box. (B) Correlative AFM images of the green boxed area in A. Compared with the control (C), the anti-opsin antibodies conjugated with the secondary antibodies appear to have a complicated shape and bind heavily to disks in the ROS. The disk arrangement is also apparent behind the labelling at higher magnifications (inset). (C) Control AFM image incubated with non-immune IgG.
	Atomic force micrograph of the cryosection of a rabbit psoas muscle. (A) Phase contrast light microscopic image of cryosections placed on a glass slide and soaked in PBS. Striations consisting of the anisotropic band and isotropic band were found. (B) AFM images of the anisotropic band in a sarcomere (a part of A) showing the interaction between thin (actin) and thick (myosin) filaments clearly. (C) High power AFM image of the anisotropic band showing thick filaments swelled in the centre of the H zone (M-line) and cross bridges (an arrow) between thin and thick filaments. (D) High magnification image of the Z-disk showing attachment of actin filaments. The main feature of the Z-disk remains ambiguous. (E) A freeze-etching EM image for reference. Half-length of a sarcomere is depicted. Z: Z-disk; I: isotropic band; A: anisotropic band.
	Atomic force imaging of the cryosections of a pigment epithelial cell in retina. The cytoplasm in the perinuclear region is occupied by a complicated network of membranous structures (likely smooth endoplasmic reticulum). A similar expanse of membranous structures has been not detected with thin-section or freeze-etching EM.
	Figure 1. An illustration showing the difference in imaging between resin sections (A) and cryosections (B) with AFM. A: Polymerized resin is hard to remove; therefore, the cantilever just traces the flat surface of the resin section. B: For the cryosection, the embedding medium (sucrose) was removed by immersing the sections in PBS. The fine structures in cells and tissues were then exposed, which enabled the cantilever to trace along any undulations that appeared.
	Figure 2. AFM images of the cryosections of photoreceptor rod outer segments after removing the sucrose embedding medium (A,B,C). Double-headed arrows indicate the scanning direction of the cantilever. (A) Highly ordered piling disks were observed, but the luminal spaces of the disks were almost collapsed, except at the incisures and the peripheral region (compared to the thin section EM image, D). (B) When enlarging the images of part of the disks, the luminal spaces look like slits. The unit membrane structure of the disks that appeared in D was not observed, but the structure appears to contain globular substances. (C) The marginal surfaces of the disk membranes are exposed but partially covered with cell membrane (asterisk) when the sections were near the peripheral region. (D) Thin-section EM image of the ROS inserted for a comparative analysis and better understanding of the ROS structure. The insets in A and D are Fourier transforms of A and B, respectively. The inset in A shows periodicity of disks piling up, but a high-resolution zone was obscured by another random structure, and there seems to be subtle information in the lateral direction of the disk membranes (arrows). Although the inset in D shows highly ordered periodicity of disks due to the contrast enhanced by the staining effect as though it were a line drawing, there is no information in the lateral direction of the disks.
	Figure 3. The AFM images of the connecting region between the outer and inner segment showing the outside and inside of the connecting cilium (CC). (A) The ciliary necklaces (an arrow) are exposed to the external surface of the connecting cilium because of the removal of sucrose. The upper section area contained microtubules (MT) that should be doubled in size. The finer structure of the microtubules is not observable. (B) The same image from A at a higher magnification. The external surfaces of the connecting cilium and microtubules are coloured in green and yellow, respectively. (C) A conventional thin-section EM image included as a reference.
	Figure 4. Atomic force micrographs of the synaptic active zone of photoreceptor cells (A,B,C). (A) The synaptic ribbons (SR) are tethered onto the cytoplasmic surface (green) of the pre-synaptic cell membrane with short filaments (arrow). The arrowheads indicate the characteristic circular structure on the cytoplasmic surface. (B) The post-synaptic membrane facing the pre-synaptic active zone contains several trans-membrane particles (arrow). An asterisk shows the cytoplasmic surface of the post-synaptic membrane. SR: synaptic ribbon. (C) The external surface of the post-synaptic membrane facing the active zone contains highly ordered particles (arrow). (D) A conventional thin section EM image in the synaptic active zone is provided in the inset for comparison. SR: synaptic ribbon.
	Figure 5. AFM immunocytochemistry identifying anti-opsin antibody binding (A,B). The anti-opsin antibody was used to evaluate whether AFM visualized the antibody binding because opsin is the major constituent protein that localizes to ROS. A: Immunofluorescence images of ROS labelled with the anti-opsin antibody and the secondary antibody conjugated to Alexa 568. The inset shows an enlargement of the white box. (B) Correlative AFM images of the green boxed area in A. Compared with the control (C), the anti-opsin antibodies conjugated with the secondary antibodies appear to have a complicated shape and bind heavily to disks in the ROS. The disk arrangement is also apparent behind the labelling at higher magnifications (inset). (C) Control AFM image incubated with non-immune IgG.
	Figure 6. Atomic force micrograph of the cryosection of a rabbit psoas muscle. (A) Phase contrast light microscopic image of cryosections placed on a glass slide and soaked in PBS. Striations consisting of the anisotropic band and isotropic band were found. (B) AFM images of the anisotropic band in a sarcomere (a part of A) showing the interaction between thin (actin) and thick (myosin) filaments clearly. (C) High power AFM image of the anisotropic band showing thick filaments swelled in the centre of the H zone (M-line) and cross bridges (an arrow) between thin and thick filaments. (D) High magnification image of the Z-disk showing attachment of actin filaments. The main feature of the Z-disk remains ambiguous. (E) A freeze-etching EM image for reference. Half-length of a sarcomere is depicted. Z: Z-disk; I: isotropic band; A: anisotropic band.
	Figure 7. Atomic force imaging of the cryosections of a pigment epithelial cell in retina. The cytoplasm in the perinuclear region is occupied by a complicated network of membranous structures (likely smooth endoplasmic reticulum). A similar expanse of membranous structures has been not detected with thin-section or freeze-etching EM.

References [+] :

Ando, High-speed AFM imaging. 2014, Pubmed