Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations

Abstract

Fenestrations are pores in liver sinusoidal endothelial cells that filter substrates and debris between the blood and hepatocytes. Fenestrations have significant roles in aging and the regulation of lipoproteins. However their small size (<200 nm) has prohibited any functional analysis by light microscopy. We employed structured illumination light microscopy to observe fenestrations in isolated rat liver sinusoidal endothelial cells with great clarity and spatial resolution. With this method, the three-dimensional structure of fenestrations (diameter 123+/-24 nm) and sieve plates was elucidated and it was shown that fenestrations occur in areas of abrupt cytoplasmic thinning (165+/-54 nm vs. 292+/-103 nm in non-fenestrated regions, P<0.0001). Sieve plates were not preferentially co-localized with fluorescently labeled F-actin stress fibers and endothelial nitric oxide synthase but appeared to occur in primarily attenuated non-raft regions of the cell membrane. Labyrinthine structures were not seen and all fenestrations were short cylindrical pores. In conclusion, three-dimensional structured illumination microscopy has enabled the unlimited power of fluorescent immunostaining and co-localization to reveal new structural and functional information about fenestrations and sieve plates.

Fig. 1

(A) Conventional deconvolution microscopy of LSECs stained with CellMask Orange, used to stain the cell membrane, showed that LSECs were intact and growing in a near- confluent monolayer. (Scale bar 20 μm). A selected cell of interest is examined at higher magnification below. (B) The selected cells from the image above were visualized using fluorescence deconvolution. (C) SIM image of the same cells showing improved resolution including fenestrations (circled). (Scale bar 2 μm, N nucleus, G gap).

Fig. 2

(A, B, C) SIM images of representative sieve plates showing clusters of fenestrations. (D) In comparison, conventional microscopy cannot resolve sieve plates. (E, F) Scanning electron microscopy of representative sieve plates. (Scale bar 200 nm)

Fig. 3

SIM of LSEC showing fenestrations clustered in sieve plates (the circle identifies the same sieve plate in each image). The image has been rotated so that Fig. 3A shows the top surface while Fig. 3D shows the underneath surface of the LSEC. (Scale bar 2 μm, G gap). A 3D movie is shown in the supplementary files.

Fig. 4

3D isosurface rendering of the sieve plate revealing that the lumen of the fenestrations span right through the cells. The sieve plate lies in an area of cytoplasmic attenuation. The 3D reconstruction can be rotated in all directions as shown. (Scale bar 300 nm). 3D movies are shown in the supplementary files.

Fig. 5

(A, B) SIM fluorescence images of phalloidin-labeled isolated LSEC showing F-actin stress fibers surrounding the sieve plates and at higher magnification (B) actin fibers can be seen forming a lace-like network around individual fenestrations. (Scale bars A: 2 μm, B: 500 nm). (C, D) SIM fluorescence images of an isolated LSEC labeled with Cell Mask Orange (orange) and eNOS (green). A low magnification image of the LSEC reveals scattered eNOS staining all over the cell membrane of the LSEC. A higher magnification image of the boxed area in C is shown as D. This highlights that there is only limited staining for eNOS in the sieve plates and there does not appear to be significant colocalization of eNOS with fenestrations. (Scale bars C: 5 μm, D: 500 nm) (E, F) SIM fluorescence images of an isolated LSEC stained for both F-actin and eNOS. The pattern of F-actin distribution is shown in orange-stress fibers and large gaps are apparent around liver sieve plates and scattered eNOS staining is seen in green. The boxed area in E is magnified and shown as F. At higher resolution, actin staining is apparent around individual fenestrations but eNOS does not appear to be colocalized with fenestrations. (Scale bars E:2 μm, F: 500 nm)