Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells

Abstract

Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells

“…The passive transport of solutes through LSEC is primarily facilitated by fenestrations, which are 50–350 nm diameter, transcellular pores gathered in sieve plates. Fenestrations are present in the flat regions of the cell, occurring from the perinuclear cell zone to its periphery [ 4 , 5 ].…”

Far-Field Super-Resolution Microscopy Using Evanescent Illumination: A Review

“…Modulation of evanescent structured illumination on Si 3 N 4 waveguide has also been demonstrated by Balpeet Singh et al [61] who have been working on the development of chip-based microscopy for a long time [61][62][63][64][65][66]. They implemented a two-dimensional SIM method based on a Si 3 N 4 chip, which is named cSIM, as shown in Figure 4a,b.…”

Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria

“…Furthermore, LSECs incubated under 20% oxygen tension produced more lactate but did not consume more glucose. Since the publication of these results, various research groups have used conditions of 5% [46,78] or atmospheric [20,79,80] oxygen tension in their studies of LSEC biology.…”