Abstract
AbstractSuper-resolution fluorescence microscopy, also known as nanoscopy, has provided us with a glimpse of future impacts on cell biology. Far-field optical nanoscopy allows, for the first time, the study of sub-cellular nanoscale biological structures in living cells, which in the past was limited to electron microscopy (EM) (in fixed/dehydrated) cells or tissues. Nanoscopy has particular utility in the study of “fenestrations” – phospholipid transmembrane nanopores of 50–150 nm in diameter through liver sinusoidal endothelial cells (LSECs) that facilitate the passage of plasma, but (usually) not blood cells, to and from the surrounding hepatocytes. Previously, these fenestrations were only discernible with EM, but now they can be visualized in fixed and living cells using structured illumination microscopy (SIM) and in fixed cells using single molecule localization microscopy (SMLM) techniques such as direct stochastic optical reconstruction microscopy. Importantly, both methods use wet samples, avoiding dehydration artifacts. The use of nanoscopy can be extended to the in vitro study of fenestration dynamics, to address questions such as the following: are they actually dynamic structures, and how do they respond to endogenous and exogenous agents? A logical further extension of these methodologies to liver research (including the liver endothelium) will be their application to liver tissue sections from animal models with different pathological manifestations and ultimately to patient biopsies. This review will cover the current state of the art of the use of nanoscopy in the study of liver endothelium and the liver in general. Potential future applications in cell biology and the clinical implications will be discussed.
Highlights
Super-resolution fluorescence microscopy, known as nanoscopy, has provided us with a glimpse of future impacts on cell biology
Several superresolution optical microscopy methodologies have been developed that allow optical resolution beyond the diffraction limit [41,42,43,44,45]. They can be classified into three broad classes: those based upon structured illumination microscopy (SIM) (Figure 2C and D), those based upon point spread function (PSF) engineering, and those based upon localization of individual fluorophores, commonly referred to as single molecule localization microscopy (SMLM) (Figure 2E and F)
The standard error in the fitted fluorophore position, i.e. the localization precision, can be written as σ / N, where σ is the standard deviation of the PSF of the experimental setup that is frequently approximated by a Gaussian function, and N is the number of detected photons emitted by the fluorophore
Summary
The liver is the largest internal organ of the body representing approximately 2–5% of the total body weight. A form of capillarization (fenestration loss, basement membrane deposition) of the liver sinusoids occurs during aging, termed “pseudocapillarization” [12, 25,26,27,28,29,30,31,32] These age-related morphological changes are accompanied by altered expression of many vascular proteins including von Willebrand’s factor, ICAM-1, laminin, caveolin-1, and various collagens [33]. The samples must undergo a series of fixation and dehydration steps that generate artifacts, such as shrinkage of the specimen and alteration of tissue structure [35] This may explain why the average diameter of the fenestrations, measured to be 150–175 nm by transmission electron microscopy (TEM), was found to be 105–110 nm by scanning electron microscopy (SEM) in the same study [36]. They can be classified into three broad classes: those based upon structured illumination microscopy (SIM) (Figure 2C and D), those based upon point spread function (PSF) engineering, and those based upon localization of individual fluorophores, commonly referred to as single molecule localization microscopy (SMLM) (Figure 2E and F)
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