Cost-efficient nanoscopy reveals nanoscale architecture of liver cells and platelets

Abstract

Single-molecule localization microscopy (SMLM) provides a powerful toolkit to specifically resolve intracellular structures on the nanometer scale, even approaching resolution classically reserved for electron microscopy (EM). Although instruments for SMLM are technically simple to implement, researchers tend to stick to commercial microscopes for SMLM implementations. Here we report the construction and use of a “custom-built” multi-color channel SMLM system to study liver sinusoidal endothelial cells (LSECs) and platelets, which costs significantly less than a commercial system. This microscope allows the introduction of highly affordable and low-maintenance SMLM hardware and methods to laboratories that, for example, lack access to core facilities housing high-end commercial microscopes for SMLM and EM. Using our custom-built microscope and freely available software from image acquisition to analysis, we image LSECs and platelets with lateral resolution down to about 50 nm. Furthermore, we use this microscope to examine the effect of drugs and toxins on cellular morphology.

The Hepatic Sinusoid in Aging and Disease: Update and Advances From the 20th Liver Sinusoid Meeting

“…Work by Karolina Szafranska (Arctic University of Tromsø)supported the detrimental effect of unbalanced BCAAs on LSEC morphology, showing reduced fenestration porosity and increased scavenging capacity. This group also presented on the ability of sildenafil and its analogues to increase porosity (confirming previous data (7,8) ), without any alteration in endocytic capacity. Building on previous work demonstrating the toxic effects of palmitic acid (a FFA) on hepatic toxicity, (9) Yana Geng (University of Groningen, Groningen, the Netherlands) presented data on the toxicity of oleate and palmitate on in vitro rat LSECs.…”

“…(73) However, impressive data derived from direct stochastic optical reconstruction, structured illumination, atomic force, and stimulated emission depletion microscopy presented by Peter McCourt (Arctic University of Tromsø) demonstrated that it is possible to stain and visualize the LSECs as they move between defenestrated, fenestrated, and ultrafenestrated states. (8,74) These techniques allow for the effects of therapeutic agents on the ultrastructure of LSECs to be viewed in real time. (8,67) With age, there is an accelerated development of fibrosis in the liver.…”

“…(8,74) These techniques allow for the effects of therapeutic agents on the ultrastructure of LSECs to be viewed in real time. (8,67) With age, there is an accelerated development of fibrosis in the liver. (66,75) Natalie Török (Stanford University, Sacramento, CA) presented data on the age-related changes in 52 kDa isoform Src homology 2 domain containing (p52Shc) in the liver and its contribution to accelerated fibrosis in aging.…”

The wHole Story About Fenestrations in LSEC

“…Sildenafil also increased cGMP levels, NO synthesis and levels of phosphorylated nitric oxide synthase (pNOS). Mao et al (2019) also challenged LSEC (from young mice) and found that the actin rings (which delineate fenestrations) and actin stress fibers became more pronounced. In contrast to Hunt et al (2019) and Mao et al (2019) found that sildenafil increased fenestration diameter on average by 30%.…”

“…NMN challenge had no apparent effects on NOS or cGMP levels in LSEC. Analysis of NMN challenged LSEC using direct stochastical optical reconstruction microscopy (dSTORM) revealed that the F-actin within LSEC was more condensed and that the actin rings delineating fenestrations became more pronounced ( Mao et al, 2019 ). The mode of NMN action in LSEC remains to be elucidated – NAD + associates with sirtuins which play a critical role in multiple cellular functions ( Imai and Yoshino, 2013 ) so the study of the role of sirtuins in fenestration biology is therefore warranted.…”