Abstract
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.
Highlights
The diffraction limit of visible light (~200 nm for blue wavelengths) prevents our use of conventional light microscopy to study a number of biological structures, such as fenestrations, mitochondrial ultrastructure, super-fine filopodia, the clustering of filamentous proteins, membrane channels, and many other structures
We evaluated the use of available objective lenses from our obsolete microscope park to reduce costs even further
Using an oil-immersion 60 × /NA = 1.40 objective lens from an obsolete Olympus microscope, individual fenestrations of mouse liver sinusoidal endothelial cells (LSECs) were resolvable with direct stochastical optical reconstruction microscopy (dSTORM) [30] (Figure 1C, inset)
Summary
The diffraction limit of visible light (~200 nm for blue wavelengths) prevents our use of conventional light microscopy to study a number of biological structures, such as fenestrations (cellular pores ≤200 nm that allow free passage of molecules through cells), mitochondrial ultrastructure, super-fine filopodia, the clustering of filamentous proteins, membrane channels, and many other structures. Such structures were only visible through the use of electron microscopy (EM) on fixed and dehydrated samples, resulting in conclusions where their relevance to hydrated or possibly even living cells is often questionable.
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