Build and operation of a custom 3D, multicolor, single-molecule localization microscope.

Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems.

Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles.

To navigate through diverse tissues, migrating cells must balance persistent self-propelled motion with adaptive behaviors to circumvent obstacles. We identify a curvature-sensing mechanism underlying obstacle evasion in immune-like cells. Specifically, we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature-sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature.

Clathrin coats partially preassemble and subsequently bend during endocytosis.

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites.

Maximum-likelihood model fitting for quantitative analysis of SMLM data.

Quantitative data analysis is important for any single-molecule localization microscopy (SMLM) workflow to extract biological insights from the coordinates of the single fluorophores. However, current approaches are restricted to simple geometries or require identical structures. Here, we present LocMoFit (Localization Model Fit), an open-source framework to fit an arbitrary model to localization coordinates.

Photon-free (s)CMOS camera characterization for artifact reduction in high- and super-resolution microscopy.

Modern implementations of widefield fluorescence microscopy often rely on sCMOS cameras, but this camera architecture inherently features pixel-to-pixel variations. Such variations lead to image artifacts and render quantitative image interpretation difficult. Although a variety of algorithmic corrections exists, they require a thorough characterization of the camera, which typically is not easy to access or perform.

Systematic Tuning of Rhodamine Spirocyclization for Super-resolution Microscopy.

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a nonfluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, and an adjustment of the equilibrium poses a challenge for the design of suitable probes.

Expanding the Cross-Link Coverage of a Carboxyl-Group Specific Chemical Cross-Linking Strategy for Structural Proteomics Applications.

Carboxyl-group specific chemical cross-linking is gaining an increased interest as a structural mass spectrometry/structural proteomics technique that is complementary to the more commonly used amine-specific chemistry using succinimide esters. One of these protocols uses a combination of dihydrazide linkers and the coupling reagent DMTMM [4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium] chloride, which allows performing the reaction at neutral pH. The reaction yields two types of products, carboxyl-carboxyl cross-links that incorporate the dihydrazide linker and zero-length carboxyl-amine cross-links induced by DMTMM alone.

Quantitative Data Analysis in Single-Molecule Localization Microscopy.

Super-resolution microscopy, and specifically single-molecule localization microscopy (SMLM), is becoming a transformative technology for cell biology, as it allows the study of cellular structures with nanometer resolution. Here, we review a wide range of data analyses approaches for SMLM that extract quantitative information about the distribution, size, shape, spatial organization, and stoichiometry of macromolecular complexes to guide biological interpretation. We present a case study using the nuclear pore complex as an example that highlights the power of combining complementary approaches by identifying its symmetry, ringlike structure, and protein copy number.

Connecting color with assembly in the fluorescent B-phycoerythrin protein complex.

Phycoerythrin is the major light-harvesting pigment protein in red algae and is nowadays widely used as a fluorescent probe in biotechnological applications such as flow cytometry and immunofluorescence microscopy. In addition, it has had substantial economic impact due to its potential as a natural food colorant. However, knowledge on the precise molecular composition of phycoerythrin is limited.